Figure 1. PKCgamma kinase activity was measured in a radiometric assay using the following reaction conditions: 60 mM HEPES-NaOH, pH 7.5, 5 mM MgCl2, 1.32 mM CaCl2, 1 mM EDTA, 1.25 mM EGTA, 0.25 μg/50 μl Phosphatidylserine, 50 ng/50 μl 1.2 Dioleyl-glycerol, 1.2 mM DTT, ATP (variable), 2.5 μg/50 μl PEG 20.000, Substrate: Histone H1, 1 μg/50 μl recombinant PKCgamma: 100 ng/50 μl.
Figure 3. Dose dependence curve of PKCgamma kinase activity: DELFIA® data generated using Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 to detect phosphorylation of substrate peptide (#1331) by PKCgamma kinase. In a 50 µl reaction, increasing amounts of PKCgamma and 1.5 µM substrate peptide were used per reaction at room temperature for 15 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 5. Staurosporine inhibition of PKCgamma kinase activity: DELFIA® data generated using Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 to detect phosphorylation of PKCgamma substrate peptide (#1331) by PKCgamma kinase. In a 50 µl reaction, 10 ng PKCgamma, 1.5 µM substrate peptide, 20 µM ATP and increasing amounts of staurosporine were used per reaction at room temperature for 15 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 4. Peptide concentration dependence of PKCgamma kinase activity: DELFIA® data generated using Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 to detect phosphorylation of substrate peptide (#1331) by PKCgamma kinase. In a 50 µl reaction, 10 ng of PKCgamma and increasing concentrations of substrate peptide were used per reaction at room temperature for 15 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 2. Time course of PKCgamma kinase activity: DELFIA® data generated using Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 to detect phosphorylation of PKCgamma substrate peptide (#1331) by PKCgamma kinase. In a 50 µl reaction, 10 ng PKCgamma and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)