Selective labeling of sulfhydryls and disulfides on blot transfers using avidin-biotin technology: studies on purified proteins and erythrocyte membranes.
Various strategies for the use of 3-(N-maleimido-propionyl) biocytin (MPB) as a general label for distinguishing between protein sulfhydryls and disulfides on blot transfers are presented. In the first approach, endogenous SH groups in proteins were labeled directly with MPB. For disulfide staining, endogenous sulfhydryls were blocked with N-ethylmaleimide, disulfides were then reduced with mercaptoethanol, and the newly formed SH groups were labeled with MPB. In this approach, all treatments were performed in vitro, and, between steps, excess reagent was removed by dialysis. The MPB-labeled proteins were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (in the presence of mercaptoethanol), the labeled proteins were transferred to nitrocellulose, and the blotted proteins were detected by avidin-biotin technology. In the second approach, MPB treatment was performed directly on blot transfers. For SH labeling, proteins were subjected to SDS-PAGE in the absence of mercaptoethanol, thus retaining the status of endogenous sulfhydryl and disulfide groups. For S-S labeling, proteins were treated with N-ethylmaleimide in vitro and then subjected to SDS-PAGE in the presence of mercaptoethanol, such that endogenous sulfhydryls were blocked and endogenous disulfides were converted to SH groups. Subsequent treatments and washings were performed on blots. In the third approach, immobilized proteins (i.e., in artificial systems or in natural systems such as membrane preparations or intact cells) were treated essentially as described in the first approach, except that washings were carried out by centrifugation. In vitro treatments were performed before SDS-PAGE (carried out in the presence of mercaptoethanol) and subsequent blot transfer. The relative merits of the three strategies are discussed.
D001360:Avidin; D001710:Biotin; D003545:Cysteine / Q000032:analysis; D004220:Disulfides / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000032:analysis; D006801:Humans; D008239:Lysine / Q000031:analogs & derivatives*; D008301:Maleimides; D008565:Membrane Proteins / Q000032:analysis; D008623:Mercaptoethanol / Q000494:pharmacology; D011506:Proteins / Q000032:analysis*; D013194:Staining and Labeling; D013438:Sulfhydryl Compounds / Q000032:analysis*
D004220:Disulfides; D008301:Maleimides; D008565:Membrane Proteins; D011506:Proteins; D013438:Sulfhydryl Compounds; D001360:Avidin; D008623:Mercaptoethanol; D001710:Biotin; C047272:3-(N-maleimidopropionyl)biocytin; D008239:Lysine; D003545:Cysteine