The assay kit is designed to measure the phosphotransferase activity of protein kinase C (PKC) in immunoprecipitates and column fractions. It is highly specific for alpha, beta and gamma. Crude cell lysates may also be used but detergents/biochemicals contained in the cell lysis buffer may inhibit PKC kinase activity. Furthermore, although two inhibitors are included with the kit, editors may suggest other unknown kinases found in crude lysates are responsible for substrate peptide phosphorylation. The assay kit is based on phosphorylation of a specific substrate peptide (QKRPSQRSKYL) using the transfer of the -phosphate of adenosine-5'-[32P] triphosphate ([ -32P] ATP) by PKC kinase. The phosphorylated substrate is then separated from the residual [ -32P] ATP using P81 phosphocellulose paper and quantitated by using a scintillation counter. The assay is linear for incubation times of up to 30 minutes and incorporation of up to 20% of total ATP. Further incubation or incorporation may not be linear and may therefore not be a true indication of PKC activity in the sample extract. The enzyme assay is rapid, convenient and fairly specific for PKC.
~200 PKC kinase assays.
1. Assay Dilution Buffer II (ASBII) containing 20mM MOPS, pH 7.2, 25mM b-glycerol phosphate, 1mM sodium orthovanadate, 1mM dithiothreitol, 1mM CaCl , 2x1ml
2. PKC Substrate Cocktail, 500uM PKC substrate peptide [QKRPSQRSKYL] in ADBII, 2x1ml
3. PKA/CaMK Inhibitor Cocktail containing 2uM PKA inhibitor peptide, and 20uM Compound R24571 in ADBII, 2x1ml
4. PKC Lipid Activator containing 0.5mg/ml phosphatidyl serine and 0.05mg/ml diacylglycerol in ADBII, 2x1ml
5. Mg/ATP Cocktail containing 75mM magnesium chloride and 500um ATP 20mM MOPS, pH 7.2, 25mM b-glycerophosphate, 5mM EGT A, 1mM Na3VO4, 1mM dithiothreitol, 2x1ml. (Before starting the assay, 90ul of the Mg2+/ATP cocktail should be added to 100uCi (10ul) of the [g-32P]ATP (?3000Ci/mmol).)
6. p81 squares, 1x200 each
Storage and Stability:
Store components at -20 degrees C. Stable for 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.