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Casein Kinase II (CK2) |
 | | | | Casein Kinase II (CK2) | | | Will be discontinued as of April 1, 2009 | |
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Prices are in US dollars and valid only for US orders.
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- Protein serine/threonine kinase
- Human, recombinant (E. coli)
Description:
Casein Kinase II (CK2) is a constitutively active serine/threonine protein kinase composed of two catalytic (α) and two regulatory β-subunits to form stable heterotetramers. CK2 holoenzyme undergoes autophosphorylation at two serine residues (S2/S3) of its β-subunit. Recently it has been shown that CK2 α-subunits undergo intermolecular tyrosine-autophosphorylation at Y182, which may represent a specific regulatory mechanism. Also, CK2 is able to phosphorylate, under special circumstances, tyrosyl residues in proteins. CK2 is implicated in a variety of cellular functions (1,2).
Recognition Determinants:
The CK2 substrate specificity is invariably determined by multiple acidic residues located at positions between -2 and +5 relative to the target amino acid (mostly Ser and rarely Thr). The general recognition motif for phsophorylation by CK2 is SXXE/D, although SXE/D and S/D, and variations of these sequences are also phosphorylated. Polyanionic compounds, like heparin, inhibit CK2 activity with a Ki of 1.4 nm (4,5).
Source:
Isolated from a strain of E. coli expressing both α and β CK2 subunits derived from a human glioblastoma cDNA library (kindly provided by Dr. D. Marshak) (3).
Reagents Supplied:
CK2 Reaction Buffer (10X)
Enzyme Properties
Protein Kinase Substrate Recognition
Specific Activity:
500,000 units/mg
Reaction & Storage Conditions
Reaction Conditions:
1X CK2 Reaction Buffer
Supplemented with 200 μM ATP and 300 μCi/μmol gamma-labeled ATP
Incubate at
30°C.
1X CK2 Reaction Buffer:
20 mM Tris-HCl
50 mM KCl
10 mM MgCl2
pH 7.5 @ 25°C
Unit Definition:
One unit is defined as the amount of CK2 required to catalyze the transfer of 1 pmol of phosphate to CK2 Peptide Substrate, RRRADDSDDDDD (100 µM, NEB #P6012), in 1 minute at 30°C in a total reaction volume of 25 µl (4,5).
Concentration:
500,000 units/ml
Storage Conditions:
20 mM Tris-HCl
350 mM NaCl
2 mM DTT
1 mM EDTA
0.1% Triton X-100
pH 7.5 @ 25°C
Storage Temperature:
-70°C
Avoid repeated freeze/thaw cycles.
Notes
Molecular Weight: α-subunit (45 kDa), β-subunit (25 kDa). The apparent molecular weight of the α-subunit estimated by SDS-PAGE is about 42 kDa.General notes:- For short term storage (two weeks or less) CK2 can be stored at -20°C.
- 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 CK2 Reaction Buffer, supplement with 200 µM ATP and gamma-labeled ATP to a final specific activity of 100-500 µCi/µmol. (CK2 will also accept GTP as a phosphoryl donor in place of ATP).
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 Casein Kinase II (NEB# P6010) should be used?
- What is the consensus sequence for this CK2 (P6010)?
- Can you recommend inhibitors for CK2 (P6010)?
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:
CK2 contains no detectable protease or phosphatase activities.
Protease Activity:
After incubation of 5,000 units of Casein Kinase II (CK2) 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 5,000 units of with 50 mM p-nitrophenyl phosphate for 2 hours at 30ºC, no
phosphatase activity could be detected by spectrophotometric analysis.
References
- Chester, N. and Marshak, D.R. (1993) Anal. Biochem., 209, 284-290.
- Donella-Deana, A. et al. (2001) Biochem J., 357, 563-567.
- Marin, O. et al. (1999) J. Biol. Chem., 274, 29260-29265.
- Marin, O. et al. (1994) BBRC, 198, 898-905.
- Sarno, S. et al. (1996) J. Biol. Chem., 271, 10595-10601.
Reagents Sold Separately
CK2 Reaction Buffer
Companion Products
Adenosine 5´-Triphosphate (ATP)
CK2 Peptide Substrate
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