Figure 1. IKKepsilon 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, 1 µM ATP, 2.5 µg/50 µl PEG20.000, Substrate: Casein, 10 µg/50 µl, and recombinant IKKepsilon: 100 ng/50 µl.
Figure 3. Dose dependence curve of IKKepsilon kinase activity: DELFIA® data generated using Phospho-(Ser/Thr) Phe Antibody #9631 to detect phosphorylation of substrate peptide (#1134) by IKKepsilon kinase. In a 50 µl reaction, increasing amounts of IKKepsilon 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 IKKepsilon kinase activity: DELFIA® data generated using Phospho-(Ser/Thr) Phe Antibody #9631 to detect phosphorylation of IKKepsilon substrate peptide #1134 by IKKepsilon kinase. In a 50 µl reaction, 50 ng IKKepsilon, 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 IKKepsilon kinase activity: DELFIA® data generated using Phospho-(Ser/Thr) Phe Antibody #9631 to detect phosphorylation of substrate peptide (#1134) by IKKepsilon kinase. In a 50 µl reaction, 50 ng of IKKepsilon 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 IKKepsilon kinase activity: DELFIA® data generated using Phospho-(Ser/Thr) Phe Antibody #9631 to detect phosphorylation of IKKepsilon substrate peptide #1134 by IKKepsilon kinase. In a 50 µl reaction, 50 ng IKKepsilon and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)