Figure 1. Jak2 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: Tetra (LRRWSLG), 5 µg/50 µl, recombinant Jak2: 50 ng/50 µl.
Figure 3. Dose dependence curve of Jak2 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1305) by Jak2 kinase. In a 50 µl reaction, increasing amounts of Jak2 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 Jak2 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of Jak2 substrate peptide (#1305) by Jak2 kinase. In a 50 µl reaction, 100 ng Jak2, 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 Jak2 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) to detect phosphorylation of substrate peptide (#1305) by Jak2 kinase. In a 50 µl reaction, 100 ng of Jak2 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 Jak2 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) to detect phosphorylation of Jak2 substrate peptide (#1305) by Jak2 kinase. In a 50 µl reaction, 100 ng Jak2 and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)