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

Abstract

AbstractA 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 μm 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 soluble polymers. The washed gels were impregnated 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 efficient 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 values of 500–900 V/cm and thus, at a given 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.