¹²⁵I-wheat germ agglutinin blotting: Increased sensitivity with polyvinylpyrrolidone quenching and periodate oxidation/reductive phenylamination

Two substantial improvements in sensitivity in the identification of ¹²⁵I-wheat germ agglutininbinding glycoproteins on nitrocellulose blots of sodium dodecyl sulfate-polyacrylamide gels are reported. The major improvement in sensitivity (about 30-fold) derives from the use of 2% ( w v) polyvinylpyrrolidone (average M_r 40,000) instead of bovine serum albumin or denatured hemoglobin as the quenching agent (or carrier) during incubation with ¹²⁵I-wheat germ agglutinin in detergent-free, phosphate-buffered saline. Under these conditions, specific labeling with ¹²⁵I-wheat germ agglutinin is observed for orosomucoid derivatives that display N-acetylglucosamine or sialic acid residues at the nonreducing termini of their oligosaccharides, as well as for a number of glycoprotein components of a rat hepatocyte plasma membrane fraction. An additional improvement in sensitivity (up to 10-fold) results from an increase in the binding of ¹²⁵I-wheat germ agglutinin to sialic acid-containing glycoproteins after treatment of the blots with 5 mm sodium metaperiodate followed by 5 mm aniline in the presence of 30 mm sodium cyanoborohydride. This treatment appears to cause the sequential oxidation and reductive phenylamination of the side chain of glycoprotein sialic acid residues.