Update profile
    Change password
    Sign out

O-GlcNAc Western Blot Detection Kit


Detect of beta-O-linked N-acetylglucosamine proteins.

Pierce O-GlcNAc Western Blot Detection Kit

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.

Highlights:

  • Highly specific monoclonal antibody against O-GlcNAc modification
  • Speed and sensitivity of chemiluminescent detection
  • Complete kit contains cell lysis and detection reagents

Product Details

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.

 Specific Western blot detection of O-GlcNAc proteins with Thermo Scientific Pierce Kit
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.

Related Resources:

Protein glycosylation technical guide

References:

  1. 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.
  2. 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.
  3. Comer, F.I., et al. (2001). Characterization of a mouse monoclonal antibody specific for O-linked N-Acetylglucosamine. Anal. Biochem. 293: 169-177.

Ordering Information
 
Product # Description Pkg. Size Instructions MSDS CofA Price
24565 O-GlcNAc Western Blot Detection Kit
Formulation: Multi-component kit
Sufficient For: 10 mini blots (8 x 10cm each)

Kit Contents:
M-PER Mammalian Protein Extraction Reagent, 25mL
Dilution/Blocking Buffer (10X), 2 x 50mL
PBS Packs (each makes 500mL), 17 packs
Surfact-Amps 20 Detergent Solution, 3 x 10mL
O-GlcNAc Western Blot Positive Control, 100ng
Anti-O-GlcNAc Monoclonal Antibody (MAb CTD 110.6) in ascites, 100µL
Goat anti-Mouse IgM(µ), HRP Conjugate 75µg
SuperSignal West Dura Luminol/Enhancer Solution, 50mL
SuperSignal West Dura Stable Peroxide Solution, 50mL
255mL kit Product Instructions for product #24565 O-GlcNAc Western Blot Detection Kit MSDS for product #24565 O-GlcNAc Western Blot Detection Kit $520.00

Add to cart

Instructions | MSDS | CofA
Product Instructions | MSDS | CofA  

Share This Page
 
 

Follow Us
  Email Sign-up  Email Announcements

 



Antibodies  |   Molecular Biology   |   Cell Biology   |  Thermo Scientific  |   * Trademarks  |   Privacy Statement
© 2014 Thermo Fisher Scientific Inc.   |   3747 N Meridian Rd, Rockford, IL USA 61101   |   1-800-874-3723  or  815-968-0747