MitoSciences (MS1081)

GSK3 beta Total + pSer9 Human In-Cell ELISA Kit (Colorimetric) (ab129731)

Overview

  • Product nameGSK3 beta Total + pSer9 Human In-Cell ELISA Kit (Colorimetric)
  • Detection methodColorimetric
  • Tests
    1 x 96 well plate
  • Sample type
    Adherent cells, Suspension cells
  • Assay typeCell-based (quantitative)
  • Assay durationMultiple steps standard assay
  • Species reactivity
    Reacts with: Human
  • Product overview

    ab129731 is an In-Cell ELISA single plex assay panel that uses quantitative immunocytochemistry to measure GSK3 beta protein levels or post-translational modification (phospho S9) in cultured cells.

     

    Cells are fixed in a microplate and targets of interest are detected with highly specific, well-characterized monoclonal antibodies. HRP labeled Secondary antibodies are employed for quantitation and development of the peroxidase can be observed as end-point at 450nm after the addition of stop solution.

     

    Glycogen synthase kinase 3 is a proline directed serine, threonine kinase originally identified due to its ability to phosphorylate and inactivate glycogen synthase and later found to be of key importance in signaling pathways, cell fate determination, energy metabolism, transcription regulation, neuronal development and body pattern formation. There are two isoforms of GSK-3 (alpha and beta) which show a high degree of homology within their catalytic domains.

     

    Activity of GSK3 beta is regulated by phosphorylation of serine 9 (inactivating), phosphorylation of tyrosine 216 (activating) and by protein complex formation which can activate (Axin, APC, B-catenin) or inactivate (Frat 1 and 2) the enzyme. GSK3 beta regulates by phosphorylation the activity of numerous metabolic and signaling proteins such as glycogen synthase, ATP citrate lyase, cyclic-AMP-dependent protein kinase, acetyl CoA carboxylase, cyclin D1, insulin receptor substrate-1 and pyruvate dehydrogenase amongst others. Furthermore, it phosphorylates structural cytoskeletal proteins (MAPs and tau), making it a key component of neuronal structure and plasticity. GSK3 beta is also important for the regulation of transcription factors that modulate cell survival such as activator protein-1, cyclic AMP response element binding protein (CREB), Myc and NFkB.

     

    Plates are available in our ICE (In-Cell ELISA) Support Pack (ab111542) which can be bought seperately.

  • Tested applicationsIn-Cell ELISA more details
  • PlatformMicroplate

Properties

  • RelevanceConstitutively active protein kinase that acts as a negative regulator in the hormonal control of glucose homeostasis, Wnt signaling and regulation of transcription factors and microtubules, by phosphorylating and inactivating glycogen synthase (GYS1 or GYS2), EIF2B, CTNNB1/beta-catenin, APC, AXIN1, DPYSL2/CRMP2, JUN, NFATC1/NFATC, MAPT/TAU and MACF1. Requires primed phosphorylation of the majority of its substrates. In skeletal muscle, contributes to insulin regulation of glycogen synthesis by phosphorylating and inhibiting GYS1 activity and hence glycogen synthesis. May also mediate the development of insulin resistance by regulating activation of transcription factors. Regulates protein synthesis by controlling the activity of initiation factor 2B (EIF2BE/EIF2B5) in the same manner as glycogen synthase. In Wnt signaling, GSK3B forms a multimeric complex with APC, AXIN1 and CTNNB1/beta-catenin and phosphorylates the N-terminus of CTNNB1 leading to its degradation mediated by ubiquitin/proteasomes. Phosphorylates JUN at sites proximal to its DNA-binding domain, thereby reducing its affinity for DNA. Phosphorylates NFATC1/NFATC on conserved serine residues promoting NFATC1/NFATC nuclear export, shutting off NFATC1/NFATC gene regulation, and thereby opposing the action of calcineurin. Phosphorylates MAPT/TAU on 'Thr-548', decreasing significantly MAPT/TAU ability to bind and stabilize microtubules. MAPT/TAU is the principal component of neurofibrillary tangles in Alzheimer disease. Plays an important role in ERBB2-dependent stabilization of microtubules at the cell cortex. Phosphorylates MACF1, inhibiting its binding to microtubules which is critical for its role in bulge stem cell migration and skin wound repair. Probably regulates NF-kappa-B (NFKB1) at the transcriptional level and is required for the NF-kappa-B-mediated anti-apoptotic response to TNF-alpha (TNF/TNFA). Negatively regulates replication in pancreatic beta-cells, resulting in apoptosis, loss of beta-cells and diabetes. Phosphorylates MUC1 in breast cancer cells, decreasing the interaction of MUC1 with CTNNB1/beta-catenin. Is necessary for the establishment of neuronal polarity and axon outgrowth. Phosphorylates MARK2, leading to inhibit its activity. Phosphorylates SIK1 at 'Thr-182', leading to sustain its activity.
  • Cellular localizationCell Membrane, Cytoplasmic and Nuclear. Note: 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.
  • Alternative names
    • C86142
    • Glycogen synthase kinase 3 beta
    • GSK-3
    • GSK-3beta
    • GSK3
    • Serine/threonine-protein kinase GSK3B
    see all
  • Database links

Applications

Our Abpromise guarantee covers the use of ab129731 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Notes
In-Cell ELISA Use at an assay dependent concentration.

GSK3 beta Total + pSer9 Human In-Cell ELISA Kit (Colorimetric) images

  • Validation of antibodies by WB. Western blot was run on a 4-20% gradient acrylamide gel. Membrane blocking and incubation of primary and secondary antibodies was carried out with 1X blocking buffer (ab126587) included in this kit as 10X for anti-GSK3B and anti-GSK3B pSer9. Actin was targeted with ab8224 following recommendations on product sheet.

  • Specificity - Assay specificity was demonstrated by using HeLa cells grown in DMEM media supplemented with 10% FCS and exposed to 100nM Calyculin in 0F for 3 hours and compared to DMSO treated cells for the same period of time. Phosphorylation was with 100nM of Calyculin for 3 hours in 0F media. The plate was fixed following the steps specified in this protocol.

  • Antibody specificity demonstrated by immunocytochemistry ICC was carried out on HeLa cells treated with DMSO for 3 hours in 0F media with anti-GSK3B phospho S9 and anti-GSK3B and all buffers and reagents as supplied in this kit. Labeling was carried out with Goat anti-Mouse IgG H&L Dylight® 488 (ab96871), Goat anti-Rabbit IgG H&L Dylight® 594 (ab96885) and DAPI. In this image, the vehicle control shows the absence of GSK3B phospho S9 induction.
  • Antibody specificity demonstrated by immunocytochemistry ICC was carried out on HeLa cells treated with 100nM Calyculin for 3 hours in 0F media with anti-GSK3B phospho S9 and anti-GSK3B and all buffers and reagents as supplied in this kit. Labeling was carried out with Goat anti-Mouse IgG H&L Dylight® 488 (ab96871), Goat anti-Rabbit IgG H&L Dylight® 594 (ab96885) and DAPI. This image shows induction of GSK3B phospho S9 which is absent from the vehicle control.

Protocols

References for GSK3 beta Total + pSer9 Human In-Cell ELISA Kit (Colorimetric) (ab129731)

ab129731 has not yet been referenced specifically in any publications.

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