Up-regulation and Antibody-Peptide Competition: Peptide Competition. Extracts of 3T3L1 cells stimulated with 100 nM insulin for 10 minutes were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was blocked with a 5% BSA-TBST buffer for one hour at room temperature and either left untreated (1-4) or treated with lambda phosphatase (5), and then incubated with the GSK-3a [pY279]/beta [pY216] antibody (Product # OPA1-03083) for two hours at room temperature in a 1% BSA-TBST buffer, following its prior incubation with: the phosphopeptide immunogen (1), no peptide (2), the non-phosphopeptide corresponding to the phosphopeptide immunogen (3), or a generic phosphotyrosine-containing peptide (4). After washing, the membrane was incubated with goat F(ab')2 anti-rabbit IgG HRP conjugate (Product # ALI4404), and signals were detected using the Pierce SuperSignal™ method. The data show that only the phosphopeptide corresponding to GSK-3a [pY279]/beta [pY216] blocks the antibody signal, demonstrating the specificity of the antibody. The data also show that phosphatase stripping eliminates the signal, further verifying that the antibody is phospho-specific.
|Tested species reactivity||Human, Mouse|
|Published species reactivity||Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Synthetic phosphopeptide derived from regions of human GSK3alpha/beta that contain tyrosine 279/216.|
|Storage buffer||Dulbecco's PBS, pH 7.3, with 1mg/ml BSA, 50% glycerol|
|Contains||0.05% sodium azide|
|Storage Conditions||-20° C, Avoid Freeze/Thaw Cycles|
|Tested Applications||Dilution *|
|Western Blot (WB)||Assay-Dependent|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
|Western Blot (WB)||See 1 publications below|
OPA1-03083 detects the alpha and beta isoforms of phospho-GSK3 from human and mouse tissues. This antibody is expected to cross-react with rat, frog, and zebra fish GSK-3 due to 100% sequence homology.
OPA1-03083 has been successfully used in Western blot procedures. By Western blot, this antibody detects an ~51 kDa protein and an ~47 kDa protein from stimulated 3T3-L1 cells representing tyrosine 279 phosphorylated GSK3alpha and tyrosine 216 phosphorylated GSK3beta, respectively. Positive control: serum starved mouse 3T3-L1 cells +/- insulin (100 nM, 10 minutes), or background extracts +/- human recombinant GSK-3â.
OPA1-03083 immunizing phosphopeptide was derived from a regions of human GSK3alpha/beta that contain tyrosine 279/216.
Glycogen synthase kinase-3 (GSK3) is a protein kinase that was originally identified as a regulator of 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. GSK3 and quote;s substrate specificity is unique in that phosphorylation of substrate only occurs if a phosphoserine or phosphotyrosine is present four residues C-terminal to the site of GSK phosphorylation. There exists two isoforms of GSK3, GSK3alpha and GSK3beta, and they are strictly regulated via phosphorylation. Phosphorylation of GSK3beta on Ser9 (Ser21 in GSK3alpha) by protein kinase B (PKB) causes its inactivation is the primary mechanism responsible for growth factor inhibition of this kinase. Activation of GSK3beta is dependent upon the phosphorylation of Tyr216 (Tyr279 in GSK3alpha). Upon activation, it has been shown to phosphorylate a number of different cellular proteins, including p53, c-Myc, c-Jun, heat shock factor-1 (HSF-1), and cyclin D1. GSK3beta also has been shown to phosphorylate aberrant sites on the microtubule associated protein tau, which is critical for the progression of Alzheimer and quote;s disease.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
The intersection of genetic and chemical genomic screens identifies GSK-3¿ as a target in human acute myeloid leukemia.
OPA1-03083 was used in western blot to study the potential of GSK3-alpha as a therapeutic target in human acute myeloid leukemia using a combination of genetic and small molecule screens
|Banerji V,Frumm SM,Ross KN,Li LS,Schinzel AC,Hahn CK,Kakoza RM,Chow KT,Ross L,Alexe G,Tolliday N,Inguilizian H,Galinsky I,Stone RM,DeAngelo DJ,Roti G,Aster JC,Hahn WC,Kung AL,Stegmaier K||The Journal of clinical investigation (122:935)||2012|