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  • Calmodulin-dependent Protein Kinase II (CaMKII)

    Description

    Calmodulin-Dependent Protein Kinase II (CaMKII) is a serine/threonine kinase. It is a Ca2+/calmodulin-dependent, truncated monomer (1-325 amino acid residues) of the α subunit. Autophosphorylation of threonine 286 in the presence of Ca2+ and calmodulin activates CaMKII and produces substantial Ca2+/calmodulin-independent activity (1,2). 

    Highlights

    • Protein serine/threonine kinase
    • Rat, recombinant (S. frugiperda Sf9)

    Product Source

    Isolated from Spodoptera frugiperda (Sf9) cells infected with recombinant baculovirus carrying the rat truncated CaMKII (kindly provided by Dr. H. Shulman).

    Recognition Determinant

    The minimal recognition motif for phosphorylation by CaMKII is RXXS/T. A more recent report suggests the presence of positive determinants at the -5, -2 and +1 positions in addition to the -3R. Thus, CaMKII preferentially phosphorylates substrates with motifs: HydXRXXS/T and HydXRNBXS/T, respectively, where Hyd represents a hydrophobic, X any, and NB a non-basic amino acid residue (3).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBuffer for Protein Kinases (PK)-2010X
    CaCl2 (10X)20 mM
    Adenosine-5'-Triphosphate (ATP)10 mM
    Calmodulin12 μM

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of activated CaMKII required to catalyze the transfer of 1 pmol of phosphate from ATP(200mM) to Autocamtide-2 (CaMKII Peptide Substrate), KKALRRQETVDAL (50 µM), in 1 minute at 30°C in a total reaction volume of 30 µl (5).

    Reaction Conditions

    1X NEBuffer for Protein Kinases (PK)
    Supplement with 200 μM ATP, 1.2 μM Calmodulin and 2 μM CaCl2 (activation)
    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

    Storage Temperature

    -70°C

    Storage Conditions

    50 mM HEPES
    100 mM NaCl
    1 mM DTT
    0.1 mM EDTA
    50% Glycerol
    0.01% Brij 35
    pH 7.5 @ 25°C

    Heat Inactivation

    65°C for 20 min

    Molecular Weights

    Apparent: 33 kDa
    Theoretical: 36 kDa

    Specific Activity

    5,000,000 units/mg

    Storage Notes

    • Avoid repeated freeze/thaw cycles.

    Quality Control

    Quality Assurance Statement

    • CaMKII contains no detectable protease or phosphatase activities.

    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.

    Notes

    1. Optimal incubation times and enzyme concentrations must be determined empirically for each particular substrate.
    2. 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 (4). Apparent Km values of ATP for most protein kinases are below 100 μM.
    3. 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.
    4. CaMKII has been purified to >95% homogeneity as determined by SDS-PAGE and Coomassie Blue staining.
    5. Avoid freeze/thaw cycles. Can be stored for 2 weeks or less at -20°C.

    References

    1. Takeuchi-Suzuki, E. et al. (1992). Protein Expr. Purif.. 3, 160-164.
    2. Yang, E. and Schulman, H. (1999). J. Biol. Chem.. 274, 26199-26208.
    3. Hanson, P.I. et al. (1989). Neuron. 3, 59-70.
    4. Hardie, D.G. (1993). Protein Phosphorylation: A Practical Approach.

    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.

    Datacards

    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.

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