Identification and characterization of protein glycosylation using specific endo- and exoglycosidases

Methods Mol Biol. 2012;801:189-211. doi: 10.1007/978-1-61779-352-3_13.

Abstract

Enzymatic deglycosylation followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples. Specific glycosidases were used to remove sugars from glycoproteins in a controlled fashion leaving the protein core intact; the resulting change in molecular weight could be detected as shifts in gel mobility. Alternatively, glycan-sensitive reagents were used to visualize the intensity of glycoprotein bands before and after enzyme treatment. The ease of use of these techniques, which require only basic laboratory instrumentation and reagents, makes them the methodology of choice for initial glycobiology studies. These protocols are also well suited to screen for optimal expression conditions, since multiple glycoprotein samples can be processed at once.

MeSH terms

  • Acetylglucosamine / metabolism
  • Acetylglucosaminidase / metabolism
  • Animals
  • Antibodies, Monoclonal / immunology
  • Electrophoresis, Polyacrylamide Gel
  • Glycoprotein Hormones, alpha Subunit / chemistry
  • Glycoprotein Hormones, alpha Subunit / genetics
  • Glycoprotein Hormones, alpha Subunit / isolation & purification
  • Glycoprotein Hormones, alpha Subunit / metabolism
  • Glycoproteins / metabolism*
  • Glycoside Hydrolases / metabolism*
  • Glycosylation
  • HeLa Cells
  • Humans
  • Membranes, Artificial
  • Mice
  • Polysaccharides / metabolism
  • Polyvinyls / chemistry
  • Protein Denaturation
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Substrate Specificity
  • alpha-Crystallins / chemistry
  • alpha-Crystallins / genetics
  • alpha-Crystallins / isolation & purification
  • alpha-Crystallins / metabolism

Substances

  • Antibodies, Monoclonal
  • Glycoprotein Hormones, alpha Subunit
  • Glycoproteins
  • Membranes, Artificial
  • Polysaccharides
  • Polyvinyls
  • Recombinant Proteins
  • alpha-Crystallins
  • polyvinylidene fluoride
  • Glycoside Hydrolases
  • Acetylglucosaminidase
  • Acetylglucosamine