Product Pathways - Kinases
CaMKII-α Kinase #7358
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Description
Purified recombinant full length human CaMKII-α kinase, supplied as a GST fusion protein.
Source / Purification
The GST-Kinase fusion protein was produced using a baculovirus expression system using sf9 cells and a recombinant virus encoding full-length human CaMK2α (Met1-His478) (GenBank Accession No. NM_171825) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using GSH-agarose.
Gel Staining
Figure 1. The purity of the GST-CaMKII-α fusion protein was analyzed using SDS/PAGE followed by Coomassie stain.
Kinase Assay - Radiometric
Figure 2. CaMKII-α kinase activity was measured in a radiometric assay using the following reaction conditions: 5 mM MOPS, pH 7.2, 2.5 mM β-glycerophosphate, 1 mM EGTA, 0.4 mM EDTA, 5 mM MgCl2, 0.5 mM CaCl2, 0.03 mg/ml calmodulin, 0.05 mM DTT, 50 μM ATP, Substrate: Autocamtide (KKALRRQETVDAL) 300 ng/μL, and recombinant CaMKII-α: variable.
Quality Control
The theoretical molecular weight of the GST- CaMK2α fusion protein is 74 kDa. The purity of the kinase was assessed using SDS-PAGE followed by Coomassie stain [Fig.1]. CaMK2α kinase activity was determined using a radiometric assay [Fig.2].
Background
CaMKII is an important member of the calcium/calmodulin-activated protein kinase family, functioning in neural synaptic stimulation and T-cell receptor signaling (1,2). CaMKII has catalytic and regulatory domains. The binding of Ca2+/calmodulin to its regulatory domain releases its autoinhibitory effect and activates the kinase (3). The activated CaMKII further autophosphorylates at Thr286 to render the kinase constitutively active (3). The threonine phosphorylation state of CaMKII can be regulated through PP1/PKA. PP1 (protein phosphatase 1) dephosphorylates phospho-CaMKII at Thr286. PKA (protein kinase A) prevents this dephosphorylation through its inhibitory effect on PP1 (4).
- Hughes, K. et al. (2001) J. Biol. Chem. 276, 36008-36013.
- Barria, A. et al. (1997) Science 276, 2042-2045.
- Means, A.R. (2000) Mol. Endocrinol. 14, 4-12.
- Makhinson, M. et al. (1999) J. Neurosci. 19, 2500-2510.
Application References
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Companion Products
This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.
