Cell Biolabs’ Checkpoint Kinase Activity Immunoblot Kit utilizes recombinant Cdc25C as checkpoint kinase substrate. After incubating the substrate with checkpoint kinase samples (such as purified kinase, cell lysate or immunoprecipitate), the phosphorylated Cdc25C is detected by western blot analysis using an anti-phospho-Cdc25C (Ser216).
CHK1 Activity Immunoblot Assay. 1X Kinase Buffer containing 0.2 mM ATP and 8 µg of recombinant Cdc25C was incubated with (lane 2) and without (lane 1) 10 ng active CHK1 for 60 minutes at 30ºC. Kinase was stopped by adding SDS-PAGE sample buffer. Phosphorylation of Cdc25C substrate was detected by phospho-specific antibody provided.