Elimination of non-specific binding in western blots from non-reducing gels.
The reaction of some antibodies with Western blots of protein shows strong non-specific binding especially at a region that corresponds to about 70-90 kDa. This binding is independent of protein concentration. Further analysis indicated that the factor responsible for the non-specific binding is 2-mercaptoethanol in the gel sample buffer. Gel electrophoresis of total tissue homogenates in the absence of this reducing agent resulted in dramatic elimination of the non-specific background binding without affecting the mobility of the two proteins we studied.
Hanukoglu
I
I
Department of Hormone Research, Weizmann Institute of Science, Rehovot, Israel.
D000302:Adrenal Cortex / Q000201:enzymology; D000325:Adrenodoxin / Q000032:analysis*; D000818:Animals; D000918:Antibody Specificity / Q000187:drug effects*; D015153:Blotting, Western; D002417:Cattle; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis*; D008623:Mercaptoethanol / Q000494:pharmacology*
D000325:Adrenodoxin; D008623:Mercaptoethanol; D003577:Cytochrome P-450 Enzyme System