PREPARATIVE ELECTROPHORESIS ON LINEAR POLYACRYLAMIDE-AGAROSE COMPOSITE GELS

A preparative method for isolating centigram quantities of high molecu lar weight polypeptide chains with high resolution and recovery uses l inear polyacrylamide/agarose composite (LPAC) gels as electrophoretic media from which the polypeptides can be easily extracted. The composi tes are prepared in a manner yielding linear copolymers of acrylamide and 1-allyloxy-2.3-propanediol within 2% agarose gels. After electroph oresis in sodium dodecyl sulfate (SDS), protein bands were rapidly vis ualized for excision by briefly immersing the gel in cold 0.1 M KCl wh ich precipitates the protein-associated SDS. The gel slices Sire then freeze-thawed to disrupt the agarose matrix and promote syneresis of f luid upon centrifugation. The polypeptides are then separated froth th e polyacrylamide in the supernatant solution by precipitating with eit her acidic isopropanol, trichloroacetic acid, ammonium sulfate or othe r general protein precipitants. As determined with polypeptide chains of fibrinogen and its cross-linked derivatives. recoveries were virtua lly complete (95.4% +/- 2.2%), and were independent of molecular weigh ts over the range tested (10(4)-10(6)).