The Maxam and Gilbert method
In this method the single-stranded DNA fragment to be sequenced is end-labelled by treatment with alkaline phosphatase to remove the 5' phosphate, followed by reaction with 32P-labelled ATP in the presence of polynucleotide kinase, which attaches 32P to the 5' terminal. The labelled DNA fragment is then divided into four aliquots, each of which is treated with a reagent which modifies a specific base as follows. Aliquot 1 is treated with dimethyl sulphate, which methylates guanine residues....
Scintillation counters
Some substances, known as fluors or scintillants, respond to the ionizing effects of alpha and beta particles by emitting flashes of light or scintillations . While they do not respond directly to gamma rays, they do respond to the secondary ionization effects that gamma rays produce and, as a result, provide a valuable detection system for all emissions. A range of scintillants is available, many designed for maximum efficiency with specific isotopes. Crystals of sodium iodide containing a...
Immobilized enzymes
It is possible to bind enzymes to an insoluble matrix by a variety of methods and still retain their catalytic activity. The reusable nature of immobilized enzymes can significantly reduce costs and provides a convenient source of enzymes for performing substrate assays. Such preparations often show a greater stability and reduced inhibition effects than do soluble enzymes, although occasionally optimum pH values may be altered slightly. Covalent linking to a variety of polymers using...
Immunoprecipitation in solution
The number and size of the immune complexes formed by the reaction of antigen with antibody is dependent on the relative concentrations of the reactants Figure 7.5 . The formation of antigen-antibody complexes may be observed by measuring the apparent absorption of light when incident light is scattered by the complexes or by direct measurement of the scattered light. The reaction is carried out in an excess of antibody and a calibration curve is prepared by measuring the turbidity of a series...
Solvent extraction
Lipids can be extracted from biological samples using a variety of organic solvents. A chloroform-methanol solvent is suitable for all lipids but it is possible to extract different classes of lipid selectively on the basis of their solubility in different organic solvents. This may be achieved by the addition of a solvent that will effect either the precipitation or the extraction of the lipids of interest. An example of the former is the precipitation of high concentrations of phospholipids...
General principles of analytical biochemistry
The selection of a valid method of analysis gt A qualitative method enables only the presence of the substance to be detected. gt A quantitative method t-nahles the amount of substance present in a sample to be determined. Analytical biochemistry involves the use of laboratory methods to determine the composition of biological samples and it has applications in many widely differing areas of biological science. The information gained from an analysis is usually presented as a laboratory...
Info Ptv
Very widely used for one-way runs or as a first solvent in two-way chromatography Very widely used for one-way runs or as a first solvent in two-way chromatography Gives a large spread of RF values for a range of amino acids. Must always be used second in any two-way runs. The removal of the phenol is time consuming. Precautions must be exercised in its use because of the toxic and corrosive nature of phenol Useful for the separation of basic amino acids and must always be used second in any...
BeerLambert relationship
Lambert's law states that the proportion of radiant energy absorbed by a substance is independent of the intensity of the incident radiation. Beer's law states that the absorption of radiant energy is proportional to the total number of molecules in the light path. Beer's law describes the basic relationship between the concentration of the absorbing substance and the measured value of absorbed radiation. Lambert's law is of major significance in the manner in which measurements are made and...
Dry chemistry analysers
Advances in the technology associated with the production of immobilized reagents in thin layers has resulted in the development of a range of analytical systems in which the assays are carried out by the addition of a liquid sample to the reagents in a dry form. This method of analysis requires no reagent preparation and can be conveniently performed without the usual laboratory facilities. A major application is in the analysis of a range of blood serum components for clinical diagnostic...
Inhibition of enzymes
A substance that decreases the rate of an enzyme-catalysed reaction is known as an inhibitor and its effects may be permanent or transient. The inhibition of some reactions by substances which may be products of either that reaction or a subsequent reaction provides a control mechanism for cellular metabolism, while the selective inhibition of enzymes is the basis of many aspects of pharmacology and chemotherapy. Some inhibitors structurally resemble the substrate and are bound by the enzymes...
Info Hrh
Figure 11.9 Biuret reaction. The coordination complex formed in alkaline solution between cupric ions and the nucleophilic nitrogen atoms in four molecules of biuret. Figure 11.10 Absorption spectrum of the protein-copper complex of the biuret reaction. of the protein-copper complex. The biuret reagent consists of an alkaline solution of copper sulphate with either sodium potassium tartrate or sodium citrate added to prevent the precipitation of cupric ions as the hydroxide. The method is...
Fluorescamine
All primary amines react with fluorescamine under alkaline conditions pH 9-11 to form a fluorescent product Figure 10.12 excitation maximum, 390 nm emission maximum, 475 nm . The fluorescence is unstable in aqueous solution and the reagent must be prepared in acetone. The secondary amines, proline and hydroxyproline, do not react unless they are first converted to primary amines, which can be done using iV-chlorosuccinimide. Although the reagent is of interest because of its fast reaction rate...
Good laboratory practice
Good laboratory practice GLP is a set of procedures within which the overall performance of a laboratory can be monitored. It is applicable to the organization and functioning of any laboratory but it is particularly relevant to the pharmaceutical industry. Compliance with GLP may be required for accreditation of a laboratory by an external regulating agency. GLP involves all aspects of the organization which is involved in generating an analytical result, from senior management to the bench...
