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

Abreviews (8)Q&A (10)Specific References (31)
Customer reviews and FAQs

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

  • 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.

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

  • Immunocytochemistry/ Immunofluorescence - Anti-O-Linked N-Acetylglucosamine antibody [RL2] (ab2739)
    Immunocytochemistry/ Immunofluorescence - Anti-O-Linked N-Acetylglucosamine antibody [RL2] (ab2739)

    ab2739 stained in MCF7cells. 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.

  • Western blot - Anti-O-Linked N-Acetylglucosamine antibody [RL2] (ab2739)
    Western blot - Anti-O-Linked N-Acetylglucosamine antibody [RL2] (ab2739)
    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. 

  • Western blot - Anti-O-Linked N-Acetylglucosamine antibody [RL2] (ab2739)
    Western blot - Anti-O-Linked N-Acetylglucosamine antibody [RL2] (ab2739)This image is courtesy of an anonymous Abreview
    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

  • Western blot - O-Linked N-Acetylglucosamine antibody [RL2] (ab2739)
    Western blot - O-Linked N-Acetylglucosamine antibody [RL2] (ab2739)
    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 minuteThe antibody was tested against the immunogen (isolated rat liver nuclear envelopes, which contain 8 O-linked glycoproteins in the nuclear pore complex).

Protocols

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

This product has been referenced in:
  • Zhang X  et al. The essential role of YAP O-GlcNAcylation in high-glucose-stimulated liver tumorigenesis. Nat Commun 8:15280 (2017). WB, IP, IF, IHC-P ; Human . Read more (PubMed: 28474680) »
  • Magescas J  et al. Spindle pole cohesion requires glycosylation-mediated localization of NuMA. Sci Rep 7:1474 (2017). IHC-P ; Mouse . Read more (PubMed: 28469279) »

See all 31 Publications for this product

Publishing research using ab2739? Please let us know so that we can cite the reference in this datasheet.

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18 Abreviews or Q&A

10
Votes: Highest First

Western blot abreview for Anti-O-Linked N-Acetylglucosamine antibody [RL2]

 Excellent
Abreviews
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
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Abcam user community

Verified customer

Submitted Jun 01 2016

Western blot abreview for Anti-O-Linked N-Acetylglucosamine antibody [RL2]

 Excellent
Abreviews
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
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Dr. guoqing zhu

Verified customer

Submitted Jan 20 2016

Western blot abreview for Anti-O-Linked N-Acetylglucosamine antibody [RL2]

 Excellent
Abreviews
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
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Abcam user community

Verified customer

Submitted Feb 12 2015

Western blot abreview for Anti-O-Linked N-Acetylglucosamine antibody [RL2] - ChIP Grade

 Excellent
Abreviews
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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Abcam user community

Verified customer

Submitted May 20 2014

Western blot abreview for Anti-O-Linked N-Acetylglucosamine antibody [RL2] - ChIP Grade

 Excellent
Abreviews
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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Abcam user community

Verified customer

Submitted Apr 15 2014

Western blot abreview for Anti-O-Linked N-Acetylglucosamine antibody [RL2] - ChIP Grade

 Excellent
Abreviews
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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Abcam user community

Verified customer

Submitted Oct 11 2013

Thank you for the reply and the recommendations. I have one question though. We are not looking for an antibody to detect the enzyme B3GNT6. We are looking to detect core 3 0-glycan(this glycosylation is done by B3GNT6). If I am understanding your emai...

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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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We are trying to get an antibody against core 3 structure of o- glycans. I checked abcam site and found a few antibodies against the enzyme B3GNT6 which synthesizes the core 3 structure. Could you tell me if abcam carries any antibody against the glyco...

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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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RL2 antibody I am trying to perform immunofluorescence assays using the conditions specified on the antibody webpage (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 / ...

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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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Here is the reply from customer .   1. Do you boil your samples? If so, how long and at which temperature? Yes. 5 minutes at 98-100 degree centigrade.   2. Are you checking the transfer with Ponceau? Yes. We also use prestained markers which ...

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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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