The Thermo Scientific Pierce O-GlcNAc Western Blot Detection Kit contains a specific antibody for the O-GlcNAc post-translational modification (PTM), as well as detection reagents needed to perform an complete detection experiment.
The Pierce O-GlcNAc Western Blot Detection Kit contains a monoclonal antibody which has specific affinity for the beta-O-linked serine or threonine GlcNAc modification. There is no cross-reactivity with the alpha-O-GlcNAc linkage, the α/β-O-GalNAc modification or other N-linked oligosaccharides. The kit also includes cell lysis reagent, secondary antibody and chemiluminescent substrate needed to perform a complete detection experiment.
- Highly specific monoclonal antibody against O-GlcNAc modification
- Speed and sensitivity of chemiluminescent detection
- Complete kit contains cell lysis and detection reagents
Glycosylation and phosphorylation are two of the most extensively studied post-translational modifications because of their important role in the signaling pathways that regulate cellular response. The O-glycosidically linked modifications of serine or threonine residues are found on proteins from essentially all eukaryotic cells. Proteins in both the cytoplasm and nucleus can be found with this specific posttranslational modification. Although not precisely known, the function of the β-O-linked N-acetylglucosamine (O-GlcNAc) modification may serve as an alternative to phosphorylation, as a modulator of protein interactions in multimeric complexes or as a fortification against proteolytic attack.
The O-GlcNAc modification is seemingly related to phosphorylation in many respects. Notably, the modification is controlled similarly to that of phosphorylation. All O-GlcNAc-modified proteins currently known are also phosphoproteins. In addition, there is an apparent reciprocal relationship between these two important posttranslational modifications within a single protein. Therefore, there is considerable interest in the O-GlcNAc modification.
|Thermo Scientific Pierce O-GlcNAc Western Detection Kit analysis of O-GlcNAc-modified proteins after SDS-PAGE. Jurkat cells were grown in suspension in RPMI 1640 medium supplemented with 10% fetal bovine serum. Cells were treated for 3 hours with PUGNAc [O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino N-phenylcarbamate] and glucosamine at final concen-trations of 50µM and 4mM, respectively. Extracts were prepared using M-PER Mammalian Protein Extraction Reagent (Part No. 78501) to which protease inhibitors (Part No. 78410) were added. Protein extract (25µg) was separated on a 10% SDS-PAGE gel. The proteins were visualized with GelCode Blue Stain Reagent (Part No. 24590) (right panel). The proteins were electroblotted to nitrocellulose membrane, probed with the CTD 110.6 (an O-β-GlcNAc-specific monoclonal antibody) and detected by chemiluminescence with SuperSignal West Dura Substrate after incubation with the appropriate secondary antibody:HRP conjugate (left panel). Lanes 1-4 are proteins from the Jurkat cell extract. Lanes 5, 6 and 7 are the negative controls: ovalbumin (5µg), fetuin (5µg) and O-β-GalNAc-modified BSA (10ng). Lane 8 is O-β-GlcNAc-modified BSA (5ng, positive control). The (+) and (-) refer to plus and minus treatment with PUGNAc and glucosamine, and M = Molecular weight marker.
An overview of glycosylation and methods to analyze glycoproteins and glycopeptides
- Rex-Mathes, M., et al. (2002). Immunological detection of O-GlcNAc. In Posttranslational Modification of Proteins. Tools for Functional Proteomics, Kannicht, C., Ed.; Humana Press: Totowa, N.J., pp.73-87.
- Comer, F.I. and Hart, G.W. (2000). O-Glycosylation of nuclear and cytosolic proteins. Dynamic interplay between O-GlcNAc and O-phosphate. J. Biol. Chem. 275(38): 29179-29182.
- Comer, F.I., et al. (2001). Characterization of a mouse monoclonal antibody specific for O-linked N-Acetylglucosamine. Anal. Biochem. 293: 169-177.