Characterization of a mouse monoclonal antibody specific for O-linked N-acetylglucosamine

Anal Biochem. 2001 Jun 15;293(2):169-77. doi: 10.1006/abio.2001.5132.


beta-O-linked N-acetylglucosamine (O-GlcNAc) is an abundant posttranslational modification of resident nuclear and cytoplasmic proteins in eukaryotes. Increasing evidence suggests that O-GlcNAc plays a regulatory role in numerous cellular processes. Here we report on the production and characterization of a highly specific mouse monoclonal antibody, MAb CTD110.6, that specifically reacts with O-GlcNAc. The antibody recognizes O-GlcNAc in beta-O-glycosidic linkage to both serine and threonine. We could detect no cross-reactivity with alpha-linked Ser/Thr-O-GlcNAc, alpha-linked Ser-O-linked N-acetylgalactosamine (O-GalNAc), or N-linked oligosaccharides on ovalbumin and immunoglobulin G. The monosaccharide GlcNAc, but not GalNAc, abolishes immunoreactivity, further demonstrating specificity toward O-GlcNAc. Furthermore, galactose capping of O-GlcNAc sites also inhibits CTD110.6 immunoreactivity. Enrichment of GlcNAc-containing glycoproteins using the lectin wheat germ agglutinin dramatically enriches for CTD110.6-reactive proteins. The antibody reacts with a large number of proteins from cytoplasmic and nuclear extracts and readily detects in vivo changes in O-GlcNAc modification. These studies demonstrate that CTD110.6 is highly specific toward O-GlcNAc, with no cross-reactivity toward similar carbohydrate antigens or toward peptide determinants.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acetylglucosamine / analogs & derivatives*
  • Acetylglucosamine / immunology*
  • Animals
  • Antibodies, Monoclonal / immunology*
  • Antibody Specificity / immunology*
  • Cell Extracts
  • Chromatography, Affinity
  • Electrophoresis, Polyacrylamide Gel
  • HeLa Cells
  • Humans
  • Immunoblotting
  • Immunoglobulin M / immunology
  • Jurkat Cells
  • Mice
  • Mice, Inbred BALB C
  • Precipitin Tests
  • Serine / metabolism
  • Threonine / metabolism


  • Antibodies, Monoclonal
  • Cell Extracts
  • Immunoglobulin M
  • Threonine
  • Serine
  • Acetylglucosamine