Recombinant
RabMAb

Recombinant Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y] (ab68476)

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Overview

  • Product name

    Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y]
    See all GSK3 (alpha + beta) primary antibodies

  • Description

    Rabbit monoclonal [EPR933Y] to GSK3 (alpha + beta) (phospho Y216 + Y279)

  • Host species

    Rabbit

  • Specificity

    This antibody detects both GSK alpha phosphorylated on tyrosine 279 and GSK3 beta phosphorylyated on tyrosine 216.

  • Tested applications

    Suitable for: WB, IP, IHC-P, Dot blotmore details
    Unsuitable for: Flow Cyt or ICC

  • Species reactivity

    Reacts with: Mouse, Rat, Human, Zebrafish

  • Immunogen

    Synthetic peptide within Human GSK3 (alpha + beta). The exact sequence is proprietary.
    (Peptide available as ab186139)

  • Positive control

    • WB: HEK-293 cell lysates, cell lysate from untreated, nerve growth factor-beta treated PC12 cells, Zebrafish lysates and mouse brain lysates. IHC: human brain tissue, human glioma.

  • 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.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab68476 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 1/500 - 1/2000. Detects a band of approximately 47-52 kDa (predicted molecular weight: 47-52 kDa).
IP 1/40.
IHC-P 1/50. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

This antibody may not be suitable for IHC with mouse, rat or zebrafish samples.

See IHC antigen retrieval protocols.

For unpurified use at 1/100 - 1/250.
Dot blot Use at an assay dependent concentration.
  • Application notes
    Is unsuitable for Flow Cyt or ICC.

    • Target

    • Relevance

      Glycogen synthase kinase 3 (GSK3) is a proline directed serine threonine kinase that was initially identified as a phosphorylating and inactivating glycogen synthase, a key enzyme in glycogen metabolism. Since then, it has been shown to be involved in the regulation of a diverse array of cellular functions, including protein synthesis, cell proliferation, cell differentiation, microtubule assembly/disassembly, and apoptosis. GSK3s substrate specificity is unique in that phosphorylation of substrate only occurs if a phosphoserine or phosphotyrosine is present four residues C terminal to the site of GSK phosphorylation. There exists two isoforms of GSK3, alpha and beta, and they show a high degree of amino acid homology. The two isoforms of GSK3 are strictly regulated via phosphorylation. Phosphorylation of GSK3 beta on Ser9 (Ser21 in GSK3 alpha) by protein kinase B (PKB) causes its inactivation is the primary mechanism responsible for growth factor inhibition of this kinase. Activation of GSK3 beta is dependent upon the phosphorylation of Tyr216 (Tyr279 in GSK3 alpha). Upon activation, it has been shown to phosphorylate a number of different cellular proteins, including p53, c-Myc, c-Jun, heat shock factor 1 (HSF1), and cyclin D1. GSK3 beta also has been shown to phosphorylate aberrant sites on the microtubule associated protein tau, which is critical for the progression of Alzheimer's disease. GSK3B is involved in energy metabolism, neuronal cell development, and body pattern formation.

    • Cellular localization

      Cytoplasmic and Nuclear

    • Database links

      see all

    • Alternative names

      • Factor A antibody
      • Glycogen synthase kinase 3 alpha antibody
      • Glycogen synthase kinase 3 beta antibody
      • GSK3 alpha antibody
      • GSK3 beta antibody
      • GSK3B antibody
      see all

    Images

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y] (ab68476)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y] (ab68476)

      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human glioma tissue sections labeling GSK3 (alpha + beta) with Purified ab68476 at 1:50 dilution (5.3 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

    • Western blot - Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y] (ab68476)
      Western blot - Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y] (ab68476)
      All lanes : Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y] (ab68476) at 1/1000 dilution (purified)

      Lane 1 : Mouse brain lysates.
      Then the membrane was incubated with phosphatase
      Lane 2 : Untreated Mouse brain lysates

      Lysates/proteins at 15 µg per lane.

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

      Predicted band size: 47-52 kDa



      Blocking and diluting buffer: 5% NFDM/TBST

    • Western blot - Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y] (ab68476)
      Western blot - Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y] (ab68476)
      All lanes : Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y] (ab68476) at 1/500 dilution (unpurified)

      Lane 1 : SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysates
      Lane 2 : SH-SY5Y (Human neuroblastoma epithelial cell) treated with nerve growth factor at 100ng/ml for 5 minutes. Whole cell lysates.
      Lane 3 : SH-SY5Y (Human neuroblastoma epithelial cell) treated with nerve growth factor at 100ng/ml for 5 minutes. Whole cell lysates. Then the membrane was incubated with phosphatase.

      Lysates/proteins at 15 µg per lane.

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

      Predicted band size: 47-52 kDa
      Observed band size: 47-52 kDa


      Exposure time: 10 seconds


      Blocking and diluting buffer and concentration: 5% NFDM/TBST.

    • Western blot - Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y] (ab68476)
      Western blot - Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y] (ab68476)
      All lanes : Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y] (ab68476) at 1/1000 dilution (purified)

      Lane 1 : Zebrafish lysates
      Then the membrane was incubated with phosphatase
      Lane 2 : Untreated Zebrafish lysates

      Lysates/proteins at 15 µg per lane.

