Table of contents :

Laboratory instruments Estimating affinity and kinetics Osmometer Voltammeter Purification chromatography HPLC distillation Conway cell or microdiffusion apparatus centrifugation Structure modeling Nucleic acid analysis

Protein analysis Polyketide synthesis Testing for other molecules detections purification

• clinical laboratory : a laboratory for measurement and examination of materials derived from the human body (e.g., fluids, tissues, cells) for the purpose of providing information on diagnosis, prognosis, prevention, or treatment of disease.

• workbench
• cabinet
• vertical laminar air flow cabinet with UV
• vial : a small bottle
• phial : a vial or small bottle
• 1.5 mL microcentrifuge (microfuge) tube (Eppendorf)
• 2 mL microcentrifuge (microfuge tube)
• laboratory glassware refers to a number of tools used by chemists and biologists in performing scientific experiments. They include :
• test-tube : a thin glass tube closed at one end, used for various procedures in chemistry and for the growth of bacterial or viral cultures. A test tube is a kind of laboratory glassware, composed of a fingerlike length of glass tubing, open at the top, usually with a rounded lip at the top, and a rounded 'U' shaped bottom. They range in size from a couple inches to several inches long, from a few millimeters to a couple centimeters in diameter. They are designed to allow easy heating of samples, to be held in a flame, and often are made of expansion resistant glasses, such as Pyrex^®. Tests tubes are often preferred above beakers when multiple small chemical or biological samples have to be handled and/or stored. Vacutainers are a type of test tube that can be used for both collection and storage of blood.
• culture tube
• flask : container, such as a narrow-necked vessel of glass for containing liquid
• Erlenmeyer conical flask : a glass flask with a conical body, broad base, and narrow neck. It is a type of laboratory glassware which consists of an inverted conical base with a cylindrical neck. The main advantages in an Erlenmeyer flask are that is less likely to tip over than a beaker and the smaller neck which slows evaporative loss. It is named after the German chemist Richard Erlenmeyer. Sometimes to anchor an Erlenmeyer flask, a 'C' shaped lead or iron weight will be placed over the outside, to keep the flask firmly planted.
• Florence boiling flask : a round or flat-bottom flask with a long neck. It is designed for even heating and is produced in a number of different glass thicknesses to stand different types of use. The flask is named after Florence, Italy.
• round-bottom flask :
• vacuum flask :
• volumetric flask : a narrow-necked vessel of glass calibrated to contain or deliver an exact volume at a given temperature.
• beaker : a form of glass cup, usually with a lip for pouring, used by chemists and pharmacists. A beaker is a type of laboratory glassware which consists of a cylindrical cup with a notch on the top to allow for the pouring of liquids. They are about as wide as they are tall. This makes beakers very stable and easy to handle. They may be made of plastic, glass, or Pyrex^®. Some beakers have graduated markings, to allow an easy rough measure of liquid volume. As a means to make solutions, they are probably the most used piece of laboratory glassware. Coupled with a good magnetic stirrer, they see frequent heavy use in a laboratory.
• test glass : a small glass vessel, resembling a beaker, used in a chemical laboratory
• syringe : an instrument for injecting liquids into or withdrawing them from any vessel or cavity.
• probe syringe : a syringe whose point may be used also as a probe; used mostly in treating the lacrimal passages.
• Anel's syringe : a delicate syringe for the treatment of the lacrimal passages.
• fountain syringe : an apparatus which injects a liquid by the action of gravity
• gas syringe
• hypodermic syringe : a syringe, usually of small caliber, by means of which drugs in solution or other liquids are injected through a hollow needle of small bore into the subcutaneous tissues.
• Luer-Lok syringe : a glass syringe for intravenous and hypodermic use, with a metallic tip and locking device to hold the needle firmly in place.
• microsyringe : a syringe fitted with a screw-thread micrometer head for the accurate control of minute measurements
• dish : a shallow vessel of glass or other material for laboratory work.
• dappen dish : a small, heavy, solid glass, octagonal dish with a shallow depression to hold a few drops of medicaments or filling material.
• evaporating dish : a laboratory vessel, usually wide and shallow, in which material is evaporated by exposure to heat.
• culture dish : a shallow glass vessel for making bacterial cultures.
• Petri dishes : a Petri dish is a round, shallow, flat-bottomed transparent glass or plastic cylindrical dish with vertical sides and a similar but slightly larger dish that forms a cover, that biologists use to culture microorganisms on solid media and for tissue cell cultures. It was named after the German bacteriologist J.R. Petri (1852-1921) who invented it in 1877 when working as an assistant to Robert Koch. Usually, the dish is partially filled with hot liquid agar along with a particular mix of nutrients, salts and amino acids that match the metabolic needs of the microbe being studied (technically referred to as a "selective medium"). After the agar solidifies, the dish is ready to receive a microbe-laden sample (although to grow some microbes it is often necessary to apply the sample with the hot agar). Modern Petri dishes often have rings on the lids and bases which allow them to be stacked so that they do not slide off of one another.
• Stender dishes : vessels of various forms and sizes, used in preparing and staining histologic specimens.
• graduated cylinder : used for measuring the volumes of liquids in a semiquantitative manner. They consist of a long cylindrical tube, much taller than they are wide, with markings on the side to denote volumes. The top usually has a small curled lip to allow easy pouring of liquids, and the bottom is usually anchored with a wide base, to keep the cylinder from easily tipping. The volumes of liquids graduated cylinders can handle ranges from a few milliliters to many liters.
• pipette / pipet : a glass or transparent plastic tube used for measuring or transferring small quantities of liquid or gas. There are 2 kinds of pipettes, a kind with a bulbous middle section that has a single mark for the quantitative delivery of a single volume of liquid each time, and another that has many graduated marks, much like a graduated cylinder, that can deliver moderately accurate volumes (to within a few %) of liquids in variable amounts.
• Pasteur pipette are glass pipettes used to transfer fluids from one place to another. They are not graduated and are therefore not used to measure volumes. Like graduated pipettes they should be plugged with cotton wool and sterilized before use.
• disposable pipette
Micropipettes use plastic disposable tips. For ease of use, tips are usually packed into plastic boxes that can be autoclaved. Make sure the tips you are using will fit tightly onto the end of the pipette. Treat micropipettes very gently as they are precision instruments. Keep upright when in use to prevent liquids running inside the shaft of
the pipette. Do not leave pipettes lying on the workbench where they can be knocked off and damaged. Do not allow pipettes to come into contact with corrosive chemicals. Before use, make sure the volume has been correctly set. Adjust the volume before use. Most pipettes have a digital display of volume. Some brands have a micrometer setting, which can be difficult to read.  Check all tips are securely fitted to pipette. Draw liquid up. Check that the liquid drawn up has reached the expected level in the tip and there are no air bubbles in the tip. When using multichannel pipettes, check that the volume of liquid is the same in each tip. If necessary, expel the liquid and manually tighten the tips onto the pipette. Draw up the liquid and check again. Micropipettes have 3 positions:
• rest position
• first stop
• second stop

Fit the tip to the end of the shaft. Press down and twist slightly to ensure an airtight seal. Hold the pipette in a vertical position. Depress the plunger to the first stop. Air equal to the volume of the setting (e.g. 100 µL) is
displaced. Immerse the tip into the liquid. Release the plunger back to the rest position. Wait a second for liquid to be sucked up into the tip. The volume of liquid in the tip will equal the volume of the setting of the micropipette.
Place the tip at an angle (10-45°) against the wall of the vessel receiving the liquid, for example a well of a microwell plate. Depress the plunger to the first stop, wait one second, press the plunger to the second stop to expel all the liquid. Move the end of the tip away from the liquid. Release the plunger to the rest position. Graduated pipettes are calibrated and marked with graduation lines that allow the measurement of more than one volume. The volume is read by eye by reading the value indicated on the scale at the bottom of the meniscus. Disposable plastic graduated pipettes are available and are useful for pipetting toxic or viscous substances. Graduated pipettes made of glass can be washed and reused. The pipettes should be plugged with cotton wool on the top and sterilized before use to minimize contamination of fluids being measured. 1 mL and 10 mL graduated pipettes are most commonly used in the laboratory practices. Before using graduated pipettes check the volume scale and note: Does the pipette empty from full volume to zero or from zero to full volume? Is the pipette designed to be emptied by gravity with the tip in contact with the vessel or to be expelled by blowing out with a pipette filler? Pipetting by mouth is not an acceptable laboratory practice. Fluids are drawn up into pipettes using pipette fillers. There are several options. A simple rubber bulb is suitable for a 1 mL pipette : use the thumb and index finger to squeeze the bulb and the middle fingers to support the pipette. For 10 mL pipettes, use a triple valve rubber bulb, a hand operated pump or an electronic pipette filler.
• cuvette : a kind of laboratory glassware, usually a small square tube, sealed at one end, made of plastic, glass, or optical grade quartz and designed to hold samples for spectroscopic experiments. A good cuvette might hold 3 mL of liquid. Cuvettes may be completely open to the atmosphere or have caps (glass or Teflon^®) to seal them shut. Some cuvettes have a glass barrier that extends 2/3 of the way inside, so that measurements can be taken with 2 solutions separated, and again when they are mixed.
• burette / buret : a vertical cylindrical piece of laboratory glassware with a volumetric graduation on its full length and a precision tap, or stopcock, on the bottom. It is used to dispense known amounts of a liquid reagent in a titration experiment. It is extremely precise, and accurate to 0.5 ml. The meniscus of the liquid rests on the amount you wish to measure from.
• microburet : a buret with a capacity of the order of 0.1 to 10 mL, with graduated intervals of 0.001 to 0.02 mL.
• condensers : in systems involving heat transfer, a condenser is a chamber which converts a substance from its gaseous to its liquid state. In so doing, energy is given up by the substance, and the condenser will get hot. For example a refrigerator uses a condenser to get rid of the heat extracted from the interior of the unit to the outside air. Condensers are widely used in many types of system.
Often customized pieces of glassware are needed, and for this reason most large laboratories and research facilities keep a glassblower on staff
• glassblowing is the process of forming glass into useful shapes while the glass is in a molten, semi-liquid state. While the first evidence of man-made glass occurs in Mesopotamia in the Late-Third/Early-Second Millennium BCE, the actual "blowing" of glass using a tube did not occur until the First Century BCE. This advancement transformed the material's usefulness from a time-consuming process in which the medium was hot-formed around rough cores of mud and dung into a mass-producible material which could be quickly inflated into large, leakproof vessels.
• slide : glass plate on which objects are placed for microscopic examination
• cover glass / coverglass : a thin glass plate used to cover an object for microscopic examination
• burner : the part of a lamp, stove, or furnace from which the flame issues.
• Argand burner : a burner for oil or gas, with an inner tube for supplying air to the flame.
• Bunsen burner : a gas burner in which the gas is mixed with air before ignition, in order to give complete oxidation.
• carboy :
• tray : a flat-surfaced utensil for the conveyance of various objects or material.
• microtiter tray :
• centrifuge tube :
• vortexer :
• orbital shaker :
• tube rack :
• receptaculum : a receptacle or container; that which serves for receiving or containing something.

• CAPS buffer : pH 11, is a buffer formulated without glycine that is commonly used in sequencing and amino acid analysis applications where residual glycine will interfere with interpretation of results. Because the pH is 11, this buffer can be used for the blotting of proteins with a pI >8.5. CAPS buffer is recommended for tank transfer systems. In semi-dry transfer, it may produce variable transfer efficiency.
• 0.1 M phosphate buffer (PB) :
• Na₂HPO₄  10.24 g
• NaH₂PO₄  3.4 g
• Dissolve in 1 liter of distilled H₂O.
• pH to 7.4
• 10x phosphate-buffered saline (PBS, pH 7.2)
• sodium chloride (NaCl; M_r = 58.44) 137 M  8 g
• potassium dihydrogen orthophosphate (KH₂PO₄; M_r = 136.09) 1.76 M 0.2 g
• disodium hydrogen orthophosphate (Na₂HPO₄; M_r = 141.96) 8.1 M 1.15 g
• potassium chloride (KCl; M_r = 74.56) 2.7 M 0.2 g
• double-distilled water to final volume  1000 mL
• NaH₂PO₄ H₂O  32.1 g
• Na₂HPO₄  206.5 g
• NaCl  1318 g
• dissolve in 15 liters of distilled H₂O.
Note: to make a working solution, dilute the stock buffer 1:10 with distilled H₂O.
• lysis buffer
• sucrose 54.5 g
• Tris HCl 1M pH 7.4 5 mL
• MgCl₂ 1M 5 mL
• Triton X-100 5 mL
• 10 mM Tris HCl, pH 8.3
• 50 mM potassium chloride
• 2.5 mM MgCl₂
• 0.45% NP-40
• 0.45% Tween 20
• 100 mg/ml proteinase K
• TNN lysis buffer
• 50 mM Tris pH 8
• 250 mM NaCl
• 0.5% NP-40
• RBC lysis buffer
• 320 mM sucrose
• 10 mM Tris HCl pH 7.5
• 5 mM MgCl₂
• 1% Triton X-100
• 10X PCR reaction buffer (no MgCl₂):
• 500 mM KCl
• 100 mM Tris HCl, pH 8.3
• 10% DMSO
• 10% glycerol
• 10X heteroduplex annealing buffer :
• 1000 mM NaCl
• 100 mM Tris HCl, pH 7.8
• 20 mM EDTA
• physiological buffer
• NaCl 2.19 g
• disodium EDTA 3.65 g
• 10X TAE gel electrophoresis buffer
• 400 mM Tris-acetic acid
• 10 mM EDTA
1 L of 10X stock is prepared by dissolving in deionized H₂O : 48.8 g Tris base, 11.42 ml glacial acetic acid, 20 ml of 0.5 M EDTA (pH 8.0)
• 10X TBE gel electrophoresis buffer
• 50 mM Tris base (pH 8.5)
• 50 mM boric acid
• 0.5 mM EDTA
• 880 mM Tris base, pH 8.0
• 890 mM boric acid
• 20 mM EDTA
• 1 L of 10X stock is prepared by dissolving in deionized H₂O : 108 g of Tris base, 55 g of boric acid and 40 ml of 0.5 M EDTA (pH 8.0)
• TE buffer :
• 10 mM Tris (pH 8.0)
• 1 mM EDTA
• TN buffer
• 50 mm Tris HCl
• 300 mm NaCl
• pH 8.0
• alkaline transfer buffer
• 0.4 M NaOH
• 1 M NaCl
• GST lysis buffer
• 20 mM Tris-Cl (pH 8.0)
• 200 mM NaCl
• 1 mM EDTA (pH 8.0)
• 0.5% NP-40
• 2 mg/ml aprotinin
• 1 mg/ml leupeptin
• 0.7 mg/ml pepstatin
• 25 mg/ml phenylmethylsulfonyl fluoride (PMSF)
• blocking solution
• normal goat serum  10%
• bovine serum albumin (BSA)  2%
• Triton X-100  0.25%
• dissolve in 0.1 M PB

• equilibrium dialysis. The method is particularly well suited for studying the affinity of antibodies for haptens or small diffusable antigens. A solution of antibody (at a known concentration) is placed in a dialysis bag and equilibrated with a solution of radiolabelled hapten or antigen (initially placed outside the dialysis bag). At equilibrium the concentration outside the dialysis bag corresponds to free Ag whereas the concentration inside the bag corresponds to bound + free hapten. From this, the concentration of bound antigen can be calculated.
• fluorescence quenching. Proteins contain 3 amino acids that cause UV fluorescence: Phe, Tyr and Trp. For practical purposes, however, the fluorescence emitted by Phe is not readily exploited and, therefore, estimates of protein fluorescence reflect the fluorescence of Trp and/or Tyr depending on the wavelenght for excitation and emission. There are 2 major forms of fluorescence quenching:
• static : a complex is formed between the fluorophore Ab and the quencher Ag (which have to be non fluorescent). K_a = [F-Q] / ([F] [Q]), where [F-Q] is the concentration of the complex, [F] is the concentration of the uncomplexed fluorophore and K_a is the association constant of the complex. Since [F-Q] = [F]₀ - [F], K_a = ([F]₀ - [F]) / ([F] [Q]). If the complexed species is non fluorescent, the fraction of the fluorescence that remains (f) is given by the fraction of fluorophore that is not complexed (F/F₀) : substituting the fluorophore concentrations with fluorescence intensitites the following equation is obtained: K_a = (F₀ - F) / (F [Q])
• dynamic (or collisional) : the quencher must encounter and interact with the fluorophore during the lifetime of the excited state.
• surface plasmon resonance (SPR). In this technique one species (either Ag or Ab) is immobilised onto a layer of modified dextran and the second species is allowed to flow on that surface. A sensitive detector allows estimation of the amount of the immobilised species. As the changes in the mass of chemicals present on the sensor chip are recorded in real time, a series of curves can be generated (sensorgrams) from which both association and dissociation kinetics can be derived. SPR is a very sensitive technique generally applicable to the analysis of molecular interactions and will have a profound impact on the study of antibody-interactions as it does not suffer from restrictions.

