Recombinant Human Active GSK-3 beta Protein, CF

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Product Details

Summary
Reactivity HuSpecies Glossary
Applications Bioactivity
Format
Carrier-Free

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Recombinant Human Active GSK-3 beta Protein, CF Summary

Details of Functionality
The activity of GSK-3 beta  is typically 148-201 nmol/min/mg using a synthetic peptide substrate (YRRAAVPPSPSLSRHSSPHQ(pS)EDEEE) (see Activity Assay Protocol).
Source
Spodoptera frugiperda, Sf 9 (baculovirus)-derived human GSK-3 beta protein
Accession # NM_002093
Accession #
N-terminal Sequence
Using an N-terminal GST tag
Protein/Peptide Type
Recombinant Proteins
Gene
GSK3B
Purity
>70%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane

Applications/Dilutions

SDS-PAGE
73 kDa
Publications
Read Publication using
2506-KS in the following applications:

Packaging, Storage & Formulations

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.
Buffer
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.
Purity
>70%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
Assay Procedure
  • 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.
  1. Thaw the [33P]-ATP Assay Cocktail in a shielded container in a designated radioactive work area.
  2. Thaw the Active GSK-3 beta, Kinase Assay Buffer I, Substrate, and Kinase Dilution Buffer on ice.
  3. 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
  4. 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.
  5. 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.
  6. 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.
  7. 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.
  8. Count the radioactivity on the P81 paper in the presence of scintillation fluid in a scintillation counter.
  9. 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)]

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Active GSK-3 beta Protein, CF

  • EC 2.7.11
  • EC 2.7.11.26
  • glycogen synthase kinase 3 beta
  • glycogen synthase kinase-3 beta
  • GSK3 beta
  • GSK-3 beta
  • GSK3B
  • GSK3beta isoform

Background

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).

  1. Sutherland, C. et al. (1993) Biochem. J. 296:15.
  2. Sperber, B.R. et al. (1995) Neurosci. Lett. 197:149.

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Publications for GSK-3 beta (2506-KS)(1)

We have publications tested in 1 confirmed species: Human.

We have publications tested in 1 application: Enzyme Assay.


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Bioinformatics

Gene Symbol GSK3B
Uniprot