Direct Sequence Analysis of Proteins after Electroblotting from SDS/Polyacrylamide Gel Separations

Abstract

Electrotransfer of protein bands from SDS/polyacrylamide gels onto polybrene-impregnated glass fiber filter discs (12 mm diameter) using an electrophoretic concentrator is described. Electrophoretically resolved polypeptides are detected by visualization in the gel with potassium chloride. They are then separately excised and individually electroblotted for subsequent direct gas phase sequencer analysis. Transferred and filter-immobilized polypeptides can be stored under vacuum. The method has been tested on different proteins/peptides, and overall combined yields (from sample application for electrophoresis through to recovery of phenylthiohydantoin from the sequencer) can approach 50% (average about 20%).