Figure 1. CDK1/CycB kinase activity was measured in a radioisotopic filter binding 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: Rb CTF, 5 µg /50 µl, recombinant CDK1/CycB: 100 ng/50 µl.
Figure 3. Dose dependence curve of CDK1/CycB kinase activity: DELFIA® data generated using Phospho-Rb (Ser780) Antibody #9307 to detect phosphorylation of substrate peptide (#1142) by CDK1/CycB kinase. In a 50 µl reaction, increasing amounts of CDK1/CycB 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 CDK1/CycB kinase activity: DELFIA® data generated using Phospho-Rb (Ser780) Antibody #9307 to detect phosphorylation of substrate peptide (#1142) by CDK1/CycB kinase. In a 50 µl reaction, 50 ng CDK1/CycB, 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 CDK1/CycB kinase activity: DELFIA® data generated using Phospho-Rb (Ser780) Antibody #9307 to detect phosphorylation of substrate peptide (#1142) by CDK1/CycB kinase. In a 50 µl reaction, 50 ng of CDK1/CycB 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 CDK1/CycB kinase activity: DELFIA® data generated using Phospho-Rb (Ser780) Antibody #9307 to detect phosphorylation of substrate peptide (#1142) by CDK1/CycB kinase. In a 50 µl reaction, 50 ng CDK1/CycB and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)