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

      Predicted band size: 47-52 kDa
      Observed band size: 47-52 kDa



      Blocking and diluting buffer: 5% NFDM/TBST

       

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y] (ab68476)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y] (ab68476)

      Human brain tissue stained with unpurified ab68476 at 1/100 dilution.

      Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

    • Immunoprecipitation - Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y] (ab68476)
      Immunoprecipitation - Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y] (ab68476)

      GSK3 (alpha + beta) (phospho Y216 + Y279) was immunoprecipitated from 10μg HeLa (human cervix adenocarcinoma) whole cell lysate with ab68476 at 1/50 dilution (2μg in 0.35mg lysates). Western blot was performed from the immunoprecipitate using ab68476 at 1/200 dilution (9 μg/ml). VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

      Lane 1 (Input): HeLa (human cervix adenocarcinoma) treated with 1uM staurosporine for 4h whole cell lysate 10μg
      Lane 2 (+): HeLa (human cervix adenocarcinoma) treated with 1uM staurosporine for 4h whole cell lysate
      Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab68476 in HeLa (human cervix adenocarcinoma) treated with 1uM staurosporine for 4h whole cell lysate

      Exposure Time: 30 seconds
      Blocking and diluting buffer and concentration: 5% NFDM/TBST.

       

    • Western blot - Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y] (ab68476)
      Western blot - Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y] (ab68476)
      All lanes : Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y] (ab68476) (unpurified)

      Lane 1 : PC12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
      Lane 2 : Whole cell lysate from PC12 (Rat adrenal gland pheochromocytoma cell line) cells treated with nerve growth factor-ß at 100ng/ml for 5 minutes.
      Lane 3 : Whole cell lysate from PC12 (Rat adrenal gland pheochromocytoma cell line) cells ) treated with nerve growth factor-ß at 100ng/ml for 5 minutes. Membrane incubated with phosphatase

      Lysates/proteins at 15 µg per lane.

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

      Developed using the ECL technique.

      Predicted band size: 47-52 kDa


      Exposure time: 3 seconds


      Blocking and dilution buffer: 5% NFDM/TBST

    • Dot Blot - Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y] (ab68476)
      Dot Blot - Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y] (ab68476)

      Dot Blot analysis of Lane 1: GSK3 (alpha + beta) (pY216 + pY279) phospho peptide and Lane 2: GSK3 (alpha + beta) non-phospho peptide labeling GSK3 (alpha + beta) (phospho Y216 + Y279) with ab68476 at 1/1000 dilution. 5% NFDM/TBST was used as the diluting and blocking buffer. ab97051 Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated was used as the secondary antibody at 1/100000 dilution. Exposure time: 3 minutes.

    • Western blot - Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y] (ab68476)
      Western blot - Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y] (ab68476)
      All lanes : Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y] (ab68476) at 1/2000 dilution (unpurified)

      Lane 1 : 293 cell lysate, cells untreated.
      Lane 2 : 293 cell lysate, cells treated with AP.

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : HRP conjugated Goat anti-rabbit at 1/2000 dilution

      Predicted band size: 47-52 kDa
      Observed band size: 47-52 kDa

    References

    This product has been referenced in:

    • Gao JX  et al. Overexpression of microRNA-183 promotes apoptosis of substantia nigra neurons via the inhibition of OSMR in a mouse model of Parkinson's disease. Int J Mol Med 43:209-220 (2019). Read more (PubMed: 30431059) »
    • Pang J  et al. Vorapaxar stabilizes permeability of the endothelial barrier under cholesterol stimulation via the AKT/JNK and NF-?B signaling pathways. Mol Med Rep 19:5291-5300 (2019). Read more (PubMed: 31059055) »
    See all 11 Publications for this product

    Publishing research using ab68476? Please let us know so that we can cite the reference in this datasheet.

    Customer reviews and Q&As

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    1-3 of 3 Abreviews or Q&A

    Immunohistochemistry - Free Floating abreview for Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y]

     Excellent
    Abreviews
    abreview image
    Application
    Immunohistochemistry free floating
    Sample
    Mouse Tissue sections (Brain)
    Specification
    Brain
    Read More

    Abcam user community

    Verified customer

    Submitted Jan 14 2016

    Immunohistochemistry - Free Floating abreview for Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y]

     Excellent
    Abreviews
    abreview image
    Application
    Immunohistochemistry free floating
    Sample
    Mouse Tissue sections (Hippocampus CA1 mouse Brain)
    Specification
    Hippocampus CA1 mouse Brain
    Read More

    Abcam user community

    Verified customer

    Submitted Jan 19 2016

    Immunohistochemistry (Frozen sections) abreview for Anti-GSK3 (alpha + beta) (phospho Y279 + Y216) antibody [EPR933Y]

     Poor
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Frozen sections)
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
    Sample
    Zebrafish Tissue sections (retina)
    Specification
    retina
    Permeabilization
    Yes - triton X
    Fixative
    Paraformaldehyde
    Read More

    Dr. Ryan Macdonald

    Verified customer

    Submitted May 22 2014

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