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Recombinant Human Active GSK-3 beta Protein, CF

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The approximate molecular weight is 73 kDa and the average purity is 90%.
The approximate molecular weight is 73 kDa and the average purity is 90%.

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 specific activity of GSK-3 beta was determined to be 224 nmol/min/mg using a synthetic peptide substrate (YRRAAVPPSPSLSRHSSPHQ(pS)EDEEE).
Source
Spodoptera frugiperda, Sf 9 (baculovirus)-derived human GSK-3 beta protein
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

Dilutions
  • Bioactivity
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 GSK3 beta (0.1 μg/μL) diluted with Kinase Dilution Buffer 1X and assayed as outlined in sample activity plot. Note: These are suggested working dilutions. Optimal dilutions should be determined by each laboratory for each application.
  • Kinase Assay Buffer III - 200 mM Tris-HCl, pH 7.4, 100 mM MgCl2, and 5 mg/mL BSA. Add fresh DTT prior to use to a final concentration of 250 μM.
  • Kinase Dilution Buffer IX (1x)  - Kinase Assay Buffer III diluted at a 1:4 ratio (5-fold dilution) with cold water. Add fresh DTT to the aliquot prior to use to a final concentration of 50 μM.
  • 10 mM ATP Stock Solution - Prepare the ATP Stock Solution by dissolving 55 mg of ATP in 10 mL of Kinase Assay Buffer I.
  • ADP-GloTM Kinase Assay Kit - ATP Solution, 10 mM, ADP Solution, 10 mM, ADP-GloTM Reagent, Kinase Detection Reagent.
  • Substrate - GSK3 synthetic peptide substrate (YRRAAVPPSPSLSRHSSPHQ(pS)EDEEE) diluted in distilled or deionized water to a final concentration of 1.0 mg/mL.
  1. Thaw the Active GSK3 beta , Kinase Assay Buffer III (5x), and Substrate on ice. Prepare a 15 μL enzyme dilution at the desired concentration, with Kinase Dilution Buffer IX (1x), in a pre-chilled 96-well plate.
  2. Prepare a substrate/ATP mixture as follows (25 μM example):
    a. 10 μM ATP Solution: 1.0 μL
    b. Kinase Assay Buffer III (5x): 79 μL
    c. Substrate at 1 mg/mL: 80 μL
  3. Transfer the following reaction components prepared in step 2 to a 384-well opaque plate bringing the reaction volume up to 5.0 μL:
    a. 3.0 μL of diluted Active GSK3 beta
    b. 2.0 μL of Substrate/ATP mix as prepared in Step 2. This initiates the reaction.
  4. Set up the blank control as outlined in step 2, excluding the addition of the kinase. Replace the kinase with an equal volume of Kinase Dilution Buffer IX (1x).
  5. Incubate at ambient temperature for 40 minutes.
  6. After the 40 minute incubation period, terminate the reaction and deplete the remaining ATP by adding 5.0 μL of ADP-GloTM Reagent. Spin down and shake the 384-well plate and incubate the reaction mixture for another 30 minutes at ambient temperature.
  7. Read the 384-well reaction plate using the Luminescence Module Protocol on a GloMax®-Multi Microplate Multimode Reader.
  8. Determine the corrected activity (RLU) by removing the blank control value (see step 4) for each sample and calculate the kinase specific activity as outlined below.


    Calculation of ADP Specific Activity (SA) (RLU/pmol)
    From ADP standard curve, determine RLU/pmol of ADP

    Calculation of Kinase Specific Activity (SA) (pmol/minutes/μg or nmol/minutes/mg)
    Corrected RLU from reaction / [(SA of ADP in RLU/pmol) x (Reaction time in minutes) x (Enzyme amount in μg or mg)]

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