Calmodulin-dependent Protein Kinase II (CaMKII)

  • This product was discontinued on 01/01/2017
recombinant unique buffer 30 degrees heat inactivation
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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)-2010 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.

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.

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.

Selection Charts

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. Further information regarding NEB product quality can be found here.
  • 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.

Safety Data Sheet

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

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.