Casein Kinase I (CK1)Product information
- Protein serine/threonine kinase
- Rat, recombinant (E. coli)
Product SourceIsolated from a strain of E. coli that carries a clone expressing CK1 δ derived from a rat testis cDNA library (kindly provided by Dr. P. J. Roach). Two codons, Ser-318 and Arg-319, have been changed to stop codons, resulting in a truncation of the C-terminal portion of the expressed protein (2).
Recognition DeterminantThe most effective recognition motif for phosphorylation by CK1 is pSXXS/T where Ser in the position -3 is phosphorylated (3). Also, the clusters of 3 or 4 acidic residues ending at the position -3, preferably Asp, can specify phosphorylation by CK1. However, the substrates so formed are much poorer than those containing phosphate groups (5).
The following reagents are supplied with this product:
|Store at (°C)||Concentration|
|NEBuffer for Protein Kinases (PK)||-20||10X|
Properties and Usage
Unit DefinitionOne unit is defined as the amount of CK1 required to catalyze the transfer of 1 pmol of phosphate to CK1 Phosphopeptide Substrate, KRRRALpSVASLPGL (70 µM), in 1 minute at 30°C in a total reaction volume of 25 µl.
1X NEBuffer for Protein Kinases (PK)
Supplement with 200 μM ATP
Incubate at 30°C
1X NEBuffer for Protein Kinases (PK):
50 mM Tris-HCl
10 mM MgCl2
0.1 mM EDTA
2 mM DTT
0.01% Brij 35
pH 7.5 @ 25°C
20 mM Tris-HCl
100 mM NaCl
2 mM DTT
1 mM Na2EDTA
1 mM EGTA
0.1% Triton® X-100
pH 7.0 @ 25°C
Molecular WeightTheoretical: 36 kDa
Specific Activity2,000,000 units/mg
Quality Control AssaysThe following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
- Phosphatase Activity (PNPP):
The product is tested in a reaction containing a p-nitrophenyl phosphate (PNPP), a chromogenic substrate for most phosphatases. After incubation the percent degradation is determined by spectrophotometric analysis of released p-nitrophenol at 405 nm.
- Protease Activity (SDS-PAGE):
The product is tested for protease activity by incubation with a standard mixture of proteins resulting in no detectable degradation of the proteins as determined by SDS-PAGE.
- Reaction Conditions: 1X NEBuffer for Protein Kinases (PK), supplement with 200 µM ATP and gamma-labeled ATP to a final specific activity of 100-500 µCi/µmol.
- 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.
Protein Phosphorylation: A Practical Approach (1993) ed. Hardie, D.G. IRL Press.
- 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.
- Hathaway, G.M. and Traugh, J. A. (1979). J. Biol. Chem. 254, 762-768.
- Graves, P.R., Haas, D.W., Hagedorn, C.H., DePaoli-Roach, A.A. and Roach, P.J. (1993). J. Biol. Chem. 268, 6394-6401.
- Graves, P.R., and Roach, P.J. (1995). J. Biol. Chem. 270, 21689-21694.
- Knippschild, U. et al. (2005). Onkologie. 28, 508-514.
- Iotow, H. and Poach, P.J. (1991). J. Biol. Chem. 266, 3724-3727.
- Make sure to use the appropriate protease and protein phosphatase inhibitors in the lysis buffer and to use shorter incubation time for phosphorylation if the substrate is a crude lysate.
- 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.
- 1-2 µl of enzyme is a good starting point for a 1 hr incubation of 1µg of protein.
- The most effective recognition motif for phosphorylation by CK1 is pSXXS/T where Ser in the position -3 is phosphorylated