Analysis Note

ControlJurkat cell lysate, or serum starve 3T3-L1 cells for 3 hours, then stimulate with 100 nM insulin or 100 ng/mL IGF 1 for 5 min.
Included Positive Antigen Control: Catalog # 12-303, Jurkat lysate. Add 2.5 µL of 2-mercaptoethanol/100 µL of lysate and boil for 5 minutes to reduce the preparation. Load 20 µg of reduced lysate per lane for minigels.

Application

Research Sub CategoryPI3K, Akt, & mTOR Signaling

Research CategorySignaling

This Anti-phospho-GSK3 (Tyr279/Tyr216) Antibody, clone 5G-2F is validated for use in WB for the detection of phospho-GSK3 (Tyr279/Tyr216).

Disclaimer

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

General description

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.
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. 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

Immunogen

Synthetic peptide (C-KQLLHGEPNVS[pY]ICSRY) corresponding to amino acids 203-219 derived from a region of the catalytic domain (between subdomain VII and VIII) of the Drosophila GSK3/shaggy enzyme coupled to ovalbumin.

Epitope: Surrounding pTyr214 (Drosophila GSK-3), pTyr279 (human GSK3α), pTyr216 (human GSK3β)

Legal Information

UPSTATE is a registered trademark of Merck KGaA, Darmstadt, Germany

Other Notes

Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.

Physical form

Purified mouse monoclonal IgG1κ in buffer containing 0.1 M Tris-glycine, pH 7.4, 0.15 M NaCl, 0.05% sodium azide. Frozen at -20°C.

Protein G Chromatography

Format: Purified

Quality

Routinely evaluated by immunoblot on human Jurkat cells, mouse 3T3/A31, human A431, or rat L6 cells.

Western Blot Analysis:
0.5-2 µg/mL of this lot detected phospho-GSK3 in RIPA lysates from human Jurkat cells. Previous lots detected phospho-GSK3 in mouse 3T3/A31, human A431 lysates and rat L6 cells

Specificity

Predicted to react with GSK-3 orthologs from drosophila melanogaster and many other species based on sequence homology surrounding the target phosphotyrosine residue.

Recognizes GSK-3 phosphorylated at the target tyrosine residue (human GSK-3α pY279 and GSK-3β pY216). The 17-amino acid immunogen peptide sequence differs from the corresponding human, mouse, and rat sequence by only two amino acid residues.

Storage and Stability

Stable for 1 year at -20°C from date of receipt.

Handling Recommendations:
Upon receipt, and prior to removing the cap, centrifuge the vial and gently mix the solution. Aliquot into microcentrifuge tubes and store at -20°C. Avoid repeated freeze/thaw cycles, which may damage IgG and affect product performance.

Target description

51 kDa (alpha) and 46 kDa (beta)