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 agglutinin-binding 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 Mr 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.
D000586:Amination; D000818:Animals; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000032:analysis*; D007457:Iodine Radioisotopes; D037102:Lectins; D008099:Liver / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis; D010084:Oxidation-Reduction; D010504:Periodic Acid; D011205:Povidone; D051381:Rats; D011975:Receptors, Mitogen / Q000032:analysis*; D014909:Wheat Germ Agglutinins
D006023:Glycoproteins; D007457:Iodine Radioisotopes; D037102:Lectins; D008565:Membrane Proteins; D011975:Receptors, Mitogen; D014909:Wheat Germ Agglutinins; C027503:wheat germ agglutinin receptor; D010504:Periodic Acid; D003101:Collodion; C009288:metaperiodate; D011205:Povidone