50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM glutathione, 0.1 mM EDTA, 0.25 mM DTT, 0.1 mM PMSF, 25% glycerol.
MW
102 kDa
Amino Acids
2-end
Genbank #
X06318
UniProt #
P05771
Tag(s)
N-terminal GST-tag
Background
PKCβI is a member of the PKC family (phospholipid-dependent serine/threonine kinase) and is highly related to PKCβ II. Unlike the mature PKCβII mRNA and protein, which rapidly increase following acute insulin treatment, the PKCβI mRNA and protein levels remain unchanged (1). The stable overexpression of PKCβII, but not PKCβI, leads to insulin-stimulated glucose uptake into cells. Upon stimulation of B lymphocytes and mast cells, Syk regulates Btk, and Btk selectively regulates enzymatic activity of PKCβI. Specific regulation of PKCβI by Btk is consistent with the selective association of Btk with PKCβI (2).
References
1. Cooper, D R. et al: Ectopic expression of protein kinase CbetaII, -delta, and -epsilon, but not -betaI or -zeta, provide for insulin stimulation of glucose uptake in NIH-3T3 cells. Arch Biochem Biophys. 1999 Dec 1;372(1):69-79. 2. Kawakami, Y. et al: Regulation of protein kinase CbetaI by two protein-tyrosine kinases, Btk and Syk. Proc Natl Acad Sci U S A. 2000 Jun 20;97(13):7423-8.
Storage/Stability
At least 6 months at -80°C.
Assay Conditions
Kinase activity was measured using a PKCtide peptide substrate (ERMRPRKRQGSVRRRV) diluted in distilled water at 1mg/ml.
The protein kinase was diluted to 0.1 µg/µL in buffer containing 5 mM MOPS, pH 7.2, 2.5 mM β-glycero-phosphate, 5 mM MgCl2, 1 mM EGTA, 0.4 mM EDTA and 0.05 mM fresh DTT. Increasing amounts of the protein kinase were mixed with 0.30 mg/mL substrate and a sonicated PKC lipid activator in 25 μL final volume including 5 μL of [33P]-ATP. The reaction was initiated by addition of 5 μL of [33P]-ATP diluted in kinase buffer: 6ml kinase buffer containing 1 mCi [33P]-ATP, 0.25 mM ATP, 25 mM MOPS, pH 7.2, 12.5 mM β-glycero-phosphate , 25 mM MgCl2, 5 mM EGTA, 2 mM EDTA, and 0.25 mM fresh DTT. The blank was determined from a “no substrate” sample.
After incubating for 30°C for 15 minutes, the reaction was terminated by spotting 20 μL of the mixture onto phosphocellulose paper strips that were fixed in 1% phosphoric acid and washed three times. Radioactivity was determined using a scintillation counter.
Instructions for Use
Thaw on ice and gently mix prior to use. DO NOT VORTEX. Perform a quick spin before opening. Aliquot into small volumes and flash freeze for long term storage. Avoid multiple freeze/thaw cycles.
Applications
Useful for the study of enzyme kinetics, screening inhibitors, and selectivity profiling.
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