Application of separator isoelectric focusing within the pH range 4‐6

Gill, Peter

doi:10.1002/elps.1150060607

Electrophoresis

1985

DOI: 10.1002/elps.1150060607

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Application of separator isoelectric focusing within the pH range 4‐6

Peter Gill¹

Abstract: Application of separator isoelectric focusing within theCentral Research Establishment, pH range 4-6 Home Office Forsenic Science Service, Aldermaston, Reading, BerkshireSeparator isoelectric focusing is a simple technique for creating narrow pH gradients (less than 1 pH unit wide). It has proved useful elsewhere for the enhanced separation of phosphoglucomutase, esterase D, group specific component (Gc) haemoglobin, transferrin and for distinguishing between seminal and vaginal acid phosphatases. The properti… Show more

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Cited by 6 publications

References 12 publications

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Improved esterase D typing on ultrathin isoelectric focusing gels using narrow interval Pharmalyte carrier ampholytes (pH 4.5–5.4) in conjunction with a separator and a thickness modified pH gradient

Gill

1985

Electrophoresis

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Home Office Forensic Science Service Aldermaston. Reading, Berkshire -, RG7 4PNand a thickness modified pH gradient Three common alleles are found in esterase D: EsD 1, EsD 2 and EsD 5. In addition, a number of rare variants are found. The isoelectric points of the EsD 1 and EsD 2 isoenzymes differ by only 0.02 pH units, which could lead to possible confusion between the two using conventional isoelectric focusing (IEF). Therefore, in order to ensure adequate separation of these two bands, it is necessary to use a narrow pH gradient. The separation of esterase D (EsD) has been improved on electrofocusing gels by incorporating a narrow range Pharmalyte (pH 4.5-5.4) in conjunction with the separator N~(2-hydroxyethyl)piperazine-N'-2-ethanesulphonic acid (HEPES). This mixture resultedintheformationofanarrowpHrangeofpH 4.85-5.35 (themajor typing bands of EsD fall within the range of p l 4.9-5.2). Resolution was further improved by the use of thickness modified pH gradients in ultrathin gels which flattened the pH gradient in the range ofpH 5.0-5.1. The techniques used were shown to be effective for typing bloodstains up to 1 month old.

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Improved esterase D typing on ultrathin isoelectric focusing gels using narrow interval Pharmalyte carrier ampholytes (pH 4.5–5.4) in conjunction with a separator and a thickness modified pH gradient

Gill

1985

Electrophoresis

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Preparation of rehydratable polyacrylamide gels and their application in ultrathin‐layer isoelectric focusing

et al. 1986

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A new approach to isoelectric focusing in polyacrylamide gels is described, based on the use of rehydratable gels which in dry form could be stored for extended periods and which prior to use were rehydrated with solutions of any composition. Ultrathin 60-240 pm polyacrylamide gels, with different composition (5 % T, 3 % C; 3 % T, 4 % C ; 3 % T, 20 % C), were polymerized under well-standardized conditions and, after polymerization, washed exhaustively with distilled water to remove any unreacted monomers, catalysts or solublepolymers. The washed gels wereimpregnated with suitable additives, before drying, to preserve gel functionality on storage. Polyol compounds, such as glycerol, sorbitol and dextran, as well as synthetic polymers like polyethylene glycol and polyvinylpyrrolidone, were the most efticient additives when incorporated into the gel in a concentration of 1 -10 %, either as single substances or in different combinations. Prior to isoelectric focusing the dry gels were rehydrated to the original gel volume with a solution of carrier ampholytes, in some experiments with added separators or urea. Kinetic studies have shown rehydration, depending on gel thickness, to be complete within a few minutes. Isoelectric focusing in rehydratable gels was consistently more reproducible than in wet gels by such criteria as regularity of patterns and coalescence of marker proteins, including ferritin, applied at different positions. Rehydratable gels tolerated higher field strengths at the final stage of isoelectric focusing, with typical valuesof 500-900 V/cm and thus, at agiven volt x hour product, equilibrium focusing could be attained in a shorter time, with improved resolution and sharper zones. Ultrathin-layer isoelectric focusing in rehydratable gels proved insensitive to high salt concentrations (up to 0.5 M) of the samples. Rehydratable gels represent a new generation of gels, excelling over the traditional wet gels by better standardized properties, convenient handling and unsurpassed flexibility. tetramethylethylenediamine; DHEBA, NJ-( 1,2-dihydroxyIethylene)bisacrylamide; HEPES, N-(2 hydroxyethyl)piperazine-N'-2 ethanesulfonic acid; ACES, N-(2-acetamido)-2-aminoethanesulfonic acid; BICINE, N.N-bisl2-hydroxyethyl)glycine; Vh, volt X hour; IPG. immmobilized pH gradient 'D

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Steady state electrolysis and isoelectric focusing

Hagedorn

Fuhr

1990

Electrophoresis

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The properties of pH gradients formed by stationary electrolysis of weak mobile or fixed electrolytes are analyzed. The model uses the appropriate balance equations and those of chemical equilibria. It is shown how the equation of the current density has to be modified for considering that fraction of current that is associated with the diffusion of neutral buffer molecules within a pH gradient. Furthermore it is shown that the pH gradients themselves give rise to water production within the gradient and that essential properties of the steady state are related to chemical reactions between the electrolyte constituents. The differential equations describing the gradients of the concentration of a given component, the pH, conductivity and potential are explicitly formulated in relation to those reactions. The equations are solved numerically and the significance of the results for isoelectric focusing is discussed. The experimental conditions to reach shallow and smooth pH gradients exhibiting sufficient ionic strength are formulated.

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Application of separator isoelectric focusing within the pH range 4‐6

Cited by 6 publications

References 12 publications

Improved esterase D typing on ultrathin isoelectric focusing gels using narrow interval Pharmalyte carrier ampholytes (pH 4.5–5.4) in conjunction with a separator and a thickness modified pH gradient

Improved esterase D typing on ultrathin isoelectric focusing gels using narrow interval Pharmalyte carrier ampholytes (pH 4.5–5.4) in conjunction with a separator and a thickness modified pH gradient

Preparation of rehydratable polyacrylamide gels and their application in ultrathin‐layer isoelectric focusing

Steady state electrolysis and isoelectric focusing

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