Electroblotting of individual polypeptides from SDS/polyacrylamide gels for direct sequence analysis.
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.
Bergman
T
T
Department of Chemistry I, Karolinska Institutet, Stockholm, Sweden.
Jörnvall
H
H
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D010455:Peptides; D011189:Potassium Chloride
D010455:Peptides; D011189:Potassium Chloride