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

James R. Bartles, Ann L. Hubbard

Research output: Contribution to journalArticle

Abstract

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 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.

Original languageEnglish (US)
Pages (from-to)284-292
Number of pages9
JournalAnalytical biochemistry
Volume140
Issue number1
DOIs
StatePublished - Jul 1984

Keywords

  • blotting
  • carbohydrate structure
  • electrophoresis
  • glycoproteins
  • lectin
  • lectins
  • receptors

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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