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

Abstract

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

Isoelectric focusing of proteins in rewetted ultrathin polyacrylamide gel layers

“…An intervening drying clearly adds considerable versatility, convenience, and economy to IEF, as is evident also from other studies along similar lines (4,5), and any resulting oxidizing conditions can be circumvented (3). The need for incorporating protective additives such as glycerol before drying was found in an extensive study of gels bound to polyester (4). Although a good shelf life after simple washing and drying of layers on glass was suggested by the present work, a more demanding analysis seem warranted in view of the potential value of dried gels for standardizing IEF procedures (4).…”

“…An intervening drying clearly adds considerable versatility, convenience, and economy to IEF, as is evident also from other studies along similar lines (4,5), and any resulting oxidizing conditions can be circumvented (3). The need for incorporating protective additives such as glycerol before drying was found in an extensive study of gels bound to polyester (4).…”

“…However, Robinson (2) previously obtained satisfactory focusing in relatively thick washed layers that were soaked in ampholyte solutions for only 6 h after thorough drying. This approach recently has been rediscovered in various forms (3)(4)(5) and independently in the study described here. A methodology involving ultrathin gels bound to either glass or polyester supports in semi-miniature formats is presented and its applicability to several IEF techniques demonstrated.…”

Photopatterned free-standing polyacrylamide gels for microfluidic protein electrophoresis

“…If a buffer exchange step is desired prior to PAGE, a 5 min soak in the run buffer of interest is required. 27 All devices used in this study were stored in run buffer solution prior to use. From start to finish, the fsPAG fabrication process requires ,10 min.…”

Rehydratable gel for rapid loading of nanoliter solution and its application in protein crystallization

“…PA gel could be stored in a dehydrated state after gelation and rehydrated before use to simplify the gel handling in experiments of electrophoresis-based protein separation. [17][18][19][20] The dehydrated PA gel possesses a strong affinity to water and has been used as a dehydrating reagent. 21 In our study, PA gel was produced in the microwells and dehydrated for long-term storage.…”