Avidin- or streptavidin-biotin as a highly sensitive method to stain total protein on membranes

A sensitive method for staining proteins after transfer from polyacrylamide gels to nitrocellulose paper is described. Transferred proteins are first derivatized by reaction of the nitrocellulose replica with sulfosuccinimidobiotin and are then reacted sequentially with streptavidin, rabbit anti-streptavidin, and horseradish peroxidase-conjugated goat anti-rabbit IgG antibody. Incubation with the enzyme substrate alpha-chloronaphthol, produces dark protein bands against a white background. The binding of streptavidin to the proteins is dependent on biotin derivatization as demonstrated by competition with biotinylated bovine serum albumin or 10 nM biotin. The procedure detects less than 5 ng of transferred protein in a single band and is thus 5-10 times more sensitive than horseradish peroxidase-conjugated avidin alone. For bovine serum albumin, the method is comparable in sensitivity to silver staining of protein in polyacrylamide gels.