• freezing-point osmometer : an osmometer using freezing-point depression measurement for analysis of osmotic pressure (number of particles, molecules, or ions) of solutions.
• Hepp osmometer : an osmometer in which very small quantities of material can be used and a direct reading of the osmotic pressure may be made.
• membrane osmometer : an osmometer in which diffusion through a semipermeable membrane indicates the osmotic pressure of macromolecules (number of molecules or ions) in a solution.

• anodic stripping voltammetry (ASV)
• differential pulse anodic stripping voltammetry (DPASV)

• chromatography : any of a diverse group of techniques used to separate mixtures of substances based on differences in the relative affinities of the substances for 2 different media, one (the mobile phase) a moving fluid and the other (the stationary phase or sorbent) a porous solid or gel or a liquid coated on a solid support; the speed at which each substance is carried along by the mobile phase depends on its solubility (in a liquid mobile phase) or vapor pressure (in a gas mobile phase) and on its affinity for the sorbent.
• separation methods
• ion-exchange chromatography (IEC/ IC) : that in which the stationary phase is an ion-exchange resin. The mobile phase is an aqueous buffer solution that determines the degree of ionization of the sample components and thus their affinity for the stationary phase.
• anion exchange chromatography (AIEC)
• cation exchange chromatography (CIEC)
• affinity chromatography : that based on a highly specific biologic interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand. Any of these substances, covalently linked to an insoluble support or immobilized in a gel, may serve as the sorbent allowing the interacting substance to be isolated from relatively impure samples (affinity purification); often a 1000-fold purification can be achieved in one step.
• immobilized metal affinity chromatography (IMAC)
• size exclusion chromatography (SEC)
• adsorption chromatography : that in which the stationary phase is a nonspecific adsorbent, such as silica gel, porous polymers, or charcoal.
• gel-filtration or gel-permeation chromatography / molecular exclusion or sieve chromatography : that in which the stationary phase consists of gel-forming hydrophilic beads containing pores of an accurately controlled size. As the sample is carried through the gel small molecules are frequently trapped in the pores and delayed while larger molecules pass unimpeded. Sample components are thus separated on the basis of size and shape. Exclusion chromatography can be used to separate molecules on the basis of size. The technique described here, which uses gel filtration to separate high-molecular-weight DNA from smaller molecules, is used most often to separate unincorporated labeled dNTPs from DNA that has been radiolabeled. It is also used at several stages during the synthesis of double-stranded cDNA, during addition of linkers to blunt-ended DNA, to remove oligonucleotide primers from PCR and, in general, whenever it is necessary to change the composition of the buffer in which DNA is dissolved. The 2 most commonly used gel matrices are Sephadex and Bio-Gel, both of which are available in several porosities. Sephadex G-50 and Bio-Gel P-60 are ideal for purifying DNA larger than 80 nucleotides in length. Smaller molecules are retained in the pores of the gel, whereas the larger DNA is excluded and passes directly through the column. Bio-Gel P-2 can be used to separate oligonucleotides from phosphate ions or dNTPs. Bio-Gel is supplied in the form of a gel and only needs to be equilibrated in running buffer before use. Sephadex is supplied as a powder that must be hydrated before use. Procedure^{ref1, ref2} :
• preparation of Sephadex
• 1 | Slowly add Sephadex of desired grade to distilled sterile water in a 500-ml beaker or bottle (10 g of Sephadex G-50 granules yields 160 ml of slurry; follow the manufacturer's instructions).
• 2 | Wash the swollen resin several times with distilled sterile water to remove soluble dextran. Soluble dextran can create problems by precipitating during ethanol precipitation
• 3 | Equilibrate the resin in Tris-EDTA buffer (TE; pH 7.6), autocleave at 10 p.s.i. (0.7 kg/cm²) for 15 min and store at 15-25°C.
• column chromatography :
• 4 | Prepare Sephadex or Bio-Gel columns in disposable 5-ml borosilicate glass-pipets or Pasteur pipets plugged with a small amount of sterile glass wool. Use a long, narrow pipet (for example, a disposable 1-ml plastic pipet) to push the wool to the bottom of the glass of the Pasteur pipiet
• 5 | Use a Pasteur pipet to fill the column with a slurry of the Sephadex or Bio-Gel, taking care to avoid producing bubbles. There is no need to close the bottom of the column. Keep adding gel until it packs to a level of 1 cm below the top of the column. Wash the gel with several volumes of Tris-EDTA-NaCl buffer (1X TEN; pH 8.0).
• 6| Apply the DNA sample (in a volume of 200 ml or less) to the top of the gel. Wash out the sample tube with 100 ml of 1X TEN buffer and load the wash buffer on the column as soon as the DNA sample has entered the gel. When the washing has entered the gel, immediately fill the column with 1X TEN buffer. CAUTION : columns used to separate radiolabeled DNA from radioactive precursors should be run behind Lucite screens to protect against exposure to radioactivity
• 7 | Immediately start to collect fractions (about 20 ml) in microcentrifuge tubes. If the DNA is labeled with ³²P, measure the radioactivity in each of the tubes by using either a handheld minimonitor of by Cerenkov counting in a liquid scintillation counter. Add more 1X TEN buffer to the top of the gel as required from time to time. The DNA will be excluded from the gel and will be found in the void volume (usually 30% of the total column volume). The leading peak of radioactivity therefore consists of radionucleotides incorporated into DNA and the trailing peak consists of unincorporated [³²P]dNTPs.
• 8 | Pool the (radioactive) fractions in the leading peak and store them at -20°C
• conjoint liquid chromatography (CLC)
• paper chromatography : a type of chromatography in which the stationary phase is a sheet of special-grade filter paper; it is in all other aspects similar to thin-layer chromatography
• partition or liquid-liquid chromatography : that in which the stationary and mobile phases are immiscible liquids and the sample components are separated on the basis of their partition coefficients
• thin-layer chromatography (TLC) : type of chromatography in which the stationary phase is a thin layer of an adsorbent, e.g., silica gel, SiO₂, coated on a 20 × 20 cm rigid or semi-rigid plate (normally a glass, aluminium or plastic sheet or plate; smaller sizes can also be used) and the mobile phase is an organic solvent mixture (100 mL). The sample is applied to a small spot 2 cm from a border of the plate, and then the plate is stood on end with its lower edge in solvent. As the solvent rises by capillary action through the adsorbent, the components of the sample are carried along at different rates and can be visualized as a row of spots after the plate is dried and stained or viewed under ultraviolet light. TLC is relatively inexpensive and simple to perform, and can be a powerful qualitative technique when used together with some form of sample pretreatment, such as solvent extraction. However, some separations can be difficult to reproduce. The interpretation of results can also be very difficult, especially if a number of drugs or metabolites are present. TLC of solvent extracts of urine, stomach contents or scene residues forms the basis of the drug screening procedure, and is also recommended for the detection and identification of a number of compounds. TLC can also be used as a semiquantitative technique
• preparation of TLC plates : the stationary phase is normally a uniform film (0.25 mm in thickness) of silica gel (average particle size 20 µm). Some commercially available plates incorporate a fluorescent indicator, and this may be useful in locating spots prior to spraying with visualization reagents. Prior soaking of the plate in methanolic potassium hydroxide and drying may improve the chromatography of some basic compounds using certain solvent systems but, generally, addition of concentrated ammonium hydroxide (relative density 0.88) to the mobile phase has the same effect. High-performance TLC  (HPTLC) plates have a smaller average particle size (5-10 µm) and greater efficiency than conventional plates. Reversed-phase plates, which have a hydrophobic moiety (usually C2, C8 or C18) bonded to the silica matrix, are also available. However, HPTLC and reversed-phase plates are more expensive and have a lower sample capacity than conventional plates. TLC plates can be prepared in the laboratory from silica gel containing an appropriate binding agent and glass plates measuring 20 × 20 × 0.5 cm. It is important to ensure that the plates are clean and free from grease. The silica gel is first mixed with twice its own weight of water to form a slurry. The slurry is then quickly applied  to the glass plate using a commercial spreader to form a film 0.25 mm in thickness. Small amounts of additives such as fluorescent markers can be included if required. The plates are dried in air and should be kept free of moisture prior to use. The quality of such home-made TLC plates should be carefully monitored; activation (i.e., heating at 100°C for 30 minutes prior to use) may be helpful in maintaining performance. Dipping techniques, whereby glass plates are coated by dipping into a slurry of silica and then dried, give very variable results and are not recommended. In general, home-made plates tend to give silica layers that are much more fragile than those of commercially available plates and chromatographic performance tends to be much less reproducible. Experience suggests that it is best to use one particular brand of commercially available plates. However, even with commercial plates dramatic batch-to-batch variations in retention and sensitivity to certain spray reagents may still be encountered
• sample application : some commercial plates are supplied with special adsorbent layers to simplify application of the sample. Normally, however, the sample is placed directly on to the silica-gel layer. The plate should be  prepared by marking the origin with a light pencil line at least 1 cm from the bottom of the plate - care should be taken not to disturb the silica surface in any way. A line should then be scored on the plate 10 cm above the origin to indicate the optimum position of the solvent front; other distances may be used if required. It is advisable when using 20 × 20-cm plates to score columns 2 cm in width vertically up   the plate with, say, a pencil since this minimizes edge effects. The samples and any standards should be applied carefully at the origin in the appropriate columns, using a micropipette or syringe so as to form spots no more than than 5 mm in diameter. If larger spots are produced, resolution will be impaired when the chromatogram is developed. The volume of solvent applied should be kept to a minimum; typically 5-10 µl of solution containing about 10 µg of analyte. Sample extracts reconstituted as appropriate should be applied first,  followed by the standards or mixtures of standards; this sequence minimizes the risk of cross-contamination. Glass capillaries intended for use in melting-point apparatus can easily be drawn out in the flame of a microburner to give disposable micropipettes with a very fine point. Ideally, the solvent used in applying the sample should be the same as that used to develop the chromatogram, but this is not always practicable; methanol will usually prove satisfactory. The plate may be heated with a hair-drier, for example, to increase the speed of evaporation of the spotting solvent, but it must be allowed  to cool before development starts and there is a risk of loss of volatile analytes such as amphetamines
• developing the chromatogram : glass TLC development tanks are available from many suppliers and   normally have a ground-glass rim which forms an airtight seal with a glass cover plate. A small amount of silicone lubricant jelly may be used to secure the seal. Some tanks have a well at the bottom which reduces the amount of solvent required. Smaller tanks are advantageous if smaller plates are used. All tanks should be lined with filter-paper or blotting paper on three sides and the solvent should be added at least 30 minutes before the chromatogram is to be developed. This helps to produce an atmosphere saturated with solvent vapour, which in turn aids reproducible chromatography. Some TLC mobile phases consist of a single solvent but most are mixtures; possibly the most widely used mobile phase in analytical toxicology is ethyl acetate/methanol/concentrated ammonium hydroxide (EMA). It is important to prepare mobile phases daily, since their composition may change with time because of evaporation or chemical reaction. In particular, loss of ammonia, not only from the mobile phase but also from opened reagent bottles, causes many problems. The chromatogram is developed by placing the loaded plate in the uniformly saturated tank, ensuring that the level of the solvent is above the bottom edge of the silica layer on the plate but below the level of the spots applied to the plate, and quickly replacing the lid. The chromatogram should be observed to ensure that the solvent front is rising up the plate uniformly. Usually the solvent front will show curvature at the edges of the plate; more serious curvature or bowing may be observed if the tank atmosphere is not sufficiently saturated with solvent vapour. This effect can be minimized by dividing the plate into 2-cm columns. The chromatogram should be allowed to develop for the intended distance, usually 10 cm from the origin. The plate should then be taken from the tank, placed in a fume cupboard or under a fume hood and allowed to dry. This process may be enhanced by blowing warm air (from a hair-drier) over the plate for several minutes until all traces of solvent have been removed. This can be especially important with ammoniacal mobile phases, since the presence of residual ammonia affects the reactions observed with certain spray reagents
• visualizing the chromatogram : when the chromatogram has been developed and the plate dried, the    chromatogram should be examined under ultraviolet light (at 254 nm and 366 nm) and the positions of any fluorescent compounds (spots) noted. This stage is essential if a fluorescent marker has been added to the silica, as any compounds present appear as dark areas against a fluorescent background. However, in analytical toxicology the use of chromagenic chemical detection reagents generally gives more useful information. Plates can be dipped in reagent but, unless special precautions are taken, the structure of the silica tends to be lost and the chromatogram destroyed. Thus, the reagent is normally lightly applied as an aerosol, using a commercial spray bottle attached to a compressed air or nitrogen line. Varying the line pressure varies the density of the aerosol and thus the amount of reagent reaching the chromatogram in a given time. Normally, the plate should be sprayed in an inverted position,since this avoids the risk of excess reagent being drawn up the plate by capillary action and destroying the lower part of the chromatogram.   Glass plates can be used to mask portions of the plate if columns are to be sprayed with different reagents. Alternatively, if plastic or aluminium plates are used then columns can be cut up and sprayed separately. e appearance of certain compounds may change with time, and it is important to record results as quickly and carefully as possible, noting any changes with time. A standardized recording system is valuable for reference purposes. Many spray reagents are extremely toxic - always use a fume cupboard or hood when spraying TLC plates.
• retention factor (Rf) = (distance travelled from the origin by the analyte) / (distance travelled from the origin by the solvent front). A more convenient value is Rf × 100 (hRf), especially if a standard length of chromatogram of 10 cm is always used, since then hRf is equal to the distance in millimetres travelled from the origin by the analyte. There are many factors that influence the reproducibility of hRf values including
• the TLC plate itself
• the amount of analyte applied to the plate
• the development distance
• the degree of tank saturation
• the ambient temperature
However, the influence of these factors can be minimized if standard (reference) compounds are analysed together with each sample. For unknown substances, it is a relatively simple procedure to obtain a corrected hRf value from a calibration graph constructed from experimentally observed values of sample and reference compounds. However, a further complicating factor is that the chromatography of compounds that originate from biological extracts may be different from that of the pure substances because of interferences from additional material present in sample extracts (matrix effects). Different chemicals may have the same Rf : they can be distinguished changing the TLC solvent of practicing GLC or HPLC
• column chromatography : a type of chromatography using a sorbent packed in a column. The sample, dissolved in a solvent, is poured in the top. Some components are retained in the column bound to the sorbent. They are then washed out (eluted) in successive aliquots of the same solvent (more strongly bound components being eluted later) or of different solvents.
• gas chromatography (GC) : a type of automated chromatography in which the sample, dissolved in a solvent, is vaporized by a vaporizer (whose temperature is 50° higher than that of the separation column) and carried by an inert gas (N₂ > CO₂, H, He, or Ar) through a U- or serpentine-shaped separation column (in glass, resin or metal : copper, brass or stainless steel; length from a few cm to 30 m) at T = 20-400°C packed with a sorbent to any of several types of detector. Each component of the sample, separated from the others by passage through the column, produces a separate peak in the detector output, which is graphed by a chart recorder. The sorbent may be an inert porous solid (gas-solid chromatography (GSC)) or a nonvolatile liquid coated on a solid support (gas-liquid chromatography (GLC); the commonest). The chromatogram is the plot of voltages [mV] vs. retention times. It allows contemporaneous qualitative (retention time) and quantitative (area under the peak, approximated as height x width at mid-height) analysis of a sample.
• head-space GC : as for Henry's law the concentration of analyte in the air overlying the liquid phase is the same as in the liquid phase, a sample of air is assayed for the analyte by GC
• hydrophobic interaction chromatography (HIC) is used for plasmid DNA purification. It removes impurities such as RNA, gDNA fragments, endotoxins, oligonucleotides and plasmid variants, which have similarities with plasmid DNA. By using matrices derivatised with mildly hydrophobic ligands (e.g. polypropylene glycol) it is possible to separate supercoiled and relaxed plasmid DNA from the impurities on the basis of single stranded nucleic acids that show a high exposure of the hydrophobic aromatic bases when compared with double stranded nucleic acids. In double stranded plasmid molecules the hydrophobic bases are packed and shielded inside the helix and thus hydrophobic interaction with the HIC support is minimal. On the other hand, the high content of single strands in the nucleic acid impurities (RNA and gDNA) after alkaline lysis enables hydrophobic interactions to take place. Endotoxins (e.g. LPS) interact even more strongly with HIC media via the lipidic moiety. Product quality can be controlled by HPLC, Southern slot blot, agarose gel electrophoresis, Limulus amebocyte lysate (LAL) test and protein assays.
• high-performance or high-pressure liquid chromatography (HPLC) : a type of automated chromatography in which the mobile phase is a liquid, which is forced under high pressure (100 atm) through a 25 cm column packed with a sorbent (rsin, ...). As in gas chromatography, a detector at the end of the column coupled to a chart recorder graphs the sample efflux. Various separation methods, including adsorption, gel filtration, ion-exchange, and partition, are used. The chromatogram is the plot of voltages [mV] vs. retention times. It allows contemporaneous qualitative (retention time) and quantitative (area under the peak, approximated as height x width at mid-height) analysis of a sample.

