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

    The antibody recognizes an epitope containing (serine/threonine) O-Linked N-Acetylglucosamine, which is found on hundreds of nuclear and cytoplasmic proteins, including “FG” nucleoporins of the nuclear pore complex. The sugar is a key part of the epitope. The antibody 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

    Tissue, cells or virus corresponding to O-Linked N-Acetylglucosamine. Specifically, isolated rat liver nuclear envelopes, which contain 8 O-Linked glycoproteins in the nuclear pore complex

  • 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

  • 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 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. 

  • O-GlcNAcylated proteins localize to retinal vascular plexus. A: Eye sections from P10 and 7-month-old Ins2Akita/+ mice. B: P12 and P17 oxygen-induced ischemic retinopathy (OIR) wild-type (WT) mice. O-GlcNAcylated proteins labeled with Cy3 (red, first row), vascular plexus labeled with Cy2 (green, second row) and merge images (third row). Please note the high amount of O-GlcNAcylated protein colocalization with the retinal vascular plexus in 7-month-old Ins2Akita/+ and P17 OIR eyes (arrowheads). These images are representative of images evaluated in eyes from at least six mice (original magnification x200).

    Zafer et al., Molecular vision vol., 19, 1047-59. Fig 4.; Mol Vis. 2013 May 21;19:1047-59. Print 2013.

     
  • 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:
  • Liu B  et al. The lineage stability and suppressive program of regulatory T cells require protein O-GlcNAcylation. Nat Commun 10:354 (2019). Read more (PubMed: 30664665) »
  • Sharif S  et al. Study of cross talk between phosphatases and OGA on a ZO-3-derived peptide. Amino Acids 51:739-743 (2019). Read more (PubMed: 30725225) »
See all 83 Publications for this product

Customer reviews and Q&As

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1-10 of 10 Abreviews

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (U2OS cells)
Permeabilization
Yes - NP40
Specification
U2OS cells
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 21°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Sep 07 2018

Application
Western blot
Sample
Human Cell lysate - whole cell (Vascular smooth muscle cells)
Gel Running Conditions
Reduced Denaturing
Loading amount
25 µg
Treatment
100 uM H2O2 for 2 hours
Specification
Vascular smooth muscle cells
Blocking step
Milk as blocking agent for 3 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

Abcam user community

Verified customer

Submitted Feb 12 2018

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

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

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

Abcam user community

Verified customer

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

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

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

Abcam user community

Verified customer

Submitted Oct 11 2013

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

Mr. Pijus Barman

Verified customer

Submitted Jun 10 2011

Application
Western blot
Sample
Human Cell lysate - whole cell (HepG2 human hepatoma cells)
Loading amount
30 µg
Specification
HepG2 human hepatoma cells
Treatment
10 uM PUGNAC for 24 hours
Gel Running Conditions
Reduced Denaturing (4-20 % gradient)
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Apr 08 2011

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