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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.
|Components||1 x 96 tests|
|100X (Goat anti-mouse) HRP labeled Secondary Antibody||1 x 125µl|
|100X (Goat anti-rabbit) HRP labeled Secondary Antibody||1 x 125µl|
|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 HRP Development Solution||1 x 24ml|
|30X Triton X-100 (10% solution)||1 x 1.5ml|
|400X Tween-20||1 x 2ml|
|Stop solution||1 x 24ml|
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.
|In-Cell ELISA||Use at an assay dependent concentration.|
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.
ab129731 has not yet been referenced specifically in any publications.
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"