Web resources :
• PolyLC part numbers and prices
• SDS removal guard columns
• PolyGLYCOPLEX complex carbohydrates columns
• PolyWAX LP weak anion exchange columns
• ALKYL aspartamide HIC columns
• PolySULFOETHYL aspartamide strong cation exchange columns
• Polyaspartic acid weak cation exchange columns
• HILIC and SEC columns ; polysulfoethyl and polyhydroxyethyl columns
• distillation / vaporization : the process of vaporizing (by ebollition) and then condensing (by reducing temperature or increasing pressure) a substance to purify the substance or to separate a volatile substance from less volatile substances
• destructive or dry distillation : decomposition of a solid by heating in the absence of air, which results in volatile liquid products.
• fractional distillation : that which is attended by the successive separation of volatilizable substances in the order of their respective volatility.
• vacuum distillation : distillation under reduced pressure to avoid the decomposition which might occur at atmospheric pressure
• molecular distillation : a process of purification applied to drugs and pharmaceuticals during which the crude material is evaporated under high vacuum of about one millionth of an atmosphere, and the condensate is caught on a cooled surface held close in front of the evaporating layer. The process is applied currently to vitamins A, D, and E, to animal and vegetable sterols and hormones, and to drugs and intermediates
• vapor current distillation : 200 g of organ homogenate or 100 mL of gastric content are mixed with a same water volume. Extraction > 70%; excellent purification (only water and volatile compounds); poor concentration (sample is diluted before distillation and analytes are then recovered with extraction solvents); 3-4 hours required
• for acids and neutral molecules (e.g. alcohols, chloroform, CCl4, trichloroethylene, glycols, apiol, organophosphates, ethinamate) : add 3 mL tartaric acid before extractoin
• for alkali (e.g. nicotine, amphetamines, ephedrine, ...) : add sodium hydroxide before extraction
• Conway cell or microdiffusion apparatus : liberation of a volatile compound (hydrogen cyanide in the case of cyanide salts) from a volume of test solution (too small to undergo distillation) held in the outer well. The volatile compound is subsequently trapped using an appropriate reagent (sodium hydroxide solution in the case of hydrogen cyanide) held in the inner well. The cells are normally allowed to stand for 2-5 hours at room temperature for the diffusion process to be completed. The analyte concentration is subsequently measured in a portion of the trapping solution either by spectrophotometry or by visual comparison with standards analysed concurrently in separate cells. The Conway apparatus is normally made from glass, but polycarbonate must be used with fluorides since hydrogen fluoride etches glass. The cover is often smeared with petroleum jelly or silicone grease to ensure an airtight seal. In order to carry out a quantitative assay at least 8 cells are needed: 1 blank, 3 calibration samples, 2 test samples and 2 positive controls. It is important to clean the diffusion apparatus carefully after use, possibly using an acid/dichromate mixture, rinsing it in distilled water before drying.


• centrifugation : the process of separating the lighter portions of a solution, mixture, or suspension from the heavier portions by centrifugal force.
• ultracentrifugation : subjection to the action of an ultracentrifuge (a centrifuge with an exceedingly high rate of rotation which will separate and sediment the molecules of a substance)
• density gradient centrifugation : ultracentrifugation in a liquid, such as cesium chloride solution (CsCl₂), the density of which increases along the lines of centrifugal force, the substances under test or preparation seeking their level of density.
• isopyknic centrifugation : that in which the solvent is of the same density as the substance to be isolated.
• differential centrifugation : that based on the sedimentation coefficient of the substances under investigation; applied to homogenates to derive various subcellular fractions.
• sedimentation : the act of causing the deposit of sediment, especially by the use of a centrifuge.
• erythrocyte sedimentation : the sedimentation of erythrocytes in a volume of drawn blood to calculate ESR
• Ritchie's formalin-ether sedimentation or method : a technique for detecting parasites in the feces, involving the centrifugation of diluted feces, the addition of formalin and ether to the sample, recentrifugation, and examination of the final sediment as a wet mount
• Svedberg unit (S) : a unit equal to 10^-13 second used for expressing sedimentation coefficients of macromolecules
• Svedberg flotation unit (Sf) : a unit equal to 10^-13 second used for expressing negative sedimentation coefficients of macromolecules that float rather than sink in a centrifuge, e.g., lipoproteins
• Kleinschmidt technique : rupture of the virion by osmotic shock so that viral DNA is exposed.

• Usenet bionet.structural-nmr

Polyketide synthesis : polyketides are assembled from fatty acids and include the antibiotics rifamycin and erythromycin, and the immunosuppressant FK506

• carbohydrate microarray :
• Detection
• staining
• gross fibers : scanning electron microscopy (SEM)
• oxygen (P_O2): van Slyke's device
• spectrometry : the determination of the wavelengths or frequencies of the lines in a spectrum by means of a spectrometer
• infrared spectrometer : a device that analyzes the chemical composition of a substance either by passing infrared light through a specimen and characterizing its absorption spectrum or by measuring the amount of infrared light emitted by excited atoms or molecules in a specimen; infrared light is emitted or absorbed in a given band in proportion to the concentration of the molecule characterized by the band .
• mass spectrometer (MS) or spectrograph : an analytical instrument which identifies a substance by sorting a stream of electrified particles (ions) according to their mass; the sorting is most commonly done as follows: when the stream of charged particles enters a magnetic field, the particles are deflected into semicircular paths varying with their mass and charge components, ultimately striking a photographic plate or photomultiplier tube sensor. In the postgenomic era, some resources, such as the genome sequence data that underlies functional genomics, are available to all. But MS remains mostly in the hands of the haves. Traditionally large, complex, and expensive, mass spectrometers are often found not in individual labs but instead in university core facilities. Prices are dropping to the point where a small group of scientists could pool resources to acquire one. Yet researchers continue to send their samples to the core for analysis, or collaborate with proteomics specialists willing to process the samples for them. MS is so valuable to functional genomics because it is unbiased. Microarray and RNA interference experiments must be targeted to specific genes or sequences. But MS reports data regardless of whether the experimenter is aware of a particular protein's existence. The resulting mass data can be used as a key to look up protein addresses in sequence databases and thereby identify them. The proteins don't even need to be purified; using tandem MS and a process called shotgun proteomics, it is possible to directly identify the protein components present in complex mixtures^{ref1, ref2}. These features, combined with frequent increases in the speed, sensitivity, and throughput of MS hardware, make the technique particularly amenable to the large-scale analysis of proteins in cells and subcellular compartments that is the essence of proteomics. 13 human diseases have been associated with disruptions of proteins integral to or interacting with the nuclear envelope (NE) and prior analyses have identified 13 integral membrane proteins^ref. Eric Schirmer and colleagues at the Scripps Research Institute used a subtractive proteomics method to conduct a more thorough analysis^ref. The project was particularly challenging because the outer membrane is contiguous with, and shares properties with, the endoplasmic reticulum (ER), making it difficult to know which proteins are specific to the nuclear envelope and which are specific to the ER. Schirmer's team isolated microsomal membranes containing ER and mitochondria from rodent tissues, and performed an exhaustive proteomic analysis of the recovered proteins. NEs were then isolated, extracted with 2 different strategies to ensure the sample was enriched in integral membrane proteins, and finally subjected to MS. Subtraction of the datasets revealed 67 new, integral membrane proteins. The human homologs of these proteins were identified and a representative set of the cDNAs representing the protein sequences were then epitope-tagged and localized within HeLa and COS7 cells. All the proteins tested localized to the NE, and at least 5 were found to strongly interact with the lamina, a polymer of intermediate-filament-type lamin proteins that line the inner nuclear membrane. As defects in lamin have been associated with different dystrophies and laminopathies, interactions between proteins and the lamina are of considerable interest. 23 of the proteins' genes colocalized to specific regions of the human genome associated with diseases that currently lack a known causative gene. Such associations are traditionally made through human genetic analysis, a long and tedious process. Admittedly, as each chromosomal region can be quite large and may contain several hundred genes, this analysis doesn't directly implicate any of these proteins in the disease. But it does make them interesting suspects. Schirmer's experiment was made possible by combining clever cell biological techniques with proteomic ones. On the cell biology side, the team used known fractionation techniques to remove likely protein contaminants. The analysis of integral membrane proteins, meanwhile, was made possible by shotgun proteomics, a method in which proteins are proteolytically digested prior to MS analysis rather then being isolated biochemically. In a second study, instead of isolating an organelle, researchers identified proteins involved in cytokinesis^ref. In late stages of cytokinesis a structure called the midbody forms, which contains microtubules tightly bundled by the cleavage furrow. Skop's team isolated this structure from CHO cells and analyzed its components proteomically. The group performed 4 separate enrichments, pooling the results from each analysis. Proteins were segregated by bioinformatic analysis to identify likely proteins involved in cytokinesis and their roundworm (Caenorhabditis elegans) homologs. RNAi was used to knock down the expression of the proteins in C. elegans and to observe the resulting phenotype in hermaphrodite animals expressing fluorescently tagged histone H2B and b-tubulin. Effects on embryonic divisions and gonad development were measured using video fluorescent microscopy. The majority of knockdowns (88%) resulted in observable defects. 17 proteins colocalized with tubulin, and all that were tested colocalized with the midbody. Because cytokinesis is highly conserved, these results will help researchers understand the process in other species. Though mass spectrometers are complex instruments, operation by nonexperts is possible in a highly motivated laboratory. Dissemination of proteomics technology to nonexperts is complicated, however, by the fact that the technology evolves rapidly, with new developments appearing almost monthly. Proteomics practitioners are often both technology developers and adopters in pursuit of solutions to particular biological problems – for example, identifying the many posttranslational modifications that can occur on proteins simultaneously, as well as how those modifications change over time. At the moment these experiments are technically challenging and thus difficult for nonexperts to execute, even when they have access to the necessary technology.
• time-of-flight (TOF) : separates ions of different masses, based on the time needed for the ions to travel a fixes distance
• ion trap : obtains peptide sequence information by fragmenting the peptide and measuring the ion mass/charge ratios. Mass accuracy, in general, is not as good as quadrupole time-of-flight (Q-TOF)
• matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) : analyzes subpicomole amounts of peptides, less than 6 kDa. Can be used with mixtures of proteins, peptides, salts and even some detergents
• quadrupole time-of-flight (Q-TOF) : useful for peptides up to approximately 5 kDa; similar resolution as MALDI-TOF. Has the added advantage that MS/MS experiments cann be performed to obtain sequence information on the peptides
• hybrid liquid chromatography Q-TOF (LC-Q-TOF MS-MS) : offers advantage for the identification of biotransformation pathways of drugs and biomolecules. An older technology that has made a comeback due to advances in electronics and computer control
• tandem mass spectrometry (MS/MS) : gas phase peptide ions undergo collision-induced dissociation (CID) with molecules of an inert gas such as helium or argon^ref. Other methods of dissociation have been developed, such as electron capture dissociation (ECD), surface induced dissociation (SID) and electron transfer dissociation (ETD), but gas-phase CID is the most widely used in commercial tandem mass spectrometers. The dissociation pathways are strongly dependent on the collision energy, but the vast majority of instruments use low-energy CID (<100 eV)^ref. At low collision energies fragmentation mainly occurs along the peptide backbone bonds, whereas at higher energies fragments generated by amino acid side-chains are observed^{ref1, ref2}. At low-energy CID, conditions normally used in triple quadrupole, quadrupole time of flight and ion trap (both linear and Paul) mass spectrometers, b-ions, y-ions and neutral losses of water and ammonia dominate the mass spectrum. Low-energy CID dissociates the amide bond along the peptide backbone. As a result, two fragments are produced, one containing the N terminus and the other containing the C terminus. Nomenclature denotes the N-terminal fragments with letters a, b and c and the C-terminal fragments with letters x, y and z^ref. Internal ion fragments are formed by simultaneous cleavage of N and C termini. Immonium ions are of the structure HN = CH–R^ref. A numerical subscript for each fragment ion indicates the position of the amino acid at which the bond cleavage occurs. For N-terminal fragments, the numbering starts from the N terminus, and for C-terminal fragments, the numbering starts from the C terminus. Fragmentation patterns are also strongly dependent on the chemical and physical properties of the amino acids and sequences of the peptide^{ref1, ref2}. Most algorithms assume that peptides preferentially fragment into b- and y-ions. The distribution of intensities between b- and y-ions is the subject of intensive studies, and this distribution can vary by type of instrument (for example, ion trap as compared to Q-TOF), but this information is not yet fully exploited in most matching models. A mobile proton model^ref has been proposed to explain intensity patterns observed in MS/MS, and Zhang has developed a theoretical model that predicts fragment ion intensities well^ref. As with any measurement process, tandem mass spectra may have some level of uncertainty. The accuracy of the mass-to-charge ratio (m/z) and the mass resolving power are limited, electronic and chemical noise may be present and ion signals may fluctuate as a result of changes in the concentrations of peptides entering the ion source. Given a precursor m/z value and a list of fragment ions, the goal is to match these to an amino acid sequence within the measurement and fragmentation uncertainty of the mass spectrometer. A protein mixture is digested, and the resulting peptides are analyzed by MS/MS to obtain experimental spectra. Search programs find database candidate sequences with similar precursor mass whose theoretical spectra are compared to the experimental MS/MS spectrum. The best match (highest-scoring candidate sequence) defines the identified database peptide and the corresponding database protein. Validation software then determines whether the peptide and protein identifications are true or false.

