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Casein Kinase I (CK1) |
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- Protein serine/threonine kinase
- Rat, recombinant (E. coli)
Description:
Casein Kinase I (CK1) is a serine/threonine protein kinase (1). It is a truncated monomer (1-317) of the CK1d isoform, which lacks the regulatory C-terminal domain, containing 111 amino acids (2). In vitro studies have shown that the activity of CK1δ is regulated by autophosphorylation of its C-terminal domain. Autophosphorylation of this domain on potential sites leads to inhibition of kinase activity (3). There are at least seven mammalian CK1 isoforms and their splice variants, and distinct CK1 family members have a variety of roles in eukaryotic cells (4).
Recognition Determinants:
The 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).
Source:
Isolated 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).
Reagents Supplied:
CK1 Reaction Buffer (10X)
Enzyme Properties
Protein Kinase Substrate Recognition
Molecular Weight:
Theoretical: 36 kDa
Specific Activity:
2,000,000 units/mg
Reaction & Storage Conditions
Reaction Conditions:
1X CK1 Reaction Buffer
Supplemented with 200 μM ATP and 300 μCi/μmol gamma-labeled ATP
Incubate at
30°C.
1X CK1 Reaction Buffer:
50 mM Tris-HCl
10 mM MgCl2
5 mM DTT
pH 7.5 @ 25°C
Unit Definition:
One 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, NEB #P6031), in 1 minute at 30°C in a total reaction volume of 25 µl.
Concentration:
1,000,000 units/ml
Storage Conditions:
20 mM Tris-HCl
100 mM NaCl
2 mM DTT
1 mM Na2EDTA
1 mM EGTA
50% Glycerol
0.1% Triton X-100
pH 7.0 @ 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.
- Reaction Conditions: 1X CK1 Reaction Buffer, supplement with 200 µM ATP and gamma-labeled ATP to a final specific activity of 100-500 µCi/µmol.
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.
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:
CK1 contains no detectable protease or phosphatase activities.
Protease Activity:
After incubation of 10,000 units of Casein Kinase I (CK1) 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 10,000 units of with 50 mM p-nitrophenyl phosphate for 2 hours at 30ºC, no
phosphatase activity could be detected by spectrophotometric analysis.
References
- 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.
- lotow, H. and Poach, P.J. (1991) J. Biol. Chem., 266, 3724-3727.
Reagents Sold Separately
CK1 Reaction Buffer
Companion Products
Adenosine 5´-Triphosphate (ATP)
CK1 Phosphopeptide Substrate
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