Anti-O-Linked N-Acetylglucosamine antibody [RL2] - ChIP Grade (ab2739)

Overview

  • Product nameAnti-O-Linked N-Acetylglucosamine antibody [RL2] - ChIP GradeSee all O-Linked N-Acetylglucosamine primary antibodies ...
  • Description
    Mouse monoclonal [RL2] to O-Linked N-Acetylglucosamine - ChIP Grade
  • SpecificityDetects nuclear pore complex (NPC), cytoplasmic and intranuclear O-linked glycoproteins from human, mouse, and rat tissues.
  • Tested applicationsICC/IF, IHC-Fr, ChIP/Chip, Dot Blot, WB, IP more details
  • Immunogen

    Pore complex-lamina fraction purified from rat liver nuclear envelopes.

  • General notesWe can conjugate this antibody to FITC for you (please see ab150247 for details).

Properties

Applications

Our Abpromise guarantee covers the use of ab2739 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Notes
ICC/IF Use a concentration of 5 - 10 µg/ml.
IHC-Fr Use at an assay dependent concentration. PubMed: 23734074
ChIP/Chip Use at an assay dependent concentration. PubMed: 20368426
Dot Blot 1/800.
WB Use a concentration of 1 µg/ml.
IP Use at an assay dependent concentration.

Target

  • RelevanceMany cellular proteins, including nuclear pore, oncogene, cytoskeletal, heat shock, viral and transcription regulatory proteins contain single O-linked N-acetylglucosamine (O-GlcNAc) residues attached to serine or threonine residues. It has been observed that O-GlcNAc glycosylated proteins tend to be under phosphorylated relative to unglycosylated proteins and that O-GlcNAc bearing proteins tend to be found in multimeric complexes. This has led to the suggestion that O-GlcNAc glycosylation may obscure phosphorylation sites and acts as a signaling mechanism or mediator of signaling.
  • Alternative names
    • O-GlcNAc antibody

Anti-O-Linked N-Acetylglucosamine antibody [RL2] - ChIP Grade images

  • ICC/IF image of ab2739 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal Goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab2739, 10µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 Goat anti-Mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% PFA fixed (10 min) HEK293, HepG2, and MCF-7 cells at 10µg/ml, and in 100% Methanol fixed (5 min) HeLa, Hek293, HepG2, and MCF-7 cells at 5µg/ml.
  • Anti-O-Linked N-Acetylglucosamine antibody [RL2] - ChIP Grade (ab2739) at 1 µg/ml + Rat Liver Nuclear Envelope at 10 µg

    Secondary
    Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

    Exposure time : 1 minute
  • ab2739 staining O-linked N-Acetylglucosamine in HeLa cells treated with tunicamycin from Streptomyces sp (ab120296), by ICC/IF. Decrease in O-linked N-Acetylglucosamine expression correlates with increased concentration of tunicamycin from Streptomyces sp, as described in literature.
    The cells were incubated at 37°C for 24h in media containing different concentrations of ab120296 (tunicamycin from Streptomyces sp) in DMSO, fixed with 100% methanol for 5 minutes at -20°C and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab2739 (10 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-mouse polyclonal antibody (ab96879) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

References for Anti-O-Linked N-Acetylglucosamine antibody [RL2] - ChIP Grade (ab2739)

This product has been referenced in:
  • Chu CS  et al. O-GlcNAcylation regulates EZH2 protein stability and function. Proc Natl Acad Sci U S A 111:1355-60 (2014). Read more (PubMed: 24474760) »
  • Guillaumond F  et al. Strengthened glycolysis under hypoxia supports tumor symbiosis and hexosamine biosynthesis in pancreatic adenocarcinoma. Proc Natl Acad Sci U S A 110:3919-24 (2013). Read more (PubMed: 23407165) »

See all 19 Publications for this product

Product Wall

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing (8%)
Sample Human Cell lysate - whole cell (neuroblastoma)
Specification neuroblastoma
Treatment 50 uM z-Pugnac for 24hrs
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 4% · Temperature: 16°C
Username

Abcam user community

Verified customer

Submitted Apr 15 2014

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing (4-20%)
Sample Human Cell lysate - whole cell (HaCaT)
Specification HaCaT
Treatment untreated or glucose starved
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
Username

Abcam user community

Verified customer

Submitted Oct 11 2013

You are correct that the uniprot link in the previous email (Uniprot: http://www.uniprot.org/uniprot/Q6ZMB0http://www.uniprot.org/uniprot/Q6ZMB0) is for the core 3 synthase, which is B3GNT6. Inferring from the alternative names for this synthase (Beta-...

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I can confirm that the B3GNT6 antibodies, http://www.abcam.com/b3gnt6-antibody-ab103239.html& http://www.abcam.com/b3gnt6-antibody-ab168600.html, will recognize the transferase that synthesizes the core 3 structure of the O-glycan. We also curren...

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Thank you for contacting us about this issue. Have you successfully stained other nuclear proteins using this protocol and these reagents? I think the protocol looks correct, but I am concerned the permeabilization of the nuclei may not be effective. W...

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Thank you for your swift reply. With regard of the new information I would like to suggest the following alterations in order to increase the signal strength: 1. We found that many monoclonal antibodies work better with BSA than milk as a blockin...

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Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results.
The details provided will enable us to investigate this case and will provide us with vital information for monit...

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Thanks for your inquiry. The clone number for ab2739 is RL2. It's highly probable that ab2739 would work in IHC as it has been successfully tested in ChIP,ICC/IF, IP, and WB. If you are interested, you could submit an abreview of ab2739 usin...

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Application Western blot
Sample Human Cell lysate - whole cell (B cell)
Loading amount 20 µg
Specification B cell
Gel Running Conditions Reduced Denaturing (10)
Blocking step Milk as blocking agent for 12 hour(s) and 0 minute(s) · Concentration: 2.5% · Temperature: 4°C
Username

Mr. Pijus Barman

Verified customer

Submitted Jun 10 2011

Thank you for contacting us. You may find the following reference useful: Mapping Sites of O-GlcNAc Modification Using Affinity Tags for Serine and Threonine Post-translational Modifications* Lance Wells et al, Molecular & Cellular Proteomics 1.10 ...

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