 |
|
 |
|
|
| Home >
Products >
Protein Tools >
Protein Kinases/Substrates/Inhibitors >
Calmodulin-dependent Protein Kinase II (CaMKII) |
 | | | | Calmodulin-dependent Protein Kinase II (CaMKII) | | | |
|
|
|
 |
|
Prices are in US dollars and valid only for US orders.
|
- Protein serine/threonine kinase
- Rat, recombinant (S. frugiperda Sf9)
Description:
Ca2+/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).
Recognition Determinants:
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).
Phosphorylation with CaMKII:
1. CaMKII Activation:
Dilute the desired amount of CaMKII in 1X CaMKII Reaction Buffer supplemented with 200 µM ATP, 1.2 µM calmodulin and 2 mM CaCl2. Incubate for 10 minutes at 30°C. The dilution of CaMKII should not exceed 20,000-50,000 units/ml to ensure the suggested rate of autophosphorylation.
2. Substrate Phosphorylation:
Mix the substrate with 1X CaMKII Reaction Buffer supplemented with 200 µM ATP and gamma-labeled ATP to a final specific activity of 100-500 µCi/µmol. Add the activated CaMKII. Incubate at 30°C.
Source:
Isolated from Spodoptera frugiperda (Sf9) cells infected with recombinant baculovirus carrying the rat truncated CaMKII (kindly provided by Dr. H. Shulman).
Reagents Supplied:
CaMKII Reaction Buffer Pack (10X)
ATP (50X)
CaCl2 (10X)
Calmodulin (10X)
Enzyme Properties
Protein Kinase Substrate Recognition
Heat Inactivation:
65°C for 20 minutes
Molecular Weight:
Theoretical: 36 kDa and Apparent: 33 kDa
Specific Activity:
5,000,000 units/mg
Reaction & Storage Conditions
Reaction Conditions:
1X CaMKII Reaction Buffer
Incubate at
30°C.
1X CaMKII Reaction Buffer:
50 mM Tris-HCl
10 mM MgCl2
2 mM DTT
0.1 mM Na2EDTA
pH 7.5 @ 25°C
Unit Definition:
One unit is defined as the amount of activated CaMKII required to catalyze the transfer of 1 pmol of phosphate to Autocamtide-2 (CaMKII Peptide Substrate), KKALRRQETVDAL (50 µM, NEB #P6062), in 1 minute at 30°C in a total reaction volume of 30 µl (4).
Concentration:
500,000 units/ml
Storage Conditions:
50 mM HEPES
100 mM NaCl
1 mM DTT
0.1 mM Na2EDTA
50% Glycerol
0.02% Tween-20
pH 7.5 @ 25°C
Storage Temperature:
-70°C
Avoid repeated freeze/thaw cycles.
Notes
General notes:- CaMKII has been purified to >95% homogeneity as determined by SDS-PAGE and Coomassie Blue staining.
- Avoid freeze/thaw cycles. Can be stored for 2 weeks or less 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: Prior to substrate phosphorylation, CaMKII should be activated by autophosphorylation with ATP/Mg2+ in the presence of CaCl2 and calmodulin. Neither CaCl2 nor calmodulin are required for the subsequent phosphorylation of exogenous substrate.
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:
CaMKII contains no detectable protease or phosphatase activities.
Protease Activity:
After incubation of 5,000 units of Calmodulin-dependent Protein Kinase II (CaMKII) 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
- Takeuchi-Suzuki, E. et al. (1992) Protein Expr. Purif., 3, 160-164.
- Yang, E. and Schulman, H. (1999) J. Biol. Chem., 274, 26199-26208.
- Hanson, P.I. et al. (1989) Neuron, 3, 59-70.
- White, R.R. et al. (1998) J. Biol. Chem., 273, 3166-3172.
Reagents Sold Separately
CaMKII Reaction Buffer Pack
Companion Products
Autocamtide-2 Peptide Substrate
|
|
|
 |
|
|
