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

Overview

  • Product nameAnti-O-Linked N-Acetylglucosamine antibody [RL2]
    See all O-Linked N-Acetylglucosamine primary antibodies
  • Description
    Mouse monoclonal [RL2] to O-Linked N-Acetylglucosamine
  • 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, IPmore 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 Abreviews 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] 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.
  • All lanes : Anti-O-Linked N-Acetylglucosamine antibody [RL2] (ab2739) at 1/3000 dilution

    Lane 1 : Human neuroblastoma (SH-SY5Y) whole cell lysate - treated with 50µM z-Pugnac for 24 hours
    Lane 2 : Human neuroblastoma (SH-SY5Y) whole cell lysate - untreated

    Lysates/proteins at 20 µg per lane.

    Secondary
    HRP-conjugated horse anti-mouse IgG polyclonal
    developed using the ECL technique

    Performed under reducing conditions.

    Exposure time : 30 seconds

    This image is courtesy of an anonymous Abreview

    See Abreview

  • Anti-O-Linked N-Acetylglucosamine antibody [RL2] (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
    The antibody was tested against the immunogen (isolated rat liver nuclear envelopes, which contain 8 O-linked glycoproteins in the nuclear pore complex).
  • All lanes : Anti-O-Linked N-Acetylglucosamine antibody [RL2] (ab2739) at 1 µg/ml

    Lane 1 : Jurkat cells treated with 0 uM PugNAc
    Lane 2 : Jurkat cells treated with 50 uM PugNAc (3 hours)
    Lane 3 : Jurkat cells treated with 0 uM PugNAc
    Lane 4 : Jurkat cells treated with 4 mM glucosamine and 50 uM PugNAc (3 hours)

    Lysates/proteins at 20 µg per lane.

    Secondary
    Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/50000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

    Exposure time : 12 minutes

    Jurkat cells were treated with either 50 uM PugNAc (ab144670) or 4 mM glucosamine + 50 uM PugNAc (ab144670) for three hours prior to harvest to stimulate O-linked glycosylation. The expected increase in glycosylation is observed in the treated lanes 2 & 4. 

References for Anti-O-Linked N-Acetylglucosamine antibody [RL2] (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) »
  • Zhang Q  et al. Differential regulation of the ten-eleven translocation (TET) family of dioxygenases by O-linked ß-N-acetylglucosamine transferase (OGT). J Biol Chem 289:5986-96 (2014). Read more (PubMed: 24394411) »

See all 24 Publications for this product

Product Wall

Application Western blot
Loading amount 30 µg
Gel Running Conditions Non-reduced Denaturing (8%)
Sample Human Cell lysate - whole cell (T lymphocyte)
Specification T lymphocyte
Treatment pathogen
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
Username

Abcam user community

Verified customer

Submitted May 20 2014

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-...

Read More

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...

Read More

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...

Read More

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...

Read More

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...

Read More

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...

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
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

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"