| Assay Method Information | |
| | MPS1 Kinase Activity Assay |
| Description: | The inhibitory activity of putative kinase inhibitors and the potency of selected compounds were determined using a trans-phosphorylation assay. Specific peptide or protein substrates are trans-phosphorylated by their specific ser-thr or tyr kinase in the presence of ATP traced with 33P-γ-ATP, and in the presence of their own optimal buffer and cofactors. At the end of the phosphorylation reaction, more than 98% unlabeled ATP and radioactive ATP is captured by an excess of the ion exchange dowex resin; the resin then settles down to the bottom of the reaction plate by gravity. Supernatant is subsequently withdrawn and transferred into a counting plate, then evaluated by β-counting. The buffer for MPS1 assay was composed of HEPES 50 mM, at pH 7.5, with 2.5 mM MgCl2, 1 mM MnCl2, 1 mM DTT, 3 uM Na3VO4, 2 mM β-glycerophosphate and 0.2 mg/mL BSA. The assay was run with a final concentration MPS1 of 5 nM, in the presence of 15 uM ATP and 1.5 nM 33P-γ-ATP; the substrate was P38-tide, used at 200 uM. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |