 | | | | Glycogen Synthase Kinase 3 (GSK-3) | | | |
 |
|
Prices are in US dollars and valid only for US orders.
|
- Protein serine/threonine kinase
- Rabbit skeletal muscle, recombinant (E. coli)
- Supplied with 10X Reaction Buffer
Description:
Glycogen Synthase Kinase 3 (GSK‑3) is a serine/threonine protein kinase and one of several protein kinases, which phosphorylate glycogen synthase. It is also called Factor A (FA) for its ability to activate the MgATP-dependent form of the protein phosphatase PP1 called FC (1-4). Recent studies demonstrate that GSK-3 can autophosphorylate Ser, Thr and Tyr. Ser/Thr phosphorylation causes inactivation, and Tyr phosphorylation results in increased activity (Y216 for GSK-3β). GSK-3 expressed in E. coli or insect cells is extensively phosphorylated on Tyr. Molecules lacking phosphate at this position can autophosphorylate after incubation with Mg2+ and ATP. GSK-3 phosphorylates several exogenous substrates, but not on Tyr residues (5,6).
Recognition Determinants:
The substrate specificity of GSK-3 is unique and substrate dependent. For some substrates, prior phosphorylation of the substrate to form the motif S/TXXXpS/pT is a strict requirement whereas in other substrates, no previous phosphorylation is needed. In either case, many of the GSK-3 sites have Pro residues close to the modified Ser or Thr (5,7).
Source:
Isolated from a strain of E. coli that carries a clone expressing GSK-3β derived from a rabbit skeletal muscle cDNA library (kindly provided by Dr. P. J. Roach) (5).
Reagents Supplied:
GSK-3 Reaction Buffer (10X)
Enzyme Properties
Protein Kinase Substrate Recognition
Heat Inactivation:
No
Molecular Weight:
Theoretical: 47 kDa
Specific Activity:
5,000,000 units/mg
Reaction & Storage Conditions
Reaction Conditions:
1X GSK-3 Reaction Buffer
Supplemented with 200 μM ATP
Incubate at
30°C.
1X GSK-3 Reaction Buffer:
20 mM Tris-HCl
10 mM MgCl2
5 mM DTT
pH 7.5 @ 25°C
Unit Definition:
One unit is defined as the amount of GSK-3 required to catalyze the transfer of 1 pmol of phosphate to CREB Phosphopeptide, KRREILSRRPpSYR (400 µM, NEB #P6041), in 1 minute at 30°C in a total reaction volume of 25 µl.
Concentration:
500,000 units/ml
Storage Conditions:
30 mM Tris-HCl
50 mM NaCl
5 mM DTT
1 mM EDTA
50% Glycerol
0.03% Brij 35
pH 7.5 @ 25°C
Storage Temperature:
-20°C
Notes
General notes:- If the source of protein to be phosphorylated is a crude extract of cells or tissue, it is very important to include the appropriate protease and protein phosphatase inhibitors in the lysis buffer and to use shorter incubation time for phosphorylation.
Usage notes:- Optimal incubation times and enzyme concentrations must be determined empirically for each particular substrate.
- If possible, the ATP concentration should be at or near saturation (5 -10-fold over Km). Apparent Km values of ATP for most protein kinases are below 100 μM.
However, if the objective is to measure enzyme activity using gamma-labeled ATP, it is best to use 100-200 μM ATP in order to have higher specific activity of gamma-labeled ATP (100-500 cpm/pmol). Also, an excess of substrate should be used, and the level of phosphorylation should not exceed 10% for determination of the initial rate.
To phosphorylate a protein or peptide substrate to completion, the ATP concentration should be about 5-fold over the limited substrate concentration. Higher enzyme concentration and prolonged incubation times should be employed.
Recommended reference:
Protein Phosphorylation: A Practical Approach (1993) ed. Hardie, D.G. IRL Press.
FAQs
- How much Glycogen Synthase Kinase 3 (NEB# P6040) should be used?
- What is the consensus sequence for GSK-3 (P6040)?
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
GSK-3 contains no detectable protease or phosphatase activities.
Protease Activity:
After incubation of 1,000 units of Glycogen Synthase Kinase 3 (GSK-3) with a standard mixture of proteins
for 2 hours at 30ºC, no proteolytic activity could be detected by SDS-PAGE.
Contaminating Phosphatases:
After incubation of 1,000 units of with 50 mM p-nitrophenyl phosphate for 2 hours at 30ºC, no
phosphatase activity could be detected by spectrophotometric analysis.
References
- Embi, N., Rylatt, D.B. and Cohen, P. (1980) Eur. J. Biochem., 107, 519-527.
- Hemmings, B.A., Yellowlees, D., Kernohan, J.C. and Cohen, P. (1982) Eur. J. Biochem., 119, 443-451.
- Vandenheede, J.R., Yang, S.D., Goris, J. and Merlevede, W. (1980) J. Biol. Chem., 255, 11768-11774.
- Woodgett, J. R. (1990) EMBO J., 9, 2431-2438.
- Wang, Q.M., Fiol, C.J., DePaoli-Roach, A.A. and Roach, P.J. (1994) J. Biol. Chem., 269, 14566-14574.
- Cole, A. et al. (2004) Biochem. J., 377, 249-255.
- Frame, S. and Cohen P. (2001) Biochem. J., 359, 1-16.
Reagents Sold Separately
GSK-3 Reaction Buffer
Companion Products
Adenosine 5´-Triphosphate (ATP)
CREB Phosphopeptide (GSK-3 Substrate)
|
