Validated using a knockout cell line
Recombinant
RabMAb

Anti-GSK3 beta antibody [Y174] (ab32391)

Overview

  • Product name
    Anti-GSK3 beta antibody [Y174]
    See all GSK3 beta primary antibodies
  • Description
    Rabbit monoclonal [Y174] to GSK3 beta
  • Specificity
    This antibody is specific for human GSK3 beta. It may also detect the splice isoform 2 based on sequence homology.
  • Tested applications
    Suitable for: ICC/IF, WB, IHC-P, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Human
  • Immunogen

    Synthetic peptide (Human) corresponding to the C terminal peptide

  • Positive control
    • A431 cell lysate. This antibody gave a positive result when used in the following formaldehyde fixed cell lines: DU145. IHC-P: Human breast adenocarcinoma FFPE tissue sections. ICC/IF: HeLa cell lysate
  • General notes

    Produced using Abcam's RabMAb® technology. RabMAb® technology is covered by the following U.S. Patents, No. 5, 675, 063 and/or 7, 429, 487.

    This product is a recombinant rabbit monoclonal antibody.

Applications

Our Abpromise guarantee covers the use of ab32391 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 1/100 - 1/500.

 

WB 1/5000 - 1/10000. Predicted molecular weight: 46 kDa.
IHC-P Use at an assay dependent concentration.
Flow Cyt 1/100.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

Target

  • Function
    Participates in the Wnt signaling pathway. Implicated in the hormonal control of several regulatory proteins including glycogen synthase, MYB and the transcription factor JUN. Phosphorylates JUN at sites proximal to its DNA-binding domain, thereby reducing its affinity for DNA. Phosphorylates MUC1 in breast cancer cells, and decreases the interaction of MUC1 with CTNNB1/beta-catenin. Phosphorylates CTNNB1/beta-catenin. Phosphorylates SNAI1. Plays an important role in ERBB2-dependent stabilization of microtubules at the cell cortex. Prevents the phosphorylation of APC and CLASP2, allowing its association with the cell membrane. In turn, membrane-bound APC allows the localization of MACF1 to the cell membrane, which is required for microtubule capture and stabilization. Phosphorylates MACF1 and this phosphorylation inhibits the binding of MACF1 to microtubules which is critical for its role in bulge stem cell migration and skin wound repair.
  • Tissue specificity
    Expressed in testis, thymus, prostate and ovary and weakly expressed in lung, brain and kidney.
  • Sequence similarities
    Belongs to the protein kinase superfamily. CMGC Ser/Thr protein kinase family. GSK-3 subfamily.
    Contains 1 protein kinase domain.
  • Post-translational
    modifications
    Phosphorylated by AKT1 and ILK1. Activated by phosphorylation at Tyr-216.
  • Cellular localization
    Cytoplasm. Nucleus. Cell membrane. The phosphorylated form shows localization to cytoplasm and cell membrane. The MEMO1-RHOA-DIAPH1 signaling pathway controls localization of the phosophorylated form to the cell membrane.
  • Information by UniProt
  • Database links
  • Alternative names
    • Glycogen Synthase Kinase 3 Beta antibody
    • Glycogen synthase kinase-3 beta antibody
    • GSK 3 beta antibody
    • GSK-3 beta antibody
    • GSK3B antibody
    • GSK3B_HUMAN antibody
    • GSK3beta isoform antibody
    • Serine/threonine-protein kinase GSK3B antibody
    see all

Anti-GSK3 beta antibody [Y174] images



  • Predicted band size : 46 kDa

    Lane 1: Wild type HAP1 whole cell lysate (20 µg)
    Lane 2: GSK3B knockout  HAP1 whole cell lysate (20 µg)
    Lanes 1 - 2: Merged signal (red and green). Green - ab32391 observed at 46 kDa. Red - loading control, ab8245, observed at 37 kDa.

    ab32391 was shown to recognize GSK3B when GSK3B knockout samples were used, along with additional cross-reactive bands. Wild-type and GSK3B knockout samples were subjected to SDS-PAGE. Ab32391 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 2000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

  • Immunohistochemical analysis of paraffin-embedded human breast tissue labelled with untreated ab75814 (phospho) (top-left) at a dilution of 1/1000, alkaline phosphatase treated ab75814 (phospho) (top-right) at a dilution of 1/1000, untreated ab32391 (bottom-left) at a dilution of 1/1000 and alkaline phophatase treated ab32391 (bottom-right) at a dilution of 1/1000. Ab97051 was used as secondary antibody at a dilution of 1/500 and counterstained with hematoxylin.

  • Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling GSK3 beta with purified ab32391 at 1/500. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% tritonX-100. An Alexa Fluor® 488-conjugated goat anti-rabbit IgG (ab150077) at 1/1000 dilution was used as the secondary antibody. Cells were co-stained with ab7291, a mouse anti-tubulin antibody (1/1000) using ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000) as the secondary antibody. Nuclei were counterstained with DAPI (blue).

    For negative control 1, rabbit primary antibody was used followed by anti-mouse secondary antibody (ab150120).
    For negative control 2, mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab150077) were used.

  • Overlay histogram showing HeLa cells stained with ab32391 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32391, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

  • Anti-GSK3 beta antibody [Y174] (ab32391) at 1/10000 dilution + A431 cell lysate.

    Predicted band size : 46 kDa
    Observed band size : 46 kDa
  • Immunohistochemical analysis of paraffin-embedded human breast cancer tissue labelled with untreated ab75814 (phospho) (top-left) at a dilution of 1/1000, alkaline phosphatase treated ab75814 (phospho) (top-right) at a dilution of 1/1000, untreated ab32391 (bottom-left) at a dilution of 1/1000 and alkaline phophatase treated ab32391 (bottom-right) at a dilution of 1/1000. Ab97051 was used as secondary antibody at a dilution of 1/500 and counterstained with hematoxylin.

  • IHC image of GSK3 staining in human breast adenocarcinoma formalin fixed paraffin embedded tissue section, performed on a Leica Bond system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6, epitope retrieval solution 1) for 20 minutes. The section was then incubated with ab32391, 1/200 diution, for 15 minutes at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

  • ICC/IF image of ab32391 stained DU145 cells. The cells were 4% formaldehyde fixed (10 minutes) and then incubated in 1 %BSA / 10 % normal goat serum / 0.3 M glycine in 0.1 % PBS-Tween for 1 hour to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab32391 at 1/200 dilution overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/250 dilution for 1 hour. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1 hour. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43 µM.

  • All lanes : Anti-GSK3 beta antibody [Y174] (ab32391) at 1/2500 dilution

    Lane 1 : MCF7 (human breast adenocarcinoma cell line) cell lysate
    Lane 2 : SW480 (human colorectal adenocarcinoma cell line) cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    Donkey polyclonal IRDye 800CW at 1/15000 dilution

    Predicted band size : 46 kDa
    Observed band size : 46 kDa
    Additional bands at : 37 kDa. We are unsure as to the identity of these extra bands.

    Image courtesy of an anonymous Abreview.

    See Abreview

References for Anti-GSK3 beta antibody [Y174] (ab32391)

This product has been referenced in:
  • Yu N  et al. Anti-Diabetic Effects of Jiang Tang Xiao Ke Granule via PI3K/Akt Signalling Pathway in Type 2 Diabetes KKAy Mice. PLoS One 12:e0168980 (2017). Mouse . Read more (PubMed: 28045971) »
  • Kong LY  et al. In vivo and in vitro effects of microRNA-27a on proliferation, migration and invasion of breast cancer cells through targeting of SFRP1 gene via Wnt/ß-catenin signaling pathway. Oncotarget 8:15507-15519 (2017). WB ; Human . Read more (PubMed: 28099945) »

See all 17 Publications for this product

Product Wall

Thank you very much for contacting us with your enquiry.

At this time we are not sure what the 37 kDa band represents. In the Western blot testing done in the lab, there was only a single band above 37 kDa using A431 lysate. The image showing...

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - other (breast cancer cells (MCF7) and colon cancer cells)
Loading amount
20 µg
Specification
breast cancer cells (MCF7) and colon cancer cells
Gel Running Conditions
Non-reduced Denaturing
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Aug 03 2011

Thank you for your enquiry. This antibody reacts with both the phosphorylated and non-phosphorylated forms of GSK3 beta. This antibody in fact recognizes the N-terminal region as opposed to the C-terminal region as previously detailed. I have updat...

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