Figure 1. Akt1 kinase activity was measured in a radiometric assay using the following reaction conditions: 5 mM MOPS, pH 7.2, 2.5 mM β-glycerophosphate, 1 mM EGTA, 0.4 mM EDTA, 5 mM MgCl2, 0.05 mM DTT, 50 μM ATP, Substrate: Akt-substrate peptide 200 ng/μL, and variable amount of recombinant Akt1.
Figure 3. Dose dependence curve of Akt1 kinase activity: DELFIA® data generated using Phospho-eNOS (Ser1177) (C9C3) Rabbit mAb #9570 to detect phosphorylation of substrate peptide (#1133) by Akt1 kinase. In a 50 µl reaction, increasing amounts of Akt1 and 1.5 µM substrate peptide were used per reaction at room temperature for 10 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 5. Staurosporine inhibition of Akt1 kinase activity: DELFIA® data generated using Phospho-eNOS (Ser1177) (C9C3) Rabbit mAb #9570 to detect phosphorylation of Akt1 substrate peptide (#1133) by Akt1 kinase. In a 50 µl reaction, 10 ng Akt1, 1.5 µM substrate peptide, 50 µM ATP and increasing amounts of staurosporine were used per reaction at room temperature for 10 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 4. Peptide concentration dependence of Akt1 kinase activity: DELFIA® data generated using Phospho-eNOS (Ser1177) (C9C3) Rabbit mAb #9570 to detect phosphorylation of substrate peptide (#1133) by Akt1 kinase. In a 50 µl reaction, 10 ng of Akt1 and increasing concentrations of substrate peptide were used per reaction at room temperature for 10 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 2. Time course of Akt1 kinase activity: DELFIA® data generated using Phospho-eNOS (Ser1177) (C9C3) Rabbit mAb #9570 to detect phosphorylation of Akt1 substrate peptide (#1133) by Akt1 kinase. In a 50 µl reaction, 10 ng Akt1 and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)