Recombinant Human Active GSK-3 beta Protein, CF Summary
Accession # NM_002093
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
|Formulation||Supplied in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.25 mM DTT, 10 mM glutathione, 0.1 mM EDTA, 0.1 mM PMSF, 25% glycerol.
|Shipping||The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||This product is stable at ≤ ‑70° C for up to 1 year from the date of receipt. For optimal storage, aliquot into smaller quantities after centrifugation and store at recommended temperature. Avoid repeated freeze-thaw cycles.|
- Active Kinase - Active GSK-3 beta (0.1 μg/μL) diluted with Kinase Dilution Buffer. Note: These are suggested working dilutions. Optimal dilutions should be determined by each laboratory for each application.
- Kinase Assay Buffer I, pH 7.2 - 25 mM MOPS, 12.5 mM beta -glycerolphosphate, 25 mM MgCl2, 5 mM EGTA, 2 mM EDTA. Add 0.25 mM DTT to the Kinase Assay Buffer prior to use.
- Kinase Dilution Buffer, pH 7.2 - Kinase Assay Buffer I diluted at a 1:4 ratio (5-fold dilution) with 50 ng/μL of BSA solution.
- 10 mM ATP Stock Solution - Prepare the ATP Stock Solution by dissolving 55 mg of ATP in 10 mL of Kinase Assay Buffer I.
- [33P]-ATP Assay Cocktail - Prepare 250 μM [33P]-ATP Assay Cocktail in a designated radioactive work area by combining 150 μL of 10 mM ATP Stock Solution, 100 μL of [33P]-ATP (1 mCi/100 μL), and 5.75 mL of Kinase Assay Buffer I.
- Substrate - GSK synthetic peptide substrate (YRRAAVPPSPSLSRHSSPHQ(pS)EDEEE) diluted in distilled or deionized water to a final concentration of 1 mg/mL.
- Thaw the [33P]-ATP Assay Cocktail in a shielded container in a designated radioactive work area.
- Thaw the Active GSK-3 beta, Kinase Assay Buffer I, Substrate, and Kinase Dilution Buffer on ice.
- In a pre-cooled microfuge tube, add the following reaction components bringing the initial reaction volume up to 20 μL.
a Diluted Active GSK-3 beta : 10 μL
b. Substrate (1 mg/mL Stock Solution): 5 μL
- Set up the blank control as outlined in step 3, excluding the addition of the substrate. Replace the substrate with an equal volume of distilled or deionized water.
- Initiate the reaction by the addition of 5 μL [33P]-ATP Assay Cocktail, bringing the final volume up to 25 μL. Incubate the mixture in a water bath at 30 °C for 15 minutes.
- After the 15 minute incubation period, terminate the reaction by spotting 20 μL of the reaction mixture onto individual pre-cut strips of phosphocellulose P81 paper.
- Air dry the pre-cut P81 strip and sequentially wash in a 1% phosphoric acid solution (dilute 10 mL of phosphoric acid and make a 1 liter solution with distilled or deionized water) with constant gentle stirring. It is recommended that the strips be washed a total of three times for approximately 10 minutes each.
- Count the radioactivity on the P81 paper in the presence of scintillation fluid in a scintillation counter.
- Determine the corrected cpm by removing the blank control value (see step 4) for each sample and calculate the kinase specific activity as outlined below:
Calculation of [33P]-ATP Specific Activity (SA) (cpm/pmol)
Specific Activity (SA) = cpm for 5 μL [33P]-ATP/pmole of ATP (in 5 μL of a 250 μM ATP stock solution; i.e. 1250 pmol)
Calculation of Kinase Specific Activity (SA) (pmol/minutes/μg or nmol/minutes/mg)
Corrected cpm from reaction / [(SA of 33P-ATP in cpm/pmol) x (Reaction time in minutes) x (Enzyme amount in μg or mg)] x [(Reaction volume) / (Spot Volume)]
The approximate molecular weight is 73 kDa and the average purity is 90%.
Background: GSK-3 beta
GSK-3 beta is a Serine/Threonine protein kinase that was originally identified as the kinase that phosphorylates and inhibits glycogen synthase (1). GSK-3 beta is ubiquitously present in human tissues and implicated in the regulation of several physiological processes, including the control of glycogen and protein synthesis by insulin and modulation of the transcription factors AP-1 and CREB. Transient transfection of human GSK-3 beta into Chinese hamster ovary cells stably transfected with individual human tau isoforms leads to hyperphosphorylation of tau at all the sites investigated with phosphorylation-dependent anti-tau antibodies (2).
- Sutherland, C. et al. (1993) Biochem. J. 296:15.
- Sperber, B.R. et al. (1995) Neurosci. Lett. 197:149.
Citation for Recombinant Human Active GSK-3 beta Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
1 Citation: Showing 1 - 1
A SNP in steroid receptor coactivator-1 disrupts a GSK3beta phosphorylation site and is associated with altered tamoxifen response in bone.
Authors: Hartmaier RJ, Richter AS, Gillihan RM, Sallit JZ, McGuire SE, Wang J, Lee AV, Osborne CK, O'Malley BW, Brown PH, Xu J, Skaar TC, Philips S, Rae JM, Azzouz F, Li L, Hayden J, Henry NL, Nguyen AT, Stearns V, Hayes DF, Flockhart DA, Oesterreich S
Mol. Endocrinol., 2012;26(2):220-7.
Sample Types: Recombinant Protein
Applications: Enzyme Assay
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