The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/500 - 1/2000. Detects a band of approximately 47-52 kDa (predicted molecular weight: 51 , 47 kDa).
1/100 - 1/250. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
1/250 - 1/500.
追加情報Is unsuitable for Flow Cyt.
関連性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. GSK3s 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, alpha and beta, and they show a high degree of amino acid homology. The two isoforms of GSK3 are strictly regulated via phosphorylation. Phosphorylation of GSK3 beta on Ser9 (Ser21 in GSK3 alpha) by protein kinase B (PKB) causes its inactivation is the primary mechanism responsible for growth factor inhibition of this kinase. 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. GSK3 beta also has been shown to phosphorylate aberrant sites on the microtubule associated protein tau, which is critical for the progression of Alzheimer's disease. GSK3B is involved in energy metabolism, neuronal cell development, and body pattern formation.
Dot Blot analysis of Lane 1: GSK3 (alpha + beta) (pY216 + pY279) phospho peptide and Lane 2: GSK3 (alpha + beta) non-phospho peptide labeling GSK3 (alpha + beta) (phospho Y216 + Y279) with ab68476 at 1/1000 dilution. 5% NFDM/TBST was used as the diluting and blocking buffer. ab97051 Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated was used as the secondary antibody at 1/100000 dilution. Exposure time: 3 minutes.
Western blot - GSK3 (alpha + beta) (phospho Y279 + Y216) antibody [EPR933Y] (ab68476)
All lanes : Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y] (ab68476) at 1/2000 dilution
Lane 1 : 293 cell lysate, cells untreated. Lane 2 : 293 cell lysate, cells treated with AP.
Lysates/proteins at 10 µg per lane.
Secondary HRP conjugated Goat anti-rabbit at 1/2000 dilution