Database searching algorithms :
• sescriptive models are based on a mechanistic prediction of how peptides fragment in a tandem mass spectrometer, which is then quantified to determine the quality of the match between the prediction and the experimental spectrum. Mathematical methods such as correlation analysis have been used to assess match quality.
• interpretative models are based on manual or automated interpretation of a partial sequence from a tandem mass spectrum and incorporation of that sequence into a database search. Matches between the sequence and the spectrum have been scored using probabilities or correlation methods.
• stochastic models are based on probability models for the generation of tandem mass spectra and the fragmentation of peptides. Basic probabilities of fragment ion matches are obtained from training sets of spectra of known sequence identity. Stochastic models use statistical limits on the measurement and fragmentation process to create a likelihood that the match is correct.
• statistical and probability models determine the relationship between the tandem mass spectrum and sequences. The probability of peptide identification and its significance are then derived from the model.
False positives are a perpetual concern in database searching. They can arise for several reasons. Data-dependent algorithms for large-scale acquisition of tandem mass spectra do not discriminate between peptide ions and other types of ions that may be present. Thus, search algorithms are often confronted with a collection of spectra that could be single peptide ions, chemical noise, nonpeptide molecules or mixtures of coeluting isobaric peptides, which are then matched to amino acid sequences. Good data preprocessing or a search of a library of contaminants can help remove nonpeptide spectra prior to a search. Peptides are often present at a wide range of concentrations in a sample, and peptides present at the limit of detection can produce poor quality fragmentation. The issue of sensitivity is more difficult to correct as it is heavily dependent on the limit of detection of a mass spectrometer. The effects can range from incomplete dissociation to poor ion statistics for fragment ions, making them indistinguishable from noise. In these cases incomplete fragmentation patterns or poor signal-to-noise ratios may lead to a solution that is not unique or correct. The chemistry of peptide fragmentation is also not completely understood, and thus, fragmentation models used in database searching may not accommodate aberrant fragmentation processes and result in false positives. Several statistical studies of peptide fragmentation have been performed to better understand the contributions of specific amino acids to fragmentation processes. In time, improved models will account for more of the aberrant fragmentation processes. Sequence conservation can lead to confusing results. If the same peptide sequence exists in multiple proteins, all of the proteins will be identified. Without additional peptide data it would be impossible to determine which protein produced the peptide that generated the tandem mass spectrum. Identifying this situation is straightforward, as most algorithms track all proteins that a spectrum matches. A final possibility, and perhaps of more concern, are amino acid sequences that do not produce a unique fragmentation pattern but share enough of the same fragment ions to be indistinguishable from one another. In these cases a unique amino acid sequence can not be determined directly from the fragmentation pattern and other means are required to determine the absolute identity of the peptide. In particular, small peptides, < 8 amino acids in length, may not produce a fragmentation pattern that achieves a unique result. Large-scale proteomic experiments can present big challenges for data analysis. Most database searching algorithms can confidently identify amino acid sequences from tandem mass spectra showing good fragmentation and signal-to-noise ratio. Spectra of poorer quality or those containing aberrant fragmentation processes present the greatest challenge, as often the spectra are of peptides from low-abundance proteins. What are the strategies that can be used to mine a data set most thoroughly? To limit the number of spectra and ensure an enriched set of unique spectra, poor-quality spectra could be removed and duplicates identified and eliminated^{ref1, ref2, ref3}. Spectra should be searched with at least two algorithms to take advantage of the different selectivities of algorithms (for example, SEQUEST and Mascot). Unassigned spectra should be searched for modified amino acids. Any remaining spectra can be analyzed by automated or manual sequence tagging. Lastly, automated or manual de novo analysis can be applied to the remaining unassigned spectra. To assist in the assignment of protein identifications, several algorithms have been developed to assess database searching results. Some programs simply filter the data and others assign a statistical confidence^{ref1, ref2, ref3, ref4}. These programs are essential when dealing with large data sets^ref.
Database search programs and websites where information about these programs can be obtained : Mascot; Masslynx; MS-Tag/MS-Seq; PeptideSearch; PepFrag; ProbID; SEQUEST (mirror); SpectrumMill; X!Tandem
• Mossbauer spectrometer : an instrument that detects small changes in interaction between an atomic nucleus and its environment caused by changes in temperature, pressure, and chemical state; used in chemical-physical research with applications in medicine.
• inductively coupled plasma/mass spectrometry (ICP/MS) :
• deuterium exchange/mass spectrometry (DXMS) allows scientists to infer the flexibility and "wiggle" room of small regions of protein by measuring how quickly peptide amide hydrogen atoms are exchanged for deuterium atoms when the protein is placed in heavy water. Flexible regions are very accessible to the deuterium solvent, while rigid regions are much slower to exchange their hydrogen atoms^ref. Crystallographic efforts often fail to produce suitably diffracting protein crystals
Web resources : companies offering mass spectrometry platforms and related equipment and bioinformatics solutions for proteomics research : Advion BioSciences; Agilent Technologies; Applied Biosystems; Becton, Dickinson and Company; Beckman Coulter; Biacore; Bio-Rad Laboratories; Bruker Biosciences; Cerno Bioscience; Ciphergen; Decodon; Dionex; Eksigent Technologies; Eldex; EMD Biosciences; Eprogen; ESA Biosciences; FFE Weber; GE Healthcare (formerly Amersham Biosciences); GeneBio; GenoLogics Life Sciences Software; Genomic Solutions (subsidiary of Harvard Biosciences); GenWay Biotech; Gyros; Imaxia; Invitrogen; IonSpec; Jeol; Kratos Analytical/Shimadzu; Lumicyte; Matrix Science; Merck KgaA; MDS Sciex; NextGen Sciences; New Objective; Nonlinear Dynamics; Oxford Genome Sciences ; Perkin Elmer; Proteome Sciences; Proteome Systems; Qiagen; Shimadzu; Sigma-Aldrich; Syngene; Tecan; Thermo Electron; Waters
• electron paramagnetic resonance (EPR) / electron spin resonance (ESR) : in spectrometry, a measure of electron spin as an indication of the presence and extent of activity of free radicals in an organic reaction
• spectrophotometry : the use of the spectrophotometer : an apparatus for estimating the quantity of coloring matter in solution by the quantity of light absorbed (as indicated by the spectrum) in passing through the solution
• absorption spectrophotometer : an analytical instrument for comparing the absorption of radiation of a given wavelength against a standard to identify a sample material.
• spectrophotofluorometer : an analytical instrument combining the techniques of spectrophotometry and fluorescence analysis.
Lambert-Beer law : A_sample = e_substance^. d ^. [substance]_sample
• qualitative analysis with UV rays (250-350 nm)
• quantitative analysis with visible light and standard sample with known concentration. [substance]_sample = [substance]_standard^. A_sample/A_standard
With the NanoDrop ND-1000 spectrophotometer, you simply pipette the sample directly onto the measurement surface, measure, wipe clean and you're ready for the next sample. Takes 30 seconds, tops. The natural surface tension of the sample is enought to keep it in place. So you don't risk wasting precious sample or time with tedious dilutions and expensive dcuvettes or capillaries. It has the power to determine quality and concentration of nucleic acid or proteins, measuring the fluorescent dye label concetration of microarray samples, or doing full spectrum. UV/Vis spectrophotometry. All you need is 1 mL of sample, with no dilutions required on samples up to 3700 ng/ml (dsDNA). And the NanoDrop spectrophotometer is about the size of a tissue box, so it fits easily on your lab bench.
• spectroscopy : the propagation and analysis of spectra; examination by means of a spectroscope : an instrument for developing and analyzing spectra.
• absorption lines : dark lines in the spectrum due to absorption of light by the substance (usually an incandescent gas or vapor) through which the light has passed
• absorption bands : dark bands in the spectrum due to absorption of light by the medium (a solid, a liquid, or a gas) through which the light has passed
• infrared spectroscopy : examination by means of an infrared spectrometer, to study systems or molecules by examining their interactions with infrared radiation.
• spectropolarimeter : a combined spectroscope and polariscope for determining optical rotation.
• microdensitometer : an instrument used in spectroscopy to measure lines in a spectrum by light transmission measurement.
• atomic absorption spectroscopy (AAS) : the study of absorption spectra by means of passing electromagnetic radiation through an atomic medium that is selectively absorbing; this produces pure electronic transitions free from vibrational and rotational transitions. The sample, either a liquid or a solid, is atomized in either a flame (air-acetylene or nitrous oxide-acetylene) or a graphite furnace. Upon the absorption of ultraviolet or visible light, the free atoms undergo electronic transitions from the ground state to excited electronic states. To obtain the best results in AA, the instrumental and chemical parameters of the system must be geared toward the production of neutral ground state atoms of the element of interest. A common method is to introduce a liquid sample into a flame. Upon introduction, the sample solution is dispersed into a fine spray, the spray is then desolvated into salt particles in the flame and the particles are subsequently vaporized into neutral atoms, ionic species and molecular species. All of these conversion processes occur in geometrically definable regions in the flame. It is therefore important to set the instrument parameters such that the light from the source (typically a hollow-cathode lamp or a electrodeless discharge lamp (EDL)) is directed through the region of the flame that contains the maximum number of neutral atoms. The light produced by the hollow-cathode lamp is emitted from excited atoms of the same element which is to be determined. Therefore the radiant energy corresponds directly to the wavelength which is absorbable by the atomized sample. This method provides both sensitivity and selectivity since other elements in the sample will not generally absorb the chosen wavelength and thus, will not interfere with the measurement. To reduce background interference, the wavelength of interest is isolated by a monochromator placed between the sample and the detector.
• graphite furnace atomic absorption spectroscopy (GFAAS) / electrothermal AAS (ETAAS) / flameless atomic absorption spectroscopy (FAAS) :
• flame atomic absorption spectroscopy (FAAS) :
• LASER-excited atomic fluorescence spectroscopy in a graphite furnace (LEAFS)
Journals :
• Journal of Analytical Atomic Spectrometry
• Spectrochimica Acta Part B: Atomic Spectroscopy
• energy dispersive spectroscopy (EDS) :
• atomic emission spectroscopy (AES) :
• inductively coupled plasma/atomic emission spectroscopy (ICP/AES) :
• neutron activation analysis (NAA) :
• radiochemical neutron activation analysis (RNAA) :
• electrothermal vaporization isotope dilution inductively coupled plasma mass spectrometry (ETV-ID-ICP-MS)
• Raman spectroscopy is a spectroscopic technique used in condensed matter physics and chemistry to study vibrational, rotational, and other low-frequency modes in a system. It relies on inelastic scattering, or Raman scattering of monochromatic light, usually from a laser in the visible, near infrared, or near ultraviolet range. Phonons or other excitations in the system are absorbed or emitted by the laser light, resulting in the energy of the laser photons being shifted up or down. The shift in energy gives information about the phonon modes in the system. Infrared spectroscopy yields similar, but complementary information. Typically, a sample is illuminated with a laser beam. Light from the illuminated spot is collected with a lens and sent through a monochromator. Wavelengths close to the laser line (due to elastic Rayleigh scattering) are filtered out and those in a certain spectral window away from the laser line are dispersed onto a detector. Spontaneous Raman scattering is typically very weak, and as a result the main difficulty of Raman spectroscopy is separating the weak inelastically scattered light from the intense Rayleigh scattered laser light. Raman spectrometers typically use holographic diffraction gratings and multiple dispersion stages to achieve a high degree of laser rejection. A photon-counting photomultiplier tube (PMT) or, more commonly, a CCD camera is used to detect the Raman scattered light. Raman scattering has a stimulated version, analogous to stimulated emission, called stimulated Raman scattering. Raman spectroscopy is commonly used in chemistry, since vibrational information is very specific for the chemical bonds in molecules. It therefore provides a fingerprint by which the molecule can be identified. The fingerprint region of organic molecules is in the range 500-2000 cm^-1. Another way that the technique is used is to study changes in chemical bonding, e.g. when a substrate is added to an enzyme. Raman gas analyzers have many practical applications, for instance they are used in medicine for real-time monitoring of anasthetic and respiratory gas mixtures during surgery. In solid state physics, spontaneous Raman spectroscopy is used to, among other things, characterize materials, measure temperature, and find the crystallographic orientation of a sample. As with single molecules, a given solid material has characteristic phonon modes that can help an experimenter identify it. In addition, Raman spectroscopy can be used to observe other low frequency excitations of the solid, such as plasmons, magnons, and superconducting gap excitations. The spontaneous Raman signal gives information on the population of a given phonon mode in the ratio between the Stokes (downshifted) intensity and anti-Stokes (upshifted) intensity. Raman scattering by a crystal gives information on the crystal orientation. The polarization of the Raman scattered light with respect to the crystal and the polarization of the laser light can be used to find the orientation of the crystal, if the crystal structure (specifically, its point group) is known.
• surface-enhanced resonance Raman scattering (SERRS) : the target compound is adsorbed onto a roughened metal surface, producing an enhanced vibrational spectrum of the target, characterized by multiple sharp peaks, that serves as a fingerprint. A suspension of citrate-reduced silver particles roughly 40 nanometers in diameter as metal surface and substrates consisting of 3 components—an enzyme recognition site, a benzotriazole azo dye, and an enzyme-cleavable linker that stably joins the other components - have been used to detect 0.025 mg/mL of lipase from Pseudomonas cepacia after reaction for 10 minutes, corresponding to at most 0.8 pmoles of enzyme in the 1-mL sample volume. Given the microscope lens SERRS uses only interrogates a small portion of the sample, a conservative estimate puts the actual sample volume at pL, whereas more realistically it is closer to fL, sampling reactions arising from only 500 or so molecules of enzyme at most. When free, the dye has a strong penchant for adsorbing to silver nanoparticles by displacing their citrate surface layers, and when this happens it can generate an increase of up to 10 to 14 times in the SERRS signal intensity, enough that near single-molecule detection levels of such dyes are observed. The substrates proved susceptible to hydrolysis by a wide range of hydrolases, including lipases, esterases, and proteases. In experiments, the substrates acted rapidly, screening for activity and enantioselectivity for 14 enzymes in less than 30 seconds. Extrapolating this productivity gives a potential throughput of roughly 100,000 enzymes per day per instrument, comparing favorably with other screening techniques. Because each dye produces a characteristic SERRS spectrum—akin to a fingerprint—that can easily be identified and quantified in a mixture, synthesizing substrates with different enzyme recognition sites coupled with different dyes could make it possible to monitor the action of multiple enzymes simultaneously^ref.
• reactive oxygen species (ROS), expecially O₂^-
• measuring the reduction of exogenously supplied cytochrome chas limited sensitivity and is complicated by the reoxidation of O₂^--reduced cytochrome c by contaminants and cell lysis
• measuring the uptake of O₂ from the medium (measured by the Clark electrode) is not only less sensitive than cytochrome c reduction but also requires a large number of cells (10⁶) per assay.
• 2-methyl-6(4-methoxyphenyl)-3,7-dihydroimidazol[1,2-a]pyrazin-3-one (MCLA). Limitations : artifactual O₂^- is produced in the presence of cell-derived NADH
• photoproteins that react with ROS to form a chemiluminescent product
• luminol is the most sensitive method of all; accurate readings can extend 3 orders of magnitude over the signal range. Limitations : it can act as a source of O₂^- in the presence of univalent oxidants to yield a chemiluminescent product that is SOD and catalase inhibitable: this would lead to artifactual production of O₂^-.
• pholasin from Pholas dactylus => oxypholasin. The protein reacts only once with ROS and the luminescent product then degrades.
• LPS from Gram-negative Bacteria
• Levin-Bang limulus test / Limulus amebocyte lysis (LAL) test : LPS from gram-negative bacteria in a blood sample can gellify Limulus polyphemus (a.k.a. Atlantic horseshoe crab) amoebocytes (blood cells) lysates by activating a Ser-protease that converts coagulogenin to coagulin, hence producing gelation of the extract of blood cells
• the RAW264-HIV-LTR-LUC cell line is a mouse macrophage cell line (RAW264.7) stably expressing a luciferase reporter gene under the control of an HIV long terminal repeat promoter, which is highly and rapidly responsive to NF-kB stimulation. These cells provide a sensitive bioassay for the presence of LPS^{ref1, ref2}, and is able to detect 5–10 pg/ml E. coli LPS
• TNF-a production by human PBMC is able to detect 40 pg/ml LPS
• proteins and nitrogenous compounds
• Millon's test : a solution is made of 10 g of mercury and 20 g of nitric acid; this is diluted with an equal volume of water and decanted after standing 24 hours. This reagent gives a red color with proteins and other substances, such as tyrosine, phenol, and thymol, which contain the hydroxyphenyl group.
• a-amino acids
• carbamino reaction : a-amino acids unite with CO₂ in the presence of alkalis or alkaline earths to form salts of carbamino-carboxylic acids. This reaction is used in studying the course of protein digestion
• phenylalanine
• Guthrie test (for phenylketonuria): in the presence of blood containing phenylalanine, b-2-thienylalanine does not inhibit growth of Bacillus subtilis.
• tyrosine
• Hoffmann's test : add mercuric nitrate to the suspected liquid and boil it; then add nitric acid with a little nitrous acid. A red color is produced if tyrosine is present, and a red precipitate is seen.
• Mörner's test : to a small quantity of the crystals in a test tube add a few mL of Mörner's reagent (solution of formaldehyde, 1 mL; distilled water, 45 mL; concentrated sulfuric acid, 55 mL). Heat gently to the boiling point. A green color shows the presence of tyrosine
• Peria's or Piria's test : moisten the suspected material with strong sulfuric acid and warm it; then dilute and warm it again; neutralize it with barium carbonate, filter, and add ferric chloride in dilute solution: if tyrosine is present, a violet color is seen, which is destroyed by an excess of ferric chloride.
• Udrúnszky's test : take 1 mL of the suspected substance in solution, add a drop of 0.5% aqueous solution of furfurol, and underlay with 1 mL of concentrated sulfuric acid; a pink color shows the presence of tyrosine
• Wurster's test : the suspected material is dissolved in boiling water and a little quinone; a ruby-red color will form, changing slowly to brown
• leucine
• Hofmeister's test : suspected liquid with mercurous nitrate; if leucine is present, metallic mercury is deposited
• cysteine test
• Mörner's nitroprusside test : if a protein containing cysteine is dissolved in water and 2 to 4 drops of a 4 or 5% solution of sodium nitroprusside and then a few drops of ammonia are added, a deep purple-red color appears
• Sullivan's test : to 1 or 2 mL of the unknown solution add 1 to 2 drops of a 0.5% solution of sodium b-naphthoquinone-4-sulfonate and then 5 mL of a 20% sodium thiosulfate made up in 0.25 normal sodium hydroxide. A brilliant red color indicates a free thiol group, demonstrating cysteine rather than cystine
• cystine
• Liebig's test : boil the suspected substance with a sodium hydroxide solution and a little lead sulfide; if cystine is present, the lead sulfide will form a black precipitate.
• taurine
• Lang's test : the solution to be tested is boiled with freshly prepared mercuric oxide; taurine will cause a white precipitate to appear
• proteose test : proteose does not coagulate on boiling, but gives a ring test with trichloracetic acid.
• proteins
• tripeptides and polypeptides
• biuret reaction : biuret (H₂N-CO-NH-CO-NH₂) forms a chelate having an intense violet-red color with the Cu²⁺ ion in alkaline solution; the same reaction also occurs with tripeptides and polypeptides, but not with dipeptides or amino acids and is used in colorimetric methods for total protein
• Gies' biuret test : a form of biuret test employing the following reagent: mix 25 mL of a 3 per cent solution of cupric sulfate and 975 mL of a 10% solution of potassium hydroxide
• Grigg's test : metaphosphoric acid precipitates all proteins except the peptones
• peptone test
• Hofmeister's test : mix phosphotungstic and hydrochloric acids; let the mixture stand 24 hours, and filter. With this reagent a solution containing peptones with no albumin will afford a precipitate
• triketohydrindene hydrate test : to 25 mL of water add 10 mg of aminoacetic acid. To 1 mL of this solution add a solution of 50 mg of sodium acid in 2 mL of water, then add 0.2 mL of a solution of 5 mg of triketohydrindene hydrate (Ninhydrin) in 1 mL of water. Add the suspected matter and boil for 1–2 minutes. A violet color indicates a free carboxyl and alpha-amino group in proteins, peptones, peptides, or amino acids
• Van Slyke test (for amide nitrogen) nitrous acid acting on amide nitrogen sets free nitrogen gas, which is collected and its volume determined
• Reichl's test : add 2 or 3 drops of an alcoholic solution of benzaldehyde and a quantity of sulfuric acid previously diluted to twice its volume with water; then add a few drops of ferric sulfate solution. The mixture will take on a deep-blue color if proteins are present.
• Schulte's test : remove all coagulable protein, precipitate with six volumes of absolute alcohol, dissolve the precipitate in water, and apply the biuret test
• sulfur test : the suspected liquid is heated with an excess of sodium hydroxide and a small quantity of acetate of lead; if proteins are present, a black precipitate of lead sulfide is formed
• Molisch's reaction or test / alpha-naphthol reaction : the substance is treated with a 15% alcoholic solution of a-naphthol and then with concentrated sulfuric acid; a violet color is formed if proteins are present
• albumin
• MacWilliam's test : take 20 mL of urine and add 2 drops of a saturated solution of sulfosalicylic acid: if albumin is present, a cloudiness or precipitate will be seen; if other proteolytic digestion products are present, this precipitate will disappear on boiling, but appear again on cooling.
• Rees' test : small amounts of albumin are precipitated from solution by tannic acid in alcoholic solution
• Ulrich's test : the reagent consists of saturated solution of sodium chloride, 98 mL; glacial acetic acid, 2 mL. It must be perfectly clear. Boil a few mL of this fluid in a test tube, and immediately overlay with the urine. Albumin and globulin give a white ring at the zone of contact.
• phalloidin
• rennin
• Lee's test : add 5 drops of gastric juice to 5 mL of milk; coagulation should take place in 20 minutes in the incubator
• pepsin
• Mett's (Mette's) test : tubes (Mett's tubes) of coagulated albumin are introduced into the unknown and into a standard pepsin hydrochloric acid mixture and the amount of digestion occurring in a given time is noted.
• macroglobulins
• Sia test : a simple screening test performed by adding a drop of serum to 10 to 100 mL of cold distilled water; a positive reaction is indicated by the formation of a heaving cloud of precipitate at the bottom of the container. It is not diagnostic, because it may be positive in other conditions, as in rheumatoid arthritis.
• hemoglobin
• Wagner's benzidine test : benzidine, acetic acid, and hydrogen peroxide are added to the specimen; hemoglobin catalyzes the oxidation of benzidine by hydrogen peroxide, giving a blue color. This is the most sensitive screening test for occult blood, but it is seldom used because benzidine is a carcinogen, and its use is restricted.
• guaiac test : glacial acetic acid and a solution of gum guaiac are mixed with the specimen; on addition of hydrogen peroxide, the presence of blood is indicated by a blue tint.
• Teichmann's test : the suspected liquid is put under a coverglass with a crystal of sodium chloride and a little glacial acetic acid; heat carefully without boiling and then cool. If blood is present, rhombic crystals of hemin will appear.
• hydrogen peroxide test : a 20% solution of hydrogen peroxide is added to the suspected fluid; if blood is present even in minute proportion, bubbles will rise, forming foam on the surface of the fluid
• von Zeynek and Mencki's test : precipitate the urine with acetone, extract the precipitate with acidified acetone, and examine the colored extract under the microscope for small hemin crystals.
• Weber's test : mix the sample with 30% acetic acid and extract with ether; to the ether extract add an alcoholic solution of guaiac and hydrogen peroxide. A blue color indicates blood
• fetal hemoglobin :
• Kleihauer-Betke acid elution test : air-dried blood smears on a glass slide are fixed in 80% methanol and immersed in a buffer at pH 3.3 (citric acid and sodium phosphate); all hemoglobins are eluted except fetal hemoglobin, which remains fixed in the red cells and can be detected after staining
• alkali denaturation test : a spectrophotometric method for determining the concentration of hemoglobin F, which depends on the resistance of the hemoglobin molecule to denaturation of its globin moiety when exposed to alkali.
• Apt test (for differentiating fetal from adult hemoglobin): a specimen from an infant's vomitus or stool is mixed with 5 volumes of water and centrifuged so that a clear pink supernatant separates. Sodium hydroxide solution is added to the supernatant; if hemoglobin F (fetal blood) is present, the pink color persists for more than 2 minutes, whereas if hemoglobin A (from swallowed maternal blood) is present, the supernatant turns from pink to yellow within 2 minutes.
• enzymes
• international unit of enzyme activity (U) : that amount of an enzyme that will catalyze the transformation of 1 micromole of substrate per minute under standard conditions of temperature, optimal pH, and optimal substrate concentration
• amylase : use starch agar to detect the production of amylase. A few drops of iodine solution are placed near the margin of the bacterial colonies on starch agar. If starch hydrolysis has occurred, there will be a clear brown halo at the margin of growth. If starch has not been hydrolyzed, the iodine will cause the medium to turn black or dark purple at the margin of growth.
• pancreatic lipase
• litmus milk test : add pancreatic lipase to litmus milk, incubate, and note change of color; pancreatic lipase is indicated by a pink coloration.
• cyt c oxidase : large amounts are produced by strictly aerobes, facultative aerobes and microaerophiles. 2 to 3 drops of tetramethyl-p-phenylenediamine dihydrochloride is added to a piece of Whatman No. 1 filter paper : it is a colourless synthetic substrate that turns dark purple-blue when oxidized to indophenol in the presence of cyt c oxidase and O₂.
• catalase test (for the production of catalase by bacteria): a slant culture is treated with H₂O₂ 3% The presence of gas (O₂) bubbles indicates a positive reaction. Micrococci, staphylococci, most species of Bacillus, and anaerobic diphtheroids are catalase-positive; streptococci, pneumococci, and most Actinomyces are catalase-negative.
• peroxidase : 2 H₂O₂ ==peroxidase (e.g. in RBC)==> 2 H₂O + O₂ == + benzidine ==> oxidized benzidine
• peroxidase reaction : the appearance of deep-blue granules in leukocytes of marrow origin when stained with Goodpasture's stain, distinguishing them from cells of lymphatic origin
• coagulase test : a test for coagulase activity in which bacteria are added to citrated or oxalated (human or rabbit) blood plasma; in the presence of coagulase, the plasma gels within 3 hours. Coagulase activity is also demonstrable by mixing bacteria with blood plasma on a slide; if positive, clumping occurs, with fibrin formation.
• gelatinase
• H₂S-producing enzymes : Pb-acetate or FeCl₂ + H₂S => PbS or FeS (black) + ?
• Trp ==Trp hydrolase==> indole + p-dimethylaminobenzaldehyde (Kovacs' reagent, yellow)=> ? (red/pink)
• nitrate reductase (NO₂^-, NH₃ and N₂ production) : the organism is cultured in a broth containing nitrate. The medium is tested for nitrite by mixing with solutions containing sulfanilic acid and a-naphthylamine in 5 N acetic acid; a red color indicates the presence of nitrite. The test is useful in identifying doubtful strains of Enterobacteriaceae, mycobacteria, and certain aerobic bacteria.
• Phe ==Phe deaminase==> phenylpyruvic acid == + FeCl₂ 10%==> ? (green)
• a drop of water is added to moisten a paper disk containing 1-pyrrolidonyl-b-naphthylamide (PYR reagent) ==pyrrolidonyl peptidase==> pink/cherry red color
• urease test : urease test broth is prepared in slants. After inoculation of the surface and incubation, urease-positive cultures produce an alkaline reaction (dark pink or red color) in the medium. Proteus cultures show an early urease-positive reaction; other genera may have a delayed response.
• citrate transporter
• b-galactosidase : the organism is grown in a buffered peptone medium containing D-nitrophenyl-b-D-galactopyranoside (ONPG) =b-galactosidase=> o-nitrophenol (yellow) + Gal. Used to differentiate Salmonella (positive) from Arizona (negative), and Neisseria lactamicus (positive) from N. meningitidis (negative)
