Gsk 3β

Manufactured by New England Biolabs

Sourced in Germany

GSK-3β is a protein kinase enzyme that plays a role in the regulation of various cellular processes. It is involved in the phosphorylation of a wide range of substrates, including transcription factors, structural proteins, and signaling molecules. The core function of GSK-3β is to modulate the activity and localization of its target proteins, thereby influencing important cellular pathways.

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Lab products found in correlation

Camkii, by New England Biolabs (1 mentions) C8 sep pak spe columns, by Waters Corporation (1 mentions) Halolink resin, by Promega (1 mentions) Igepal ca 630, by Merck Group (1 mentions) Halotag protein purification system, by Promega (1 mentions)

Close spelling variants of this product

Recombinant GSK-3β Anti-GSK-3β

6 protocols using gsk 3β

IKKγ Kinase Activity Assay

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Recombinant human IKKγ (Abnova, Heidelberg, Germany) or purified full-length GST-IKKγ-fusion protein (wild-type and mutants) were incubated with GSK-3β (New England BioLabs) or IKKβ at 30 °C in a total volume of 30 μl of GSK-3β kinase assay buffer containing 10 μCi of [γ-32P]ATP (5000 Ci/mmol). Phosphoprotein products were detected by PAGE (10% gel), Coomassie Blue staining, and autoradiography.

Medunjanin S., Schleithoff L., Fiegehenn C., Weinert S., Zuschratter W, & Braun-Dullaeus R.C. (2016). GSK-3β controls NF-kappaB activity via IKKγ/NEMO. Scientific Reports, 6, 38553.

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In Vitro PKM2 Phosphorylation Assay

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For in vitro kinase assays, WT-PKM2 or T328A-PKM2 recombinant proteins were incubated with GSK-3β (New England BioLabs) at 30 °C in a total volume of 30 μl of GSK-3β kinase assay buffer. Aliquots of reaction mixtures were analyzed by Western blots using anti- Ser/Thr phosphorylation antibody.

Xu Q., Tu J., Dou C., Zhang J., Yang L., Liu X., Lei K., Liu Z., Wang Y., Li L., Bao H., Wang J, & Tu K. (2017). HSP90 promotes cell glycolysis, proliferation and inhibits apoptosis by regulating PKM2 abundance via Thr-328 phosphorylation in hepatocellular carcinoma. Molecular Cancer, 16, 178.

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In vitro kinase activity assay

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In vitro kinase reactions were performed by mixing 1 μg of the recombinant active GSK3β, PKA, and Ca²⁺/calmodulin-dependent protein kinase II (CaMK II) (New England Biolabs, BioLabs, Ipswich, MA, USA), all of which were incubated at room temperature for 30 min under a reaction condition that entailed 20 μL of the kinase activity buffer (50 mM Tris HCl, 10 mM MgCl₂, 1 mM dithiothreitol, 10 μM ATP, pH 7.5); the reaction was subsequently stopped by adding 10 μL of SDS-PAGE loading buffer. After denaturation at 95 °C for 1 min, protein phosphorylation was analyzed by immunoblotting with the indicated antibodies.

Ko H.J., Chiou S.J., Wong Y.H., Wang Y.H., Lai Y.L., Chou C.H., Wang C., Loh J.K., Lieu A.S., Cheng J.T., Lin Y.T., Lu P.J., Fann M.J., Huang C.Y, & Hong Y.R. (2019). GSKIP-Mediated Anchoring Increases Phosphorylation of Tau by PKA but Not by GSK3beta via cAMP/PKA/GSKIP/GSK3/Tau Axis Signaling in Cerebrospinal Fluid and iPS Cells in Alzheimer Disease. Journal of Clinical Medicine, 8(10), 1751.

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FGF14 Phosphorylation by GSK-3β

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In vitro phosphorylation of FGF14 was performed using recombinant GSK–3β expressed from a rabbit skeletal muscle cDNA library (Cat # P6040S) with methods from New England Biolabs. Briefly, 98 nmoles (25 µL of a 7.5 mg/mL stock solution) of FGF14 peptide [KPGVTPSKSTSASAIMNGGK-NH₂] were incubated with 1× reaction buffer and 10,000 units (20 µL) of GSK–3 enzyme, and incubated for 2 h at 37 °C. Control studies were performed under identical conditions, except that experiments lacked the addition of the kinase to the reaction solution. Subsequent reduction of the peptide was performed using 10 mM DTT for 30 min at RT. The peptide was alkylated using 5 mM IAA (30 min at RT) and digested with sequencing grade Glu-C 1:00 (w/w) overnight at 37 °C. The digested samples were desalted using C₈ Sep-Pak SPE columns (Waters, Milford, MA) and the eluent was lyophilized in vacuo.

Hsu W.C., Wildburger N.C., Haidacher S.J., Nenov M.N., Folorunso O., Singh A.K., Chesson B.C., Franklin W.F., Cortez I., Sadygov R.G., Dineley K.T., Rudra J.S., Taglialatela G., Lichti C.F., Denner L, & Laezza F. (2017). PPARgamma agonists rescue increased phosphorylation of FGF14 at S226 in the Tg2576 mouse model of Alzheimer's disease. Experimental neurology, 295, 1-17.

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Tau Protein Phosphorylation Assay

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Tau proteins (0.2 mg/ml) were phosphorylated with either glycogen synthase kinase 3β (GSK3β; 2.5 U/ul; New England Biolabs, Ipswich, MA) or p42 mitogen-activated protein kinase (MAPK; 0.5 U/ul; New England Biolabs, Ipswich, MA) in 50 mM Tris, pH 7.5, 10 mM MgCl₂, 0.1 mM EDTA, 2 mM DTT, 0.1% Brij 35, 200 uM ATP for 2 h at 30 °C followed by heat inactivation at 95 °C for 5 min. Samples were diluted in SDS-sample buffer and 100 ng of each tau protein was loaded on a separate lane on SDS-polyacrylamide gels for immunoblot analysis.

Strang K.H., Goodwin M.S., Riffe C., Moore B.D., Chakrabarty P., Levites Y., Golde T.E, & Giasson B.I. (2017). Generation and characterization of new monoclonal antibodies targeting the PHF1 and AT8 epitopes on human tau. Acta Neuropathologica Communications, 5, 58.

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ETS1 Phosphorylation by GSK3β In Vitro

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Halo-tag ETS1 was purified with Halo Tag Protein purification system (Promega). In briefly, Halo-ETS1 transfected 293 cell pellets were lysed in Halo Tag purification buffer (1x PBS, 1mM DTT, 0.005% IGEPAL-CA630 (Sigma) and protease inhibitor). After freeze-thawing three times, the supernatants were added into HaloLink resin (Promega) according manufacturer instructions. Halo-ETS1 was released from the resin by using HaloTEV enzyme to cleavage TEV cutting site between Halo tag and ETS1 protein. The purified ETS1 protein was incubated with GSK3β (New England BioLabs) in GSK3 reaction buffer (20mM Tris-HCl, 10mM MgCl₂, 2mM DTT and 200μM ATP) at 3° C for 30 mins. Finally, The phosphorylation levels of purified ETS1 were performed by western blot with anti-phosphoserine/threonine antibody (Cell signaling). The reagents and antibodies used for in vitro kinase assay are shown in Supplementary Table 2.

Tsai C.L., Jung S.M., Chi L.M., Tsai C.N., Lin C.Y., Chao A, & Lee Y.S. (2021). Glycogen synthase kinase-3 beta (GSK3β)-mediated phosphorylation of ETS1 promotes progression of ovarian carcinoma. Aging (Albany NY), 13(10), 13739-13763.

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