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

Overview

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.

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

  • 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. 

  • 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).

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

This product has been referenced in:
  • de Queiroz RM  et al. Changes in O-Linked N-Acetylglucosamine (O-GlcNAc) Homeostasis Activate the p53 Pathway in Ovarian Cancer Cells. J Biol Chem 291:18897-914 (2016). Read more (PubMed: 27402830) »
  • Rønningen T  et al. Pre-patterning of differentiation-driven nuclear lamin A/C-associated chromatin domains by GlcNAcylated histone H2B. Genome Res N/A:N/A (2015). ChIP ; Human . Read more (PubMed: 26359231) »

See all 27 Publications for this product

Product Wall

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Human Cell lysate - whole cell (mast cell type)
Gel Running Conditions Non-reduced Non-Denaturing (Native)
Loading amount 10 µg
Specification mast cell type
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Jun 01 2016

Application Western blot
Sample Human Cell lysate - whole cell (Bel-7402 cells)
Gel Running Conditions Non-reduced Denaturing (gel 10%)
Loading amount 60 µg
Specification Bel-7402 cells
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Dr. guoqing zhu

Verified customer

Submitted Jan 20 2016

Application Western blot
Loading amount 30 µg
Gel Running Conditions Reduced Denaturing (8%)
Sample Human Cell lysate - whole cell (T lymphocyte (human))
Specification T lymphocyte (human)
Treatment 10,20,50 mM D-Galactopyranosyl-b-D-thiogalactopyranoside for 1hr
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 24°C
Username

Abcam user community

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Submitted Feb 12 2015

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
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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
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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
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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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