• acetylmethylcarbinol (acetoin) producing enzymes (Voges-Proskauer reaction) : acetoin + O₂ + 40% K⁺OH^- ==> diacetyl == + a-naphtol + creatin ==> ? (red)
• deoxyribonuclease (DNase) test : a nutrient agar plate containing DNA and toluidine blue is inoculated from a young agar slant; after incubation a red zone around the inoculum indicates the presence of deoxyribonuclease
• DNA
• Feulgen reaction : a specific histochemical reaction for DNA (deoxyribonucleic acid): after acid hydrolysis at 60°C, tissue sections are stained in Schiff's reagent; DNA stains magenta
• uracil and cytosine
• Wheeler and Johnson's test : to the unknown solution add bromine water until the color is permanent, but avoid excess. Then add an excess of barium hydroxide. A purple color indicates one of these substances.
• xanthine
• Hoppe-Seyler test : add the substance to be tested to a mixture of chlorinated lime in a porcelain dish; a dark-green ring is formed at first.
• Weidel's test : warm with freshly prepared chlorine water containing a trace of nitric acid until gas ceases to be produced; contact with gaseous ammonia develops a pink or purple color.
• (for xanthine bodies) dissolve in warm chlorine water, evaporate, and treat with diluted ammonia solution; a pink or purple color will form, changing to violet on the addition of sodium or potassium hydroxide solution.
• hypoxanthine
• Kossel's test : the liquid to be tested is treated with zinc and hydrochloric acid and with sodium hydroxide in excess; if hypoxanthine is present, a ruby-red color is produced.
• glycerol
• hypochlorite-orcinol test : to 3 mL of the unknown add 3 drops of N/1 sodium hypochlorite solution and boil one minute to drive off chlorine. Then add an equal volume of strong hydrochloric acid and a little orcinol. Boil, and a violet or greenish blue color indicates glycerol or a sugar, or some substance that can be oxidized to a sugar.
• pentoses
• Bial's orcinol test (for pentoses in urine): the specimen is heated with a solution of orcinol, hydrochloric acid, and ferric chloride; pentoses are converted to furfural, which reacts with orcinol to form a green product.
• glucose
• glucose test : any of various laboratory tests for glucose in the urine; many formerly common ones are no longer used.
• Benedict's test : a test for glucose in the urine using Benedict's reagent
• Rubner's test (for lactose, glucose, maltose, or fructose in urine) : add lead acetate to the urine, boil, and then add an excess of ammonium hydroxide: lactose gives a brick-red color, glucose a coffee-brown color, maltose a light-yellow color, and fructose no color at all.
• saccharimeter test : glucose in solution rotates the plane of polarized light to the right, while fructose turns it to the left
• Molisch's test
• add 2 mL of urine, 2 drops of a 15% solution of thymol, and an equal volume of strong sulfuric acid; a deep red color results
• alpha-naphthol reaction : to 1 mL of urine add 2 or 3 drops of a 5% solution of a-naphthol in alcohol, then add 2 mL of strong sulfuric acid; a deep violet color is produced, and a violet precipitate follows if water is added
• starch :
• iodine test : when a compound solution of iodine is added to starch, and especially to an acid or neutral solution of cooked starch paste, a deep-blue color is produced which disappears on heating and reappears on cooling. Erythrodextrin and glycogen give a red color with iodine
• ammonia and urea
• Benedict's test : (for urea) the urea is hydrolyzed to ammonium carbonate by potassium bisulfate and zinc sulfate made alkaline, and distilled as usual.
• Berthelot reaction : the reaction of ammonia with Berthelot's reagent to form phenol-indophenol, a stable, deep blue product; used in colorimetric methods for ammonia and urea
• xanthydrol reaction : when tissue from a uremic patient is fixed in a solution of xanthydrol in glacial acetic acid, a large deposit of xanthydrol occurs in the tissue.
• diacetyl test : the solution to be tested is mixed with concentrated hydrochloric acid and diacetyl monoxime; a yellow color develops on boiling if urea is present
• urease test : a test for urea based on the conversion of urea into ammonium carbonate by the urease of soybean.
• Schroeder's test : add a crystal of the substance to a solution of bromine in chloroform; the urea will decompose and gas will be formed
• Van Slyke test : treat the sample with urease, pass the ammonia so formed into N/50 normal acid, and titrate the excess of acid
• glycogen, epithelial mucins, neutral polysaccharides, and glycoproteins
• periodic acid–Schiff (PAS) reaction : a tissue section is exposed to periodic acid, which oxidizes hydroxyl groups on adjacent carbon atoms or adjacent hydroxyl groups and amino groups to aldehydes, and then is stained with Schiff's reagent, which forms an additional product with aldehydes to produce a red or magenta reaction product; used to test for glycogen, epithelial mucins, neutral polysaccharides, and glycoproteins
• differentiate between an exudate and a transudate
• Rivalta's reaction : utilizing acetic acid.
• Morelli's test : add a few drops of the suspected fluid to a saturated solution of mercury bichloride in a test tube; a flaky precipitate indicates a transudate, a clot indicates an exudate
• catecholamines
• chromaffin reaction : the taking on of a deep brown color by tissue of the adrenal medulla or other types that contain the catecholamines epinephrine and norepinephrine, within 12 hours after it is placed in a dichromate solution; this can be used to detect pheochromocytomas and other tumors that produce catecholamines
• Henle's reaction : a staining dark brown of the cells of the adrenal medulla on treatment with chromium salts
• sterols
• cholesterol test
• digitonin reaction : the formation of a precipitate on treating a sterol, such as cholesterol or ergosterol, with digitonin; employed to define cholesterol esters which do not precipitate in total serum cholesterol determinations
• Salkowski's test : dissolve the sample in chloroform and add an equal volume of strong sulfuric acid: if cholesterol is present, the solution becomes bluish red, and slowly changes to a violet red, the sulfuric acid becomes red, with a green fluorescence.
• Schultze's test : evaporate with nitric acid, using a porcelain dish and water bath; if cholesterol is present, a yellow deposit is formed, which changes to yellowish red when ammonia is added.
• Liebermann-Burchard test : dissolve the sample in chloroform and add acetic anhydride plus concentrated sulfuric acid; cholesterol can be quantitated by the intensity of the resulting blue-green color.
If TG < 200 mg/dl => LDL-Ch = TG - (HDL-Ch + TG/5)
If TG > 200 mg/dl => non-HDL-Ch = T-Ch - HDL-Ch = LDL-Ch + 30 mg/dl
• 17-hydroxycorticosteroids
• Porter-Silber reaction : the reaction of the dihydroxyacetone side chain of certain 17-hydroxycorticosteroids (Porter-Silber chromogens) with phenylhydrazine in acid, which produces a yellow color; an index of adrenocortical function now largely supplanted by immunoassay techniques
• estrogens
• Kober test : when estrogens are treated with a mixture of sulfuric acid and phenolsulfonic acid and then diluted with water, a clear pink color is formed; suitable for qualitative analysis.
• urobilinogen
• Ehrlich's aldehyde reaction : urobilinogen reacts with p-dimethylaminobenzene to form a red-colored substance; used for semiquantitative determination of urobilinogen in urine and feces
• Ehrlich's diazo reaction : a reaction of a pure pink or red color resulting from the action of diazotized sulfanilic acid and ammonia upon certain aromatic substances, e.g., urobilinogen, found in the urine in some conditions. This reaction has diagnostic value in hepatic disease, typhoid fever, and measles and prognostic value in tuberculosis.
• egg yellow reaction : a yellow foam appearing in Ehrlich's diazo reaction before the addition of ammonia; believed to indicate acute pneumonia
• bilirubin test
• bilirubin in urine
• Fouchet's test : a few drops of Fouchet's reagent are added to the specimen; a green color is produced if bilirubin is present
• Harrison spot test : add to 10 mL of urine 5 mL of a 10% solution of barium chloride, mix, and filter. Spread filter paper on dry filter paper. Add one to two drops of Fouchet's reagent (trichloroacetic acid 25 g, water 100 mL, and 10% solution of ferric chloride 10 mL); a positive reaction gives a blue to green color.
• van den Bergh's test : a test for bilirubin in which diazotized serum or plasma is compared with a standard solution of diazotized bilirubin. In aqueous media, Ehrlich diazoreactive (diazotized sulfanilic acid = sulfanilic acid + NaNO₂ + H⁺Cl^-) reacts directly ("direct reaction") with hydrosoluble conjugated bilirubin ("direct bilirubin") to create azabilirubin (red-violet). Further adding of a lipid solvent (MeOH in Mallow-Evelyn method, caffeine and Na⁺benzoate in Jendrassik method) allows also unconjugated hydrophobic bilirubin to leave albumin carrier and to react ("indirect reaction") : so the whole reaction shows total bilirubin, whose normal value is 0.5÷1.2 mg/dL = 8.5÷20.5 mmol/L. "indirect" bilirubin = total bilirubin (as calculated from "indirect" reaction") - "direct" bilirubin (as calculated from "direct" reaction). Normally it represents the greatest portion of total bilirubin, arising from physiological erythrocatheresis.
• ferric chloride test
• erythrocyte protoporphyrin (EP) test : a screening test for lead toxicity, in which erythrocyte protoporphyrin levels are determined by direct fluorometry of whole blood or fluorescence analysis of whole blood extracts; levels are increased in lead poisoning and iron deficiency.
• porphobilinogen
• Watson-Schwartz test : a simple qualitative procedure for differentiating porphobilinogen from urobilinogen and other Ehrlich reactors, based on the insolubility of porphobilinogen aldehyde in chloroform and butanol; it is useful in diagnosis of acute porphyria.
• bile
• bile in urine
• Trousseau's test : iodine tincture diluted with 10 parts of alcohol is added to urine in a test tube; a green ring is formed where the liquids touch if bilirubin is present.
• Tyson's test : 180 to 240 mL of urine are evaporated to dryness on the water bath. The residue is extracted with absolute alcohol, and to the extract 12 to 14 volumes of ether are added. The bile acids are precipitated, then are filtered off, dissolved in water, and the aqueous solution decolorized with animal charcoal.
• Gmelin's test : fuming nitric acid is so added to the suspected urine that it forms a layer under it. Near the junction of the two liquids, rings are formed—a green ring above, and under it a blue, violet-red, and reddish yellow. If the green and violet-red rings are absent, the reaction shows the probable presence of lutein.
• Udrúnszky's test : take 1 mL of a solution of the suspected substance, add a drop of 0.1% solution of furfurol in water, underlay with strong sulfuric acid, and cool; if bile is present, a bluish-red color is formed
• Vitali's test
• add a few drops of potassium nitrate in solution and dilute sulfuric acid. The color reactions are green, followed by blue or red and yellow
• add quinine bisulfate in solution and follow with diluted ammonia solution, sulfuric acid, a crystal of sugar, and alcohol; a violet color results
• Ultzmann's test : to 10 mL of the urine to be tested add 3 or 4 mL of a 1:3 solution of potassium hydroxide, and an excess of hydrochloric acid; bile pigments will cause an emerald-green coloration
• nitrites
• Griess test (for nitrites in the saliva): mix the saliva with 5 parts of water; add a few drops of dilute solution of sulfuric acid and a few drops of metadiamidobenzene; this produces a strong yellow color if nitrites are present.
• nitrites test (for nitrites in saliva): to the saliva add 1 or 2 drops of sulfuric acid, a few drops of potassium iodide solution, and some starch paste; a blue color indicates nitrites
• Schaffer's test (for nitrites in urine) : decolorize 4 mL of urine with animal charcoal and add to it 4 mL of 10% acetic acid and 3 drops of 5% solution of potassium ferrocyanide; an intense yellow color indicates nitrites
• nitrate reduction test
• Weyl's test (for nitric acid in the urine) : distill 200 mL of urine with 0.2 part of sulfuric or hydrochloric acid, receiving the distillate in a potassium hydroxide solution. If m-phenyldiamine is added, a yellow color will form; if there is added pyrogallic acid in aqueous solution with a little sulfuric acid, the color will be brown; but sulfanilic acid in solution, followed in ten minutes by naphthylamine hydrochlorate, produces a red tint.
• creatinine test
• Jaffe reaction : creatinine when treated with picric acid in strongly alkaline solution gives an intense red color
• Jaffe's test : to the liquid add trinitrophenol and then make alkaline with sodium hydroxide. A red color indicates presence of creatinine
• Kerner's test : acidify the suspected solution and add phosphomolybdic or phosphotungstic acid in solution; if creatinine is present, it will form a crystalline precipitate
• Salkowski's test : to the yellow solution obtained in Weyl's test add an excess of acetic acid and heat; a green color results, which turns to blue
• Thudichum's test : add to the suspected substance a dilute solution of ferric chloride; a dark-red color indicates the presence of creatinine
• von Maschke's test : to the suspected solution add a few drops of Fehling's solution, after mixing with a cold solution of sodium carbonate; an amorphous, flocculent precipitate proves the presence of creatinine
• Weyl's test : to the suspected solution add a little of a dilute solution of sodium nitroprusside, and then carefully put in a few drops of a weak solution of sodium hydroxide; a ruby red color results, changing to blue on warming with acetic acid
• Marchi's reaction : failure of the degenerated myelin sheath of a nerve to become discolored when treated with osmic acid
• Russo reaction : a reaction of the urine of typhoid patients on adding 4 drops of a solution of methylene blue to 15 mL of urine. In the first stage of typhoid, the urine becomes light green; at the height of the disease, an emerald color; and during the decline, a bluish color.
• fuchsinophil reaction : certain substances when stained with fuchsin retain the stain on being treated with picric acid alcohol.
• glycyltryptophan test : filtered gastric contents and glycyltryptophan are placed in a test tube and kept at body temperature for 24 hours; if on the addition of a few drops of bromine, a reddish violet color is formed, carcinoma of stomach is indicated.
• indole test
• Nencki's test : treat the suspected material with nitric acid and a little nitrous acid; a red color follows, and in concentrated solution a red precipitate may appear.
• nitroso-indole-nitrate test (for indole and skatole): acidify the unknown with nitric acid and add a few drops of potassium nitrite; a red color or a red precipitate indicates indole, a white turbidity indicates skatole
• pine wood test : a pine splinter moistened with concentrated hydrochloric acid is turned cherry red by a solution of indole
• Salkowski's test : to the solution to be tested add a little nitric acid, and drop in slowly a solution of potassium nitrite (2%): a red color shows that indole is present, and a red precipitate is afterward formed
• urochromogen :
• Weisz's permanganate test : to 2 mL of the urine add 4 mL of distilled water and 3 drops of a 1:1000 solution of potassium permanganate; a canary yellow color indicates urochromogen
• indican test
• Jaffe's test : to the suspected liquid are added an equal amount of concentrated hydrochloric acid, 1 mL of chloroform, and a few drops of a strong solution of chlorinated soda. The chloroform is colored blue if indican is present
• Weber's test : boil 30 mL of urine with an equal volume of hydrochloric acid containing a little nitric acid; cool it, and shake with ether; if indican is present, the ether will become red or violet and the froth will be blue
• melanin
• Thormählen's test (for melanin in urine): treat urine with a solution of sodium nitroprusside, potassium hydroxide, and acetic acid; if melanin is present, a deep-blue color will form.
• ferric chloride test
• formaldehyde
• Jorissen's test : add 0.5 mL of a 1% solution of phloroglucin in 10% sodium hydroxide to 1 mL of the urine; a bright red color indicates free formaldehyde.
• Kentmann's test : dissolve in a test tube 0.1 g of morphine in 1 mL of sulfuric acid; add, without mixing, an equal volume of the liquid to be tested; in a short time the latter will take on a reddish violet color if any formaldehyde is present.
• phenol
• Jacquemin's test : add to the suspected liquid an equal quantity of aniline and some sodium hypochlorite in solution; a blue color is produced.
• Plugge's test : a dilute solution containing phenol becomes red on mixture with a mercuric nitrate solution containing a trace of nitrous acid; mercury is also precipitated and the odor of salicylol is given off.
• ketone bodies
• acetone
• Wishart test (for acetonemia): a few drops of plasma are placed in a small test tube. Enough dry powdered ammonium sulfate is added to supersaturate, so that at the end of the test there will still be some of the solid sulfate in the bottom of the tube. A couple of drops of a fresh solution of sodium nitroprusside are next added and shaken, and finally 1 or 2 drops of diluted ammonia solution. On shaking, a purple color develops, a little more slowly than in the case of urine. The intensity of the color indicates the degree of acetonemia.
• Lange's test (for acetone in urine): 15 mL of urine are mixed with 0.5 to 1 mL of acetic acid, and a few drops of a freshly prepared concentrated solution of sodium nitroprusside added. The mixture is overlaid with ammonia. At the point of junction a characteristic violet ring is formed
• b-hydroxybutyric acid
• Külz's test : the fermented urine is evaporated to a syrupy consistency, strong sulfuric acid in equal volume is added, and the mixture is distilled. If hydroxybutyric acid is present, a-crotonic acid will be formed, which will crystallize. If, after fermentation, the urine shows dextrorotatory properties, b-hydroxybutyric acid is present.
• Osterberg's test : to 800 mg of ammonium sulfate add 0.15 mL of concentrated ammonium hydroxide solution, 2 drops of a 5% solution of nitroprusside, and 1 mL of the urine. Dilute to 50 mL and compare with a standard.
• acetoacetic acid
• Lindemann's test (for acetoacetic acid in urine): to about 10 mL of urine add 5 drops of 30 per cent acetic acid, 5 drops strong iodine solution, and 2 to 3 mL chloroform, and shake. The chloroform does not change color if diacetic acid is present, but becomes reddish violet in its absence. Uric acid also decolorizes iodine, and if much is present, double the amount of strong iodine solution should be used.
• ferric chloride test
• acid test
• pH indicators
• hydrochloric acid test
• Leo's test (for free hydrochloric acid): calcium carbonate is added to the solution, which is neutralized if the acidity is due to free acid, but not if due to acid salts
• in the stomach contents
• add MnO₂ => Cl₂ (peculiar flavour)
• Mohr's test  : dilute to a light-yellow color a solution of iron acetate, free from alkaline acetates; add a few drops of a solution of potassium thiocyanate, and then the filtered contents of the stomach: if they contain the acid, a red coloring ensues, which is destroyed by sodium acetate
• Winckler test :  filter the juice into a porcelain cell with a few drops of the 5% alcoholic solution of a-naphthol containing 1% or less of glucose. Heat carefully, and a bluish-violet zone will appear, which rapidly grows darker
• Witz's test : a 1:48 aqueous solution of methyl violet causes a violet color, changing to blue and then green.
• Uffelmann's test (for hydrochloric acid and lactic acid in the gastric contents) : to a quantity of material taken from the stomach add a few drops of a reagent containing 3 drops of a solution of ferric chloride, 3 drops of a concentrated solution of phenol, and 20 mL of water; hydrochloric acid, if present, decolorizes this solution, while lactic acid turns it yellow.
• Rabuteau's test : 1 g of potassium iodate and 0.5 g of potassium iodide are added to 50 mL of starch mucilage; filtered stomach liquids are added to it; free hydrochloric acid will render the mixture blue.
• Rabuteau's test (for hydrochloric acid in urine) : add a little indigosulfonic acid to color the urine, and sulfurous acid to decompose what hydrochloric acid may be present; the urine will be decolorized
• sulfuric acid test :
• add veratrine to gastric content => yellow color reaction.
• add a copper strip and heat => SO₂ (peculiar flavour)
• phosphoric acid
• Malot's test : a quantitative test for the determination of phosphoric acid in urine by the reaction with cochineal and a uranium salt
• lactic acid test
• MacLean test (for lactic acid in gastric juice): to 5 mL of gastric juice add 5 drops of the following reagent: ferric chloride, 5 g; concentrated hydrochloric acid, 1.5 mL; saturated solution of mercury bichloride, 100 mL. Lactic acid is indicated by a yellow coloration.
• Uffelmann's test (for hydrochloric acid and lactic acid in the gastric contents): to a quantity of material taken from the stomach add a few drops of a reagent containing 3 drops of a solution of ferric chloride, 3 drops of a concentrated solution of phenol, and 20 mL of water; hydrochloric acid, if present, decolorizes this solution, while lactic acid turns it yellow.
• uronic acid
• Tollens, Neuberg, and Schwket's test : extract the uronic acid from acidified urine with ether, add water, evaporate the ether, and perform an orcinol test
• hippuric acid
• Lücke's test : add boiling hot nitric acid, evaporate, and heat the dry residue; a strong odor of nitrobenzene proves the presence of hippuric acid.
• uric acid test
• Salkowski-Ludwig test : a solution of silver ammonionitrate and ammonium and magnesium chlorides precipitates uric acid
• Weidel's murexide test : the substance tested is treated with nitric acid, evaporated, and moistened with diluted ammonia solution; if uric acid is present, murexide will be formed, and a purple color is produced
• alkali test
• atomic absorption spectroscopy (AAS)
• alkaloids
• Mayer's test : mercury bichloride, 13 1/2 g, and potassium iodide, 50 g, are dissolved in 1000 mL of water; this is used as a test for alkaloid, with which it gives a white precipitate.
• Vitali's test :
• evaporate with fuming nitric acid and add a drop of potassium hydroxide; color reactions will occur. For atropine the color is violet, turning to red
• add sulfuric acid, potassium chlorate, and an alkaline sulfide; various color reactions will follow.  3. (for bile pigments) add a few drops of potassium nitrate in solution and dilute sulfuric acid. The color reactions are green, followed by blue or red and yellow
• Winckler test : a solution of mercury bichloride with an excess of potassium iodide is added; alkaloids will cause a white precipitate
• Wormley's test :
• treat the suspected solution with an alcoholic solution of picric acid; a yellow precipitate will be formed
• treat the suspected solution with a solution of 1 part of iodine and 2 parts of potassium iodide in 60 parts of water; a colored precipitate will be formed
• quinine
• thalleioquin test : a neutralized solution of the suspected liquid is treated with chlorine, or bromine water, and then with an excess of ammonia; a green substance, thalleioquin, will be formed
• colchicine
• Zeisel's test : dissolve in hydrochloric acid, boil with ferric chloride, and shake with chloroform; a brown or dark-red layer will form at the bottom.
• strychnine
• Sonnenschein's test : the suspected substance is dissolved in a drop of sulfuric acid, some cerosoceric oxide is added, and stirred with a glass rod; a deep blue color is formed, changing to violet, and finally to cherry red in the presence of strychnine
• Wenzell's test : treat the suspected material with a solution of 1 part of potassium permanganate in 2000 parts of sulfuric acid; strychnine, even in very small proportion, will cause color reactions.
• atropine
• Reuss' test : the substance examined is treated with sulfuric acid and oxidizing agents; if atropine is present, an odor of roses and orange-flowers is given off.
• hydrogen peroxide
• Wurster's test : test paper is saturated with the solution of tetramethylparaphenylenediamine; hydrogen peroxide turns it to a blue-violet color
• nonvolatile organic compounds :
• solvent extraction : add a strong acid or alkali (according to the researched analyte), add solvent (ether or chloroform), mix and centrifuge to rebuild the 2 phases and recover the lipophilic one, remove solvent by heating. Extraction varies from 0 to 100%, purification is poor, concentration is excellent, and 1 hour is required.
• volatile compounds
• gas chromatography
• molecular distillation
• Conway cell or microdiffusion apparatus + spectrophotometry
• ethanol
• Woodbury's test (for alcohol in the urine): to 2 mL of urine 1 mL of sulfuric acid is added, and a crystal of potassium dichromate; a green color will form
• gases :
• modern arterial blood gas (ABG) analysers are automated self diagnostic instruments requiring minimal maintenance.
• the pH electrode measures the potential difference between a known solution within the measuring electrode and the unknown (sample) solution at 37°C.
• the Paco2 electrode measures carbon dioxide tensions by allowing carbon dioxide to undergo a chemical reaction producing hydrogen ions. The hydrogen ion production causes a difference in potential that is measured by half cells similar to those of the pH electrode.
• the Pao2 electrode is a polarographic device that measures oxygen tensions by oxidation­reduction reactions, a chemical process that generates measurable electric currents.
Calibration is important since the electrodes drift over time; a 1 point calibration adjustment to a single standard is made before each analysis or every 30 minutes. A 2 point calibration using 2 standards should be done every 2­8 hours.
• carbon monoxide
• Hoppe-Seyler test (for carbon monoxide in the blood) : add to blood twice its volume of a solution of sodium hydroxide of 1.3 specific gravity: normal blood will form a dingy brown mass with a green shade if spread thin on a white surface; but if carbon monoxide is present, the mass is red, and so is the thin layer
• Preyer's test : a spectroscopic test for carbon monoxide in the blood
• Wetzel's test (for carbon monoxide in blood) : to the blood to be examined add 4 volumes of water and treat with 3 volumes of a 1% tannin solution. If CO is present, the blood becomes carmine red; normal blood slowly assumes a grayish hue.
• Zaleski's test (for carbon monoxide in blood) : to 2 mL of blood add an equal volume of water and 3 drops of a one-third saturated solution of cupric sulfate: if carbon monoxide is present, the precipitate is brick-red; otherwise it is greenish brown.
• PdCl₂ 0.22 g in 250 mL HCl 0.01 N; add H₂SO₄ 10 mL in 200 mL to break hemoglobin-CO bond; add KI 15 g in 100 mL; mix 1 mL blood and 1 mL H2 => black precipitates appears after 1 hours => spectrophotometric quantitation comparing with the unused solution :
• V%_CO = [(OD_standard - OD_sample) ^. 0.0528 ^. 420] / OD_standard
• HbCO% = (V%_CO . 73.25) / 14
• hydrogen sulfide (H₂S)
• FeCl₃ + NaCL
• hydrogen cyanide (HCN)
• thiocyanates
• Solera's test : saturate filter paper with 0.5% starch paste containing 1% of iodic acid; dry and preserve as test paper. A piece of this paper moistened with saliva will turn blue if thiocyanate is present.
• ferric chloride test : ferric chloride in acidic solution is added to a urine specimen; many substances are oxidized giving colored products. Positive reactions are given by melanin, acetoacetic acid, bilirubin, phenothiazines, salicylates, and the keto acids present in phenylketonuria, alkaptonuria, maple syrup urine disease, and oasthouse urine disease
• thymol
• Vitali's test : distill, and pass the vapor through a mixture of chloroform and potassium hydroxide solution; a red color results.
• pus
• Vitali's test (for pus in the urine) : the urine is acidified with acetic acid and filtered. On the filter paper thus obtained a small quantity of guaiacum is dropped. The paper will turn a dark blue if pus is present
• fats
• Meigs' test (for fat in milk): to 10 mL of milk in a special apparatus add 20 mL of water, 20 mL of ethyl ether, and shake. Then add 20 mL of 95% alcohol. Remove the ethereal layer, evaporate, and weigh.
• heavy metals
• Reinsch's test (for heavy metals, including arsenic, mercury, bismuth, antimony, and large amounts of selenium, tellurium, and sulfide): insert a strip of clean red copper into the suspected HCl acidified liquid or finely ground tissue, and boil fo1 hours; if one or more heavy metals are present, a black or greenish coating will form on the copper strip.
• graphite furnace atomic absorption spectroscopy (GFAAS) can't extract volatile metals evaporating at 450°C (e.g. arsenic, mercury, antimony). Extraction = 100%; excellent purification due to destruction of organic matter; excellent concentration; 6-4 hours required for analysis
• mercury
• Magpie's test (for salts of mercury): stannous chloride is added to the suspected solution; a white and gray precipitate is formed, consisting of metallic mercury and mild calomel
• Vogel and Lee's test : add 3% of hydrochloric acid and concentrate the urine to one fifth its original volume. Add a piece of clean copper wire. A silvery film indicates mercury. To confirm, place the wire in a tube with a plug of gold foil and distill the mercury over onto the gold. Sublime a crystal of iodine onto the mercury and form the red iodide of mercury.
• iodine test
• Winckler test : sodium nitrate is mixed with a starch paste; iodine gives a blue color with it
• arsenic
• Gutzeit's test : a paper is moistened with an acidulated silver nitrate solution and exposed to the fumes from the suspected liquid, which is mixed with zinc and dilute sulfuric acid. The formation of a yellow spot on the paper indicates the presence of inorganic arsenic compounds
• iron
• hemosiderin test
• Perls' Prussian blue reaction demonstrates ferric iron (Fe³⁺), the vast majority of the demonstrable iron in human tissues (including hemosiderin, which is the material usually targeted). The acid ferrocyanide reaction of Perls' Prussian blue involves treatment with acid (usually hydrochloric) to release ferric ions from tissues. These are immediately captured by replacement of the cation of potassium ferrocyanide by the ferric ion, forming insoluble ferric ferrocyanide (a.k.a. Berlin blue,Chinese blue, Hamburg blue, mineral blue, Paris blue or Prussian blue) which then precipitates. Very small amounts of iron may be demonstrated microscopically.
• Rous test : centrifuge the urine; to the sediment add 5 mL of a 2% solution of potassium ferrocyanide and 5 mL of a 1% solution of hydrochloric acid. Hemosiderin granules stain blue
• Tirman-Schmelters-Turnbull's blue reaction demostrates ferrous iron (Fe²⁺). This is similar to Perls' method. It involves replacing the cation of potassium ferricyanide with ferrous iron to make ferrous ferricyanide, or Turnbull's blue, leading to a blue-black coloration
• Tizzoni's test (for iron in tissues): treat a section of tissue with a 2% solution of potassium ferrocyanide, and then with a 0.5% solution of hydrochloric acid; the tissue will be stained a blue color if iron is present
• Purification

Please note that purifications aren't often very clean ! When the purified molecule is used for therapeutical purposes, it may contain antigens and/or infective agents (e.g. HIV-1 in factor VIII, PrP^Sc in GH).
• separation of cytoplasmic and outer membranes
• Osborn method

a) treat cells with EDTA to remove divalent ions so disaggregating LPS molecules (non-covalently cross-linked by Mg²⁺, Zn²⁺, Mn²⁺, etc).
b) treat cells with lysozyme to destroy peptidoglycan. Do this in presence of high sucrose to avoid premature lysis.
c) lyse spheroplasts by osmotic shock or sonication.
d) separate membrane fragments by ultracentrifugation on sucrose gradient . Place lysed spheroplasts on top of a gradient of 30-50% sucrose and centrifuge to equilibrium. Cytoplasmic membrane density is approx 1.14, whereas OM is found at a density of 1.22.
• LPS from Gram-negative Bacteria
• Boivin method : trichloroacetic acid (TCA) contemporaneously extracts 10-20% of proteins
• Galanos method : phenol-chloroformio-petroleum (PCP) mix
• Westphal-Luderitz method : a water solution of phenols 45% at 68°C
• Soxhlet's apparatus : an apparatus by which fatty or lipid constituents can be extracted from solid matter by repeated treatment with distilled solvent .
• C-polysaccharide from Streptococcus spp.
• bacterial suspension in H⁺Cl^- 0.2 N, 100°C for 10'
• enzymatic lysis (pepsin and trypsin)
• autoclaving at P= 1 atm for 15'
• serotypization Ag (STA) from Neisseria spp.
• Li⁺acetate^- pH 5.8, vortexing at 50°C. Then 2 hrs at 100,000g.
• Billerica, Mass.-based Millipore showcased its new Direct-Q System for delivery of pure and ultra-pure water at the 2005 Pittsburgh Conference in Orlando, Fla. The Direct-Q system is for small-volume users such as chemical, environmental, or food and beverage labs. Bottled water can become contaminated, slowing research and increasing costs. The Direct-Q system, weighing about 7 kg, will be offered in bench-style, wall-mounted, and portable versions as a compact unit that purifies 5–15 liters of tap water per day through reverse osmosis and ion exchange. The 0.6 L/min flow rate works independently of water-feed temperature, and recycles as much as 60% of rejected water to extend the pretreatment cycle. Direct-Q disposable self-service Smart-Pak filter cartridges are good for four to six months before replacement. The Direct-Q 3 model can be wall mounted or bench mounted, and is unique among other Millipore systems in that it provides both pure- and ultrapure-grade water. The American Society for Testing and Materials International defines what quality grade of water is needed for optimum research results. Grade 2 and Grade 3 (pure) water is used in media preparation, glassware rinse, and tissue culture. Grade 1 (ultrapure) water is reserved for more critical applications such as PCR and ion chromatography. Aside from affecting the quality of research, water purity can be critical for regulatory compliance. Currently, Millipore sells the Direct-Q 5, a 30-kg system designed for researchers in validated lab environments needing a high-flow rate for purified water. The unit costs around $5,100. The new Direct-Q line, designed for the small-volume user in nonvalidated labs, will have five units, including the RiOS-DI (available in July 2005), and the RiOS, Synergy, and Simplicity (available this fall). The Direct-Q 3 unit will be available in June at a cost between $1,000 and $3,000, based on customer application.