I Och
gt Cerebrosides contain a ceramide unit and either a glucose or galactose residue. Figure 12.12 Sphingomyelins. Sphingomyelins are esters of a ceramide and phospho-ryl choline. However, similar compounds are ceramide-1-phosphoryl ethanolamines and phosphono forms of sphingolipids. Ceramides W-acyl-sphingosines are amides of a long chain di- or trihydroxy base containing 12 to 22 carbon atoms, of which sphingosine 4-sphingenine is the commonest, and a long chain fatty acid whose acyl chain is...
Immunoprecipitation in gels
Gels are used in immunoprecipitation techniques to stabilize the precipitate, enabling both the position and the area of the precipitate to be measured. The point has already been made that maximum precipitation occurs when the equivalent proportions of both antigen and antibody are available. Hence, if a high concentration of antigen is permitted to diffuse into a gel that contains a uniform concentration of antibody, at some point in the concentration gradient of antigen that is gt Single...
Zeta Potential
Figure 3.23 The charge on a colloid. The charge carried by a colloid because of its chemical composition and the pH of the solution the electrochemical potential is reduced by the adsorption of ions from the solution and the resulting charge is known as the zeta potential. gt Electroendosmosis is the movement of the buffer during electrophoresis. which causes an increase in the conductivity of the electrolyte solution and this further increases the current. This rise in temperature will cause...
Selection of transformed bacteria
The procedure of inserting DNA into a vector and its subsequent introduction into the bacterial host is extremely inefficient and will result in the majority of bacteria in the culture either containing none of the required vector or containing a vector in which no DNA is inserted. However, vectors have been produced that, by placing the bacteria in specific media, allow only the growth of those containing the vector and the inserted DNA. One such plasmid vector is pBR322 Figure 13.17 . It...
Centrifugal analysers
A new concept in automated analysis was developed in the late 1960s, resulting in the introduction of centrifugal fast analysers. The development project was sponsored in the United States of America by the Institute of General Medical Sciences and the Atomic Energy Commission, and the prototype was named the GeMS AEC system. Several instruments are now available and they are all based on the original idea in which batches of samples are treated in parallel and centrifugal force is used to...
Anodic stripping voltammetry
This is a technique for trace metal analysis and is a reversal of the usual polarographic method. Using a mercury electrode, either as a hanging drop or a graphite rod coated with mercury, a negative cathodic potential is applied relative to a reference silver silver chloride electrode. Any metal ions, with discharge potentials less than that applied, will be deposited on the electrode and will form an amalgam with the mercury. It is important to control the time of electrolysis and the method...
Load Position
Figure 3.6 Diagram of a six-port sample injection valve. In the load position the loop can be filled with the sample using a simple syringe. When the valve is turned to the inject position, the sample is washed into the column. gt A diode array is a spectroscopic device for the continuous monitoring of absorbance over a specified wavelength range. A continuously monitoring detector of high sensitivity is required and those that measure absorption in the ultraviolet are probably the most...
High performance liquid chromatography 1
The use of reverse-phase columns with pre-column derivatization of the amino acids offers an acceptable alternative to the dedicated instrumentation of an amino acid analyser or separation by HPLC followed by post-column derivatization. Buffered mobile phases are used and the proportions of polar solvents e.g. methanol, tetrahydrofuran depend upon the type of derivative employed. Gradient elution is required for the resolution of complex mixtures and analysis times are less than 1 h....
Ninhydrin
Ninhydrin triketohydrindene hydrate reacts with an amino acid when heated under acidic conditions pH 3-4 to produce ammonia, carbon dioxide and a blue-purple complex. This reaction forms the basis of many widely used methods Figure 10.11 . One mole of carbon dioxide is liberated from each mole of amino acid, exceptions being the dicarboxylic amino acids, which produce two moles of carbon dioxide, and the a-imino acids, proline and hydroxyproline, which do not produce carbon dioxide. Although...
Further reading Lro
Kirkwood, E. and Lewis, C J. 1991 Understanding medical immunology, 2nd edition, Wiley-Liss, USA. Coleman, R.M. 1992 Fundamental immunology, 2nd edition, William C. Brown Co., USA. Clausen, J. 1989 Immunological techniques for the identification and estimation of macromolecules, 3rd edition, Elsevier Science, Netherlands. Goding, J.W. 1996 Monoclonal antibodies principles and practice, 3rd edition, Academic Press, UK. Price, C.P. and Newman, D J. eds 1996 Principles and practice of immunoassay,...
Coulometric titrations
When a constant current is passed through the cell, the time taken for the reaction to go to completion can be used as a measure of the amount of test substance originally present. The electrode reaction may directly involve the test ions as in constant voltage techniques or alternatively may generate another substance which will react with the test substance. Reagents can be generated at an electrode which cannot be produced in any other way because they are so unstable. The end-point can be...
Noncompetitive or immunometric immunoassay
gt Heterogeneous assays are those that require separation of the bound fraction from the free. gt The titre of an antibody in the context of immunoassay is the dilution of serum used in the assay. This approach to immunoassay is characterized by the fact that the antibody is present in excess and is generally also labelled. Because the labelled antibody is in excess there is no requirement for the setting up of an equilibrium since all of the test antigen may be sequestered by the excess of...
Chromatographie methods
gt Hydrophobic interaction chromatography is a form of reverse-phase chromatography most appropriate foi protein separations. Various chromatographic techniques may be applied to the study of protein mixtures. Column techniques have the advantage that the resulting fractions are amenable to quantitation using the general method described earlier. Gel permeation chromatography is frequently used to separate protein mixtures but it is necessary to have some prior knowledge regarding the size of...