Isoelectric focusing of proteins in rewetted ultrathin polyacrylamide gel layers

Abstract

Ultrathin layers of polyacrylamide gel bound to glass can be washed, air-dried, and stored for at least 1 year before rewetting in ampholyte solutions for isoelectric focusing. Short-term drying affects neither fluorescent banding of the ampholytes (not evident in conventional gels) nor resolution of complex protein mixtures while prolonged storage seems to have no deleterious effects. Layers are fully functional after soaking for 10 min in solutions that may contain 8 m urea and 10% sorbitol. Rewetting allows the rapid survey of different ampholytes, gradient stabilizers, separator compounds, or protein reagents and is adaptable to concentration modification of the pH gradient (alone or with a gel overlay), to focusing in a transverse urea gradient, and to electrophoresis across a preformed pH gradient. The procedure avoids protein modification by residual polymerizing reagents while adding to the convenience and economy of using ultrathin layers in relatively small formats.