• microfluidics : going small is becoming increasingly big business in the life sciences, as the desire for quicker results, higher throughput and lower costs is creating a booming market for microfluidics technology—which involves extremely small volumes of liquid. Originally a scientific curiosity of physicists and chemists, microfluidics now appears ready to transform traditional assay systems in academia and biotech as well as in big pharma and hospitals, with devices labelled as 'pinhead petri dishes' and 'Lab-on-a-chip'. At its most fanciful, microfluidics could revolutionize the way in which doctors diagnose disease and monitor treatment—with portable devices for rapid automated analysis of body fluids or tissue samples, going from sample preparation to data readout in minutes or even seconds. Meanwhile, laboratory scientists are beginning to find themselves as the giant operators of a Lilliputian world in which tens or hundreds of experiments take place in parallel on 'microchips' no bigger than a credit card, reducing sample size and consumption of solvents and reagents^{ref1, ref2, ref3}. According to a recent market analysis^ref, microfluidics applications in the life sciences have a global market of around 500 million €, which could rise to around 1.4 billion € by 2008, particularly if sales in drug discovery, medical diagnostics and therapeutic devices live up to their promise. Evolution may have gotten there first with the proboscis of a mosquito for drawing up blood, but microfluidics engineers are creating their own narrow-gage devices through which tiny volumes of fluid can be propelled by laminar flow, electrophoresis or centrifugal force. The small size of microfluidic channels means that viscosity and surface tension tend to impede flow far more than for larger volumes of liquid. As a result, any of 3 different types of forces may be chosen to drive the flow of microfluidic devices^ref :
• to drive electro-osmosis, or the movement of charged molecules across an electrical current, Micronit's custom-designed microreactors, for example, use a high voltage to create an electrical potential across microfluidic channels. Electro-osmosis also forms the basis for BioRad's Experion automated benchtop electrophoresis system, and Agilent's 2100 Bioanalyzer and 5100 automated Lab-on-a-Chip. Caliper Life Sciences' microfluidic devices use both electrokinetic and pressure-driven flow.
• laminar flow devices, in which fluids flow smoothly and without turbulence, require pumps to deliver high pressures, up to 100 bars. Micronics, for example, offers Microflow, a software-controlled ultra-low-pulse pump system for injection and analysis of submicroliter volumes of samples and reagents in microfluidic Lab Cards. The advantage of pressure-driven flow is that it can be used to move both charged and uncharged molecules, cells and beads.
• meanwhile, the Swedish company Gyros makes use of centrifugal force in its compact disc (CD)-style solution, called Gyrolab, for customers in proteomics and the large instruments research market. Liquid is drawn into distribution channels by capillary action and then spun inside a workstation, driving precise nanoliter volumes through hundreds of individual microstructures simultaneously. The system enhances reproducibility and allows parallel processing, thus reducing reaction time, and avoids pipetting errors. CDs with customized surface chemistries have been developed for applications such as protein and cytokine quantification by immunoassay (with detection by fluorescence), and peptide concentration measurement. The CD can be cut into pieces and placed directly into certain brands of mass spectrometers for analysis, such as those supplied by Applied Biosystems Inc., Bruker and Shimatsu. Gyros is seeking partners to develop this and other applications, including in vitro diagnostics.
Nature's had millions of years to move liquids through small capillaries. Microfluidics technology gives us the ability to work with relatively small amounts of samples. This capability, of course, has applications in a variety of areas—from disease diagnosis to drug discovery, and the plethora of laboratory techniques for which smaller means better, faster or more convenient. Industry pundits predict that clinical diagnostics will be one of the biggest target areas for microfluidics applications, in which miniaturized bioassays can be performed more quickly and at lower cost than with conventional methods. This is driving the development of portable devices for use at patients' bedsides or in doctors' offices, thus skipping the need for more time-consuming tests in specialized laboratories. There are few products on the market now, but it seems to be a promising field for the future—many companies are interested in this. The Liquid Handling Competence Centre (LICOM)—an EU-funded consortium that provides microfluidics R&D services for the life sciences. Companies developing microfluidics 'point-of-care' tools and products include HandyLab and Micronics. Micronics offers a laminar flow-based microfluidics Lab Card prototyping service with Active, integrating various types of clinical chemistries, immunoassays and nucleic acid assays for a range of applications. This already forms the basis for an ABO blood typing card, licensed to Pall Corporation, which achieves a result in just ten seconds from a finger-prick drop of blood. All reagents are on the card. You only have to introduce the sample. Micronics also plans to produce its own brand of devices for infectious disease and cancer diagnosis, involving either immunoassay or rapid (7-min) PCR, and has developed a portable Microcytometer for cell counting and cell sorting applications in healthcare or for sampling of water, soil and air for microbes. The widespread use of PCR in DNA and RNA analysis makes this an especially attractive target for microfluidics techno-logy. Companies with PCR-on-a-card technology include Roche, Caliper Life Sciences and Cepheid. Cepheid's GeneXpert and SmartCycler extracts and tests DNA and RNA from blood, microbial spores and other biological samples. At its core is a module the size of a deck of cards, which replicates DNA and optically measures the results. The technology can reduce analysis time from around two days to just 30 minutes, for applications including diagnosis of infectious disease, cancer and the detection of biothreats such as anthrax. Its sensitivity is comparable to lab-based PCR. For PCR, as for other microfluidics applications, sample preparation remains a potential bottleneck. There's this notion that samples magically get there on the chip. For PCR, extraction and preparation of DNA can take hours. Microfluidic Systems (MFSI) offers a solution with its first commercial product, BioLyser, an automated system that lyses cells, spores and viruses using ultrasonic energy, and then extracts DNA, all within disposable closed capsules. The system obviates the need for special buffers and avoids cross-contamination of samples. The next step is to link this with PCR, and MFSI have just delivered to the US Federal Bureau of Investigation a prototype forensics analysis kit that can perform extraction and separation of DNA from different cell types—for example to distinguish the DNA of rapists and their victims—followed by PCR amplification, analysis and readout. If approved, it could become a conveniently portable tool for police investigators. Meanwhile, Microchip Biotechnologies of Fremont, California, are developing a microbead capture-based platform for a next generation DNA sequencer that performs DNA library construction and amplification, fluorescent labelling and affinity capture, separation and sizing by microcapillary electrophoresis. The system is intended to reduce the cost of shotgun sequencing and resequencing by requiring only nanoliter volumes, reducing reagent consumption by 100-fold. The use of microfluidic devices based on electrophoresis has the potential to replace traditional one- and two-dimensional gel electrophoresis for the separation of biological molecules, such as proteins, RNA and DNA. Agilent Technologies' 2100 Bioanalyzer performs electrophoretic separation and quantification of DNA, RNA or proteins, all within a fingernail-sized glass chip. One application now being explored is its use in mitochondrial DNA analysis, a technique that is particularly useful in genetic typing of small or degraded samples for victim identification and forensic DNA profiling. Other applications include genomics, proteomics, drug development and manufacture, food safety and homeland security. The chip can hold and analyze up to 12 samples in just 30 minutes. The more recent Agilent Technologies' 5100 automated Lab-on-a-Chip platform fully automates the entire process of sample preparation from standard 96- or 384-well plates, analysis and digital data readout and storage. Applications include quality control of PCR fragments prior to spotting to microarrays, target validation or sequencing, and high-throughput protein purification. BioRad Laboratories' automated benchtop assay system Experion, also separates proteins and RNA and analyzes RNA for purity, with the advantages of being faster, allowing automation, and requiring smaller amounts of samples and reagents than traditional gel electrophoresis. Caliper Life Sciences has produced Lab Chip 90, an automated DNA and protein gel electrophoresis system, which produces digitized data and bypasses the need for scanning gels. And through collaboration with Agilent Technologies, its Lab Chip 3000 drug discovery system is proving popular with major pharmaceutical companies for screening organic compound libraries in enzymatic and cell-based assays. It includes proprietary 'Sipper chips' to automatically bring in samples stored in microtiter plates. Anything done with agarose gels can be done with chips, with the advantage that results are more reproducible and data can be archived and shared with colleagues. Caliper's collaborators include Predicant for the production of a microfluidics-based protein separation system for the analysis of complex protein patterns in blood, Affymetrix for applications in pharmacogenomics and personalized medicine, and Bio-Rad Laboratories. The pharmaceutical industry is investing substantially in microfluidics as a way to expand screening capacity at several stages in the drug discovery process. One present bottleneck is the separation of biological compounds by liquid chromatography, for studies including drug metabolism and pharmacokinetic assays. To speed up the time to results, Nanostream offers the Veloce system that performs 24 simultaneous separations by micro parallel liquid chromatography, in 24-column microfluidic Brio cartridges. An add-on software-controlled fraction collector collects each eluant at fixed time intervals ready for analysis by mass spectrometry. This avoids the delay associated with serial liquid chromatography runs, and lets the mass spectrometer go as fast as it can go. Agilent Technologies is applying the inject nozzle technology of its parent company, Hewlett Packard, to the same problem, providing a microfluidics-based interface between high-performance li-quid chromatography (HPLC) and mass spectrometry (MS). Its HPLC-Chip/MS miniaturizes HPLC to nanoscale volumes, integrating sample preparation, separation and electrospray ionization onto a single chip smaller than a credit card. The device mounts directly onto a mass spectrometer, and has the advantages of reduced sample size, and enhanced separation and sensit-ivity compared to standard HPLC. The need for functional cell-based assays causes a bottleneck during lead optimization and target validation. Sweden-based Cellectricon addresses this with Dynaflow, a computer controlled microfluidic chip with inbuilt patch-clamp and drug-dispenser technology that speeds up drug screening against whole cell ion channels. Customers include AstraZeneca, Merck and GlaxoSmithKline. One of the biggest growth markets for applying microfluidics in the life sciences is microarray technology, according to a 2003 Frost and Sullivan report, which predicted 32% annual growth rate in this area^ref. Companies developing tools in this area include Affymetrix, with a license from Caliper Life Sciences to apply microfluidics technology to nucleic acid separation, BioRad Laboratories, Invitrogen, Perkin Elmer and the European consortium LICOM. Invitrogen's three-dimensional microarray DNA chip, XeoChip, contains thousands of three-dimensional nanochambers in an area the size of a dime. Each chamber serves as a protected reaction vessel for DNA synthesis and subsequent bioassay for use with conventional microarray scanners. The same system can be adapted for the synthesis of RNA, peptides and other organic molecules. Microdrop Technologies' has an inkjet technology?inspired microfluidic nozzle system that uses brief voltage-generated pressure pulses to dispense tiny drops of between 30 and 100 ml from glass pipettes onto a target surface, at 2 m/s. This is licensed exclusively to Perkin Elmer for microarray production, using eight such nozzles in parallel to be compatible with microtiter plate systems. The nozzles are made of glass to allow the use of aggressive organic solvents and pH. Similarly, LICOM has developed a silicon chip with tiny nozzles that dispense 96 droplets of different liquids simultaneously onto a glass slide in a contact-free manner, driven by a pressure pulse. Many companies in Europe and the United States provide both off-the-shelf microfluidics components, and custom design and manufacturing services, including LioniX, Micronics Inc., Boehringer-Ingelheim's MicroParts, Silicon Valley Scientific Inc., Microdrop and LICOM. Customers are wide ranging according to Strange of ThinXXS, who target device makers in the life sciences, pharma, healthcare, environmental monitoring and food monitoring. Among them is California-based Pelikan Technologies, with a blood glucose monitoring device due to be launched in the near future, and Denmark-based Sophion Bioscience for an automated system for ion channel analysis that replaces traditional patch clamping. LICOM's collaborations include the development of microfluidic-based high-throughput screening and the manufacture of medical devices such as insulin infusion pumps, nebulizers for inhalers and other drug delivery systems. Some producers focus on using traditional materials, glass and silicon. Micronit Microfluidics, for example, produces glass and fused silica chips for capillary electrophoresis in high-throughput systems, for anyone from multibillion dollar international companies to research groups. Glass and fused silica, he says, have the benefits of being optically transparent, resistant to high temperatures and chemically inert, and are ideally suited for applications including HPLC, electrophoresis and automated high-throughput screening of drug candidates on whole cells. In contrast, Microfluidic ChipShop and ThinXXS produce plastic polymer?based microfluidic chips with the potential advantages of lower cost and ease of chemical modification compared to glass or silicon. These are ideally suited to use in diagnostics and bioanalysis, and offer the freedom to introduce fluidic fittings and structures for injecting or collecting samples. Various devices may be incorporated for the control of microfluidic flow and delivery. For example, Zurich-based Sensirion attaches electronically controlled flow sensors to the outside of microfluidic tubing for measuring pressure and flow rates down to the rate of 0.5 nl/min. This permits the detection of problems such as changes in flow caused by vibrations, clogging—owing to cells or other particles—and leakage. Customers in both research institutes and industry are using these either directly or incorporating them into complex instruments, including for the development of drug delivery systems, or, as in the collaboration with Dionex Corporation, for processes such as liquid chromatography. ABTECH Scientific, Micronit and Microdrop, also offer custom design services for the use of biosensors. Abtech Scientific's microfabricated electrodes can be placed directly into a flowing stream to detect or capture target biomolecules. Micronit also provides integrated electrodes, for example, for the creation of bead- or cell-sorting chips, or for measuring blood contamination with metals such as lithium. With such a plethora of microfluidic tools and devices already, or soon to arrive, on the market, only the sky seems to be the limit. There still remains, however, the challenge of persuading scientists to fully embrace the technology. The world is macro—there are sequencers and mass spectrometers and other big pieces of equipment. You're not going to tell them overnight that they should get rid of that! There may be some psychological barrier to switching to a microfluidic scale : people are used to doing things in a certain way and don't like change, but there are a lot more possibilities that people think.
Microchips consist of channels and chambers either etched or sand-blasted into glass or silicon, or molded into plastic polymer chips, and can incorporate valves, pumps and mixing chambers to control the flow and the mixing of reagents. Manufacturers often use proprietary technology—for example, that used by Micronit to bond together several glass layers without the need for glue—to create multiple channel depths on a single chip. The channels of microfluidic chips vary in size and shape depending on the application, and tend to be less than 100 mm in diameter. For the movement of cell suspensions, for example, channels are usually at least 50 mm in diameter to minimize the risk of clogging, whereas for DNA solutions, less than 10 mm works fine. ThinXXS produces a 'snake mixer slide' containing undulating channels that permit fluids to be mixed intentionally. In contrast, Microfluidic ChipShop's microchips consist of glass slides with H-shaped channels to permit two fluids to flow in opposite directions without mixing—for example, to clean up PCR solutions. Four microfluidic slides fit together into a standard 384-well microtiter plate frame for use in high-throughput robotic systems. Bio-Rad Laboratories' Experion chips consist of several cross-shaped channels filled with liquid polymer, into which up to ten samples can be driven by an electric field for size-based separation. Channel number also varies considerably. MicroParts, for example, produce a stamp-sized Lilliput chip for microorganism identification and antibiotic resistance assays. It consists of 96 reaction cavities, each with a volume of 1.8 ml—one hundredth of the volume of conventional microtiter plates. The cavities are connected via microchannels to a central reservoir, allowing simultaneous and automatic filling of all reaction cavities within a minute.
The recent expansion of biodefense concerns has created a market for microfluidics-based devices for the rapid detection and monitoring of infectious organisms. Together with Agilent Technologies, Invitrogen is marketing the PathAlert detection system, a PCR-based system for screening sample- and water-borne infectious agents including those responsible for anthrax, plague and tularaemia. The system combines Invitrogen's PathAlert detection kits with the Agilent 2100 Bioanalyzer or the Agilent 5100 automated Lab-on-a-chip platform, to allow higher throughput analysis, quantification and monitoring of target organisms. One of the challenges in applying microfluidics technology to biodefense screening is how to overcome the problem of low concentration of target pathogens—analysis of just a few microliters of air may not detect their presence, for example. To this end, Microfluidic Systems Inc., in collaboration with the US Department of Homeland Security and the US Army, including USAMRIID, the lead medical research laboratory for the US Biological Defense Research Program, is creating a microfluidics-based rapid pathogen detection system that concentrates and identifies viruses, bacteria and toxins in less than five minutes. The system depends upon the flow of air or liquid across a series of deep-etched pillars, 5 mm tall and 150 mm in diameter, which together present a sufficiently large surface area—on the order of centimeters—to capture DNA, proteins or cells and elute into 50-ml fractions. "That's the critical step, to go from tens of milliliters down to 50 ml. The same is true for trying to bring high-sensitivity diagnostics down to the scale of microfluidics: a single virus in a milliliter of blood could easily go undetected in microfluidic devices unless sample preparation involves a concentration step. Meanwhile, Microchip Biotechnologies is using its microbead capture technology to concentrate pathogens from dilute samples of air, water and food from large volumes to milliliter and microliter volumes, before transferring the sample into a microfluidic device for processing and analysis of DNA, RNA or proteins. Cepheid's solution is to use different kinds of filters to capture cells for analysis on GeneXpert, and a solid resin for concentrating nucleic acids, allowing larger volumes of sample to be used.
Web resources :
• companies offering products and services using microfluidics : Abbott Laboratories, ABTECH Scientific Inc., Advalytix, Advanced Liquid Logic, Affymetrix, Inc., Agilent Technologies, Applied Biosystems, Aviva Biosciences, Bartels Mikrotechnik, Biacore, Bioident, BioMicro Systems Inc., Bioprocessors, Bio-Rad Laboratories, Biotrove, Boehringer Ingelheim microParts GmbH, Caliper Life Sciences, Cellectricon AB, Cellix, Cepheid, Ciphergen, Cole-Parmer, Dionex, Eksigent Technologies, LLC, Erie Scientific Company, Evotec Technologies, Fluidigm, Genoptix, Gyros, HandyLab, Helicos Biosciences Corporation, Hewlett-Packard, Ibidi Integrated BioDiagnostics, Iq Micro Inc., Invitrogen, LioniX, Liquid Handling Competence Centre (LICOM), Liquidia Technologies, Micralyne Inc., Microchip Biotechnologies Inc, microdrop Technologies GmbH, MicroFluidic Systems, microfluidic ChipShop GmbH, Microfluidics, Micronics, Inc., Micronit Microfluidics bv, Monogram Biosciences, Nanogen, Nanostream, Nanoterra, Network Biosystems, Orchid Biosciences, Predicant Biosciences, Pyrosequencing AB, Roche 454, Sensirion AG, Shimadzu Corporation, Silicon Valley Scientific, Inc., Spin X Technologies, Surface Logix, Tecan, thinXXS, Tronics Microsystems, Zyomyx, Inc.

