Assay of glucose using glucose oxidase

The flavoprotein enzyme, glucose oxidase (EC 1.1.3.4), which may be obtained from Aspergillus niger, catalyses the oxidation of 3-n-glucose by atmospheric oxygen to produce D-gluconolactone, which is converted to gluconic acid with the production of hydrogen peroxide 3-D-glucose + 02 + H20 D-gluconic acid + H202 The oxidation of a-D-glucose occurs at less than 1 of the rate of oxidation of the f3 anomer. Because these two forms exist in solution in equilibrium in the proportion of 36 (a) and 64...

H H

Owing to this partial ionic or polar nature, water molecules tend to associate with one another to form a lattice structure stabilized by these ionic interactions. Other molecules that also show this partial ionic nature will fit more readily into the lattice structure than non-polar molecules and will dissolve in water. This attraction between two polar molecules has other effects besides affecting the solubility of the substance. If one of the substances is a solid, the binding of the other...

Isotachophoresis

Isotachophoresis or displacement electrophoresis, as it is sometimes called, is a recent development of a principle described in 1920. In this technique the electrolyte between the electrodes is not a uniform single solution but a discontinuous sequence of different electrolyte solutions (Figure 3.30), the ions of which all migrate at the same velocity. Under identical conditions of concentration and voltage, different ions have different electrophoretic mobilities and to cause all the ions to...

Continuous flow analysis

In 1957 Professor Skeggs described a method of continuous flow analysis which was developed and marketed by the Technicon Instruments Co. Ltd. under the name of 'AutoAnalyser'. The single- and dual-channel instruments had the flexibility of being able to perform a variety of different tests in laboratories where samples requiring a particular analysis could be batched and analysed together. A limited range of instruments is now available from several manufacturers and although they vary in...

Detection of specific sequences of DNA

If a single-stranded DNA molecule is placed with a complementary single DNA sequence the two molecules will hybridize. This hybridization forms the basis of a number of very powerful techniques for detecting and quantifying specific nucleic acid sequences. The hybridization may be carried out either in solution or more commonly with the DNA immobilized on nitrocellulose filters. The complementary DNA sequence is known as a cDNA probe. Probes for a large number of important nucleic acid...

The Maxam and Gilbert method

Maxam Gilbert

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....

Dry chemistry analysers

Dupont Aca Reagent Pack

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...

Fluorescamine

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...

Immunoprecipitation in gels

Single Radial Immunodiffusion Principle

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...