• My NEB
  • Print
  • PDF
  • Casein Kinase II (CK2)


    Casein Kinase II (CK2) is a constitutively active serine/threonine protein kinase composed of two 44 kDa catalytic α-subunits and two 26 kDa regulatory β-subunits in an α2β2 configuration 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).

    Product 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).

    Recognition Determinant

    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).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    CK2 Reaction Buffer-2010X

    Properties and Usage

    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), in 1 minute at 30°C in a total reaction volume of 25 µl (4,5).

    Reaction Conditions

    1X CK2 Reaction Buffer
    Supplement with 200 μM ATP
    Incubate at 30°C

    1X CK2 Reaction Buffer:
    20 mM Tris-HCl
    50 mM KCl
    10 mM MgCl2
    pH 7.5 @ 25°C

    Storage Temperature


    Specific Activity

    859,000 units/mg

    Storage Notes

    • Avoid repeated freeze/thaw cycles.

    Quality Control

    Quality Control Assays

    The 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 there is no detectable phosphatase activity as 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.


    1. 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.
    2. 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).
    3. 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.


    1. Chester, N. and Marshak, D.R. (1993). Anal. Biochem. 209, 284-290.
    2. Donella-Deana, A. et al. (2001). Biochem J. 357, 563-567.
    3. Marin, O. et al. (1999). J. Biol. Chem. 274, 29260-29265.
    4. Marin, O. et al. (1994). BBRC. 198, 898-905.
    5. Sarno, S. et al. (1996). J. Biol. Chem. 271, 10595-10601.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.


    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. Can you recommend inhibitors for CK2 (P6010)?
    2. How much Casein Kinase II (NEB# P6010) should be used?
    3. What is the consensus sequence for this CK2 (P6010)?

    Selection Tools

    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 consensus sequence is SXXE/D
    Heparin inhibits CKII at a Ki of 1.4 nM