• BioSupplyNet: Comprehensive listing of biomedical suppliers
• The Nest Group Molecular Protocols and Application Guides
• Anderson's Timesaving Comparative Guides for Biological Reagents
• Collection of Molecular Biology Protocols: Jim Brown
• Molecular Biology Protocols on WWW Horizon Press
• Molecular Biology Protocols on WWW: PCR Horizon Press
• Molecular Biology Protocols on WWW: Genetics Horizon Press
• Vector Sequence Database Anderson Unicom Group, Inc.
• Protocols on the World Wide Web--Hugh Griffin's Jump Station to Protocols, Books, Manuals, etc.
• A Selection of Molecular Biology Protocols -- A list of sites with protocols, by Hugh Griffin and published in Analytical Biochemistry 239, 120-122 (1996)
• Elsevier Trends Technical Tips Online -- Technical tips, reviewed protocols, very useful
• Recombinant DNA Isolation, Cloning, and Sequencing Protocols. Bruce Roe, Department of Chemistry and Biochemistry, University of Oklahoma (fantastic, comprehensive manual of DNA manipulation techniques)
• Protocols from the Douglas Crawford Laboratory (Molecular Biology and Biochemistry Division of the School of Biological Sciences at The University of Missouri Kansas City)
• Molecular Biology Techniques Manual. Ed Rybicki, Department of Microbiology, University of Cape Town, SA
• BIOSCI/bionet Electronic Newsgroup Network for Biology
• companies offering automated laboratory systems : Abbott Molecular, Abgene, Agilent, Applied Biosystems, Aurora Biomed, B-Bridge, BD Biosciences,

Beckman Coulter, Biometra, Bio-Rad Laboratories, BioTec, BioTek, Bruker Daltonics, Caliper Life Sciences, Corbett Life Sciences, CyBio, Deerac Fluidics, Eppendorf, Genetix, Genomic Solutions, Gentra, Gilson, Hamilton Robotics, Hudson Control Group, KBiosystems, Labcyte, Lab Services BV,
Magnetic Biosolutions, Microtec Nition, Millipore, Molecular Devices, Parallab, PerkinElmer, Promega, Protedyne, Qiagen, RoboDesign, Roche Applied Science, RTS Life Science, Sias, Tecan Group, Ltd., The Automation Partnership, Thermo Electron, Titertek, Torcon Instruments, TTP Labtech, Velocity11, Xiril, Zinsser Analytic

• Sambrook, J,; Fritsch, EF, Maniatis, T : Molecular cloning, CSHL Laboratory Press, 2nd edition, 1999

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