Figure 1. Pim-1 kinase activity was measured in a radiometric assay using the following reaction conditions: 60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 µM Na-orthovanadate, 1.2 mM DTT, ATP (variable), 2.5 µg/50 µl PEG20,000, Substrate: Histone H2B 5 µg/50 µl, recombinant Pim-1: 200 ng/50 µl.
Figure 3. Dose dependence curve of Pim-1 kinase activity: DELFIA® data generated using Phospho-Bad (Ser112) Antibody #9291 to detect phosphorylation of substrate peptide (#1342) by Pim-1 kinase. In a 50 µl reaction, increasing amounts of Pim-1 and 1.5 µM substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 5. Staurosporine inhibition of Pim-1 kinase activity: DELFIA® data generated using Phospho-Bad (Ser112) Antibody #9291 to detect phosphorylation of Pim-1 substrate peptide (#1342) by Pim-1 kinase. In a 50 µl reaction, 50 ng Pim-1, 1.5 µM substrate peptide, 20 µM ATP and increasing amounts of staurosporine were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 4. Peptide concentration dependence of Pim-1 kinase activity: DELFIA® data generated using Phospho-Bad (Ser112) Antibody #9291 to detect phosphorylation of substrate peptide (#1342) by Pim-1 kinase. In a 50 µl reaction, 50 ng of Pim-1 and increasing concentrations of substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 2. Time course of Pim-1 kinase activity: DELFIA® data generated using Phospho-Bad (Ser112) Antibody #9291 to detect phosphorylation of Pim-1 substrate peptide (#1342) by Pim-1 kinase. In a 50 µl reaction, 50 ng Pim-1 and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)