Semidry electroblotting of peptides and proteins from acid-urea polyacrylamide gels.
A semidry electrophoretic transfer method was developed for efficient electroblotting of proteins separated by acid-urea polyacrylamide gel electrophoresis (AU-PAGE). Model polypeptides ranging from 1.8 to 21.5 kDa were used to test transfer parameters that included time of transfer, power settings, transfer solutions, and membrane type. Optimized conditions were identified which allowed for transfer efficiencies of 70-100% following 5-15 min of applied current. The best transfer solution was 5% acetic acid, the same solvent used for electrophoresis. Therefore, acid-urea gels could be subjected to electrophoretic transfer without a soaking step, thereby reducing loss of band resolution and eliminating leaching of protein from the gel. The method was shown to be applicable to Western blot analysis of rat neutrophil defensins.
Wang
M S
MS
Department of Pathology, College of Medicine, University of California, Irvine, California 92697, USA.
Pang
J S
JS
Selsted
M E
ME
D019342:Acetic Acid; D000818:Animals; D001798:Blood Proteins / Q000032:analysis; D015153:Blotting, Western / Q000379:methods*; D003101:Collodion; D023082:Defensins; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005782:Gels; D008567:Membranes, Artificial; D009504:Neutrophils / Q000737:chemistry; D010455:Peptides / Q000032:analysis*; D011145:Polyvinyls; D011506:Proteins / Q000032:analysis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014508:Urea; D005465:Fluorocarbon Polymers
D019342:Acetic Acid; D001798:Blood Proteins; D003101:Collodion; D023082:Defensins; D005782:Gels; D008567:Membranes, Artificial; D010455:Peptides; D011145:Polyvinyls; D011506:Proteins; D014508:Urea; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers