Open AccessElectroblotting of individual polypeptides from SDS/polyacrylamide gels for direct sequence analysis
Author(s) -
BERGMAN Tomas,
JÖRNVALL Hans
Publication year - 1987
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1987.tb13573.x
Subject(s) - electroblotting , polyacrylamide gel electrophoresis , polyacrylamide , blot , chromatography , electrophoresis , chemistry , gel electrophoresis , coomassie brilliant blue , yield (engineering) , microbiology and biotechnology , biochemistry , staining , biology , materials science , gene , genetics , enzyme , metallurgy
A scheme for electroblotting of individual unstained protein bands from SDS/polyacrylamide gels and subsequent amino acid sequence analysis is described. Principal features are: detection of the polypeptide bands by visualization with KCl; electroblotting of excised gel pieces that correspond to the protein bands only; blotting onto polybrene‐pretreated glass‐fiber filter discs (12 mm diameter) placed in an electrophoretic concentrator. A high yield over all steps from gel application through electrophoresis, blotting, gas‐phase sequencer degradation, and phenylthiohydantoin analysis is obtained with several different types of polypeptide (combined average yield over all steps 20%, spread 10–50%). Background is low and samples can be stored under vacuum for long periods after blotting.