Recombinant
RabMAb

Recombinant Anti-O-Linked N-Acetylglucosamine antibody [EPR19847] (ab202665)

Overview

  • Product name

    Anti-O-Linked N-Acetylglucosamine antibody [EPR19847]
    See all O-Linked N-Acetylglucosamine primary antibodies
  • Description

    Rabbit monoclonal [EPR19847] to O-Linked N-Acetylglucosamine
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, Dot blot, IPmore details
  • Species reactivity

    Reacts with: Rat, Human
  • Immunogen

    Synthetic peptide within Human O-Linked N-Acetylglucosamine. The exact sequence is proprietary.

  • Positive control

    • WB: HeLa treated with DMSO (0.4%) as baseline control whole cell lysate; HeLa treated with 200 µM Ac45SGlcNAc for 24 hours whole cell lysate; HeLa treated with 200 µM Thiamet-G for 24 hours whole cell lysate; HEK-293t and HeLa whole cell lysate. Rat brain lysate. PC-12 (rat adrenal gland pheochromocytoma). SH-SY5Y (human neuroblastoma epithelial cell). IP: HeLa whole cell lysate.
  • General notes

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    This product is a recombinant rabbit monoclonal antibody.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
  • Storage buffer

    Preservative: 0.01% Sodium azide
    Constituents: PBS, 40% Glycerol, 0.05% BSA
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EPR19847
  • Isotype

    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab202665 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
WB 1/1000.
Dot blot 1/1000.
IP 1/30.

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 [EPR19847] (ab202665) at 1/1000 dilution

    Lane 1 : Rat brain lysate
    Lane 2 : PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate
    Lane 3 : SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

    Observed band size: 20-100 kDa
    why is the actual band size different from the predicted?



    Blocking buffer and concentration: 5% NFDM/TBST
    Diluting buffer and concentration: 5% NFDM/TBST

    Exposure time:

    Lane 1:8 seconds;
    Lane 2:3 seconds;
    Lane 3:26 seconds.


     

     

  • All lanes : Anti-O-Linked N-Acetylglucosamine antibody [EPR19847] (ab202665) at 1/1000 dilution

    Lane 1 : Untreated HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
    Lane 2 : HeLa (human cervix adenocarcinoma epithelial cell) treated with DMSO (0.4%) as baseline control. whole cell lysate
    Lane 3 : HeLa (human cervix adenocarcinoma epithelial cell) treated with 200 µM Ac45SGlcNAc for 24 hours whole cell lysate
    Lane 4 : HeLa (human cervix adenocarcinoma epithelial cell) treated with 200 µM Thiamet-G for 24 hours whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Observed band size: 50-100 kDa why is the actual band size different from the predicted?


    Exposure time: 3 seconds


    Blocking buffer and concentration 5% NFDM/TBST
    Diluting buffer and concentration 5% NFDM/TBST

    Ac45SGlcNAc, an inhibitor of OGT, decreases O-GlcNAc modification. Thiamet-G, an inhibitor of OGA, increases O-GlcNAc modification.

    These two chemicals were kindly provided by our collaborator Dr. Xing Chen, Peking University.

    The expression pattern is consistent with reference (PMID: 27716624)

  • All lanes : Anti-O-Linked N-Acetylglucosamine antibody [EPR19847] (ab202665) at 1/1000 dilution

    Lane 1 : HEK-293T (human embryonic kidney epithelial cell) whole cell lysate
    Lane 2 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution


    Blocking/Dilution buffer: 5% NFDM/TBST.

    Exposure times: Lane 1: 4 seconds; Lane 2: 15 seconds.

  • Dot Blot - Anti-O-Linked N-Acetylglucosamine antibody [EPR19847] (ab202665 1:1000 dilution).

    Lane 1: O-linked N-acetylglucosamine (O-GlcNAc) peptide 10 ng.

    Lane 2: Non-O-GlcNAc peptide 10 ng.

    Blocking/Dilution buffer: 5% NFDM/TBST.

    Secondary: Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051 1:100,000 dilution).

    Exposure time of 3 minutes.

    The peptides were kindly provided by our collaborator Dr. Xing Chen, Peking University.

  • O-Linked N-Acetylglucosamine was immunoprecipitated from 0.35 mg HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate with ab202665 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab202665 at 1/1,000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.
    Lane 1: HeLa whole cell lysate 10 µg (Input).
    Lane 2: ab202665 IP in HeLa whole cell lysate.
    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab202665 in HeLa whole cell lysate.
    Blocking/Dilution buffer: 5% NFDM/TBST.
    Exposure time: 3 seconds.

References

ab202665 has not yet been referenced specifically in any publications.

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