GSK3 beta Total + pSer9 Human In-Cell ELISA Kit (IR) (ab129730)
- Product nameGSK3 beta Total + pSer9 Human In-Cell ELISA Kit (IR)
- Detection methodIR
- Tests1 x 96 well plate
- Sample typeAdherent cells, Suspension cells
- Assay typeCell-based (quantitative)
- Assay durationMultiple steps standard assay
- Species reactivityReacts with: Human
- Product overview
ab129730 is an In-Cell ELISA duplex assay panel that uses quantitative immunocytochemistry to measure GSK3B 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, and levels are quantified with IRDye®-labeled Secondary Antibodies. IR imaging and quantitation is performed using a LI-COR® Odyssey® or Aerius® system.
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.
Plates are available in our ICE (In-Cell ELISA) Support Pack (ab111542) which can be bought seperately.
- Tested applicationsIn-Cell ELISA more details
- Storage instructionsStore at +4°C. Please refer to protocols.
Components 1 x 96 tests 1000X IRDye® Labeled Secondary Antibody Cocktail 1 x 24µl 100X Mouse Anti-GSK3 beta Total Primary Antibody 1 x 120µl 100X Rabbit Anti-GSK3 beta pSer9 Primary Antibody 1 x 120µl 10X Blocking Buffer 1 x 10ml 10X Phosphate Buffered Saline 1 x 100ml 1X Janus Green Stain 1 x 11ml 30X Triton X-100 (10% solution) 1 x 1.5ml 400X Tween-20 1 x 2ml
- 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.
- Glycogen synthase kinase 3 beta
- Serine/threonine-protein kinase GSK3B
- SwissProt: P49841 Human
Our Abpromise guarantee covers the use of ab129730 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|In-Cell ELISA||In-Cell ELISA|
GSK3 beta Total + pSer9 Human In-Cell ELISA Kit (IR) images
Normalized IR signal in Calyculin treated HeLa cells. HeLa cells were seeded at 25,000 and 50,000 cells per well and allowed to adhere overnight. Cells were then pre –treated with either DMSO or 100µM Ly294002 (ab120243) for 60 minutes prior to induction of phosphorylation with 100nM of Calyculin for 3 hours in 0F media. The plate was fixed following the steps specified in this protocol. Data was normalized to seeding using Janus green staining. There was a significant reduction in the specific phosphorylation levels at residue serine 9 due to LY294002 pretreatment (p = 0.0026). The variation between the two seeding densities was found at 7% for anti-GSK3B and 8% for anti-GSK3B phospho S9.
Reproducibility of signal. Reproducibility was evaluated by seeding HeLa cells at 30,000 cells per well followed by a titration of DMSO or Calyculin. Average coefficient of variation was 4% for for Anti-GSK3B and 8.8% for Anti-GSK3B phospho S9 at data points significantly greater than background. Z factors were calculated at 0.77 and 0.74 respectively.
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.
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.
All lanes : GSK3 beta Total + pSer9 Human In-Cell ELISA Kit (IR) (ab129730)
Lane 1 : GSK3B protein (ab63193) at 0.05 µg
Lane 2 : Jurkat cells treated with DMSO at 40 µg
Lane 3 : Jurkat cells treated with 100 nM Calyculin at 40 µg
Lane 4 : HeLa cells treated with DMSO at 40 µg
Lane 5 : HeLa cells treated with 100 nM Calyculin at 40 µg
References for GSK3 beta Total + pSer9 Human In-Cell ELISA Kit (IR) (ab129730)
ab129730 has not yet been referenced specifically in any publications.