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

Overview

  • Product name
    Anti-O-Linked N-Acetylglucosamine antibody [RL2]
    See all O-Linked N-Acetylglucosamine primary antibodies
  • Description
    Mouse monoclonal [RL2] to O-Linked N-Acetylglucosamine
  • Host species
    Mouse
  • Specificity
    Detects nuclear pore complex (NPC), cytoplasmic and intranuclear O-linked glycoproteins from human, mouse, and rat tissues. In Western blot many bands are expected as the O-Linked N-Acetylglucosamine modification can occur on proteins of different sizes.
  • Tested applications
    Suitable for: ICC/IF, IHC-Fr, ChIP/Chip, Dot blot, WB, IPmore details
  • Immunogen

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

  • Positive control
    • ICC-IF: MCF7 cells. WB: Jurkat cells treated with 50 uM PugNAc; SH-SY5Y) whole cell lysate - treated with 50µM z-Pugnac; Rat Liver Nuclear Envelope lysate.
  • General notes

    This antibody clone [RL2] is manufactured by Abcam.

    If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.

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

  • Relevance
    Many 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.

Images

  • All lanes : Anti-O-Linked N-Acetylglucosamine antibody [RL2] (ab2739) at 1 µg/ml

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

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : 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. 

  • ab2739 stained in MCF7 cells. Cells were fixed with 4% paraformaldehyde (10min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab2739 at 5µg/ml and ab6046 (Rabbit polyclonal to beta Tubulin - Loading Control) at 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150080 (pseudo-colored red) and ab150117 (colored green) used at 1 ug/ml for 1hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43µM for 1hour at room temperature.

  • 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
    All lanes : HRP-conjugated horse anti-mouse IgG polyclonal

    Developed using the ECL technique.

    Performed under reducing conditions.

    Exposure time: 30 seconds

    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).

Protocols

References

This product has been referenced in:
  • Brown HM  et al. Periconception onset diabetes is associated with embryopathy and fetal growth retardation, reproductive tract hyperglycosylation and impaired immune adaptation to pregnancy. Sci Rep 8:2114 (2018). IHC ; Mouse . Read more (PubMed: 29391475) »
  • Shi H  et al. Skeletal muscle O-GlcNAc transferase is important for muscle energy homeostasis and whole-body insulin sensitivity. Mol Metab N/A:N/A (2018). Read more (PubMed: 29525407) »

See all 60 Publications for this product

Customer reviews and Q&As

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, https://www.abcam.com/b3gnt6-antibody-ab103239.html& https://www.abcam.com/b3gnt6-antibody-ab168600.html, will recognize the transferase that synthesizes the core 3 structure of the O-glycan. We also curr...

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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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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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Thank you for contacting us. The following reference from 1987 describes the production and characterization of the RL2 clone, among others: Snow CM et al. Monoclonal antibodies identify a group of nuclear pore complex glycoproteins. J Cell Bio...

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Thank you for your enquiry. The concentration of this lot is 2.0 mg/ml. Please contact us again if you have any additional questions.

The concentration is 2.0 mg/ml.

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

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