| Assay Method Information | |
| | MS-Based Biochemical Assay |
| Description: | The PRP4 phosphorylation reaction was performed at room temperature with 15 nM full-length His-tagged PAK4 recombinant protein expressed in Escherichia coli (from American Research Products, Inc.), saturating concentration of ATP (2 mM), and 2.3 nM PRP4 enzyme kinase domain in an assay buffer containing 8 mM MOPS, pH 7.5, 10 mM MgCl2, and 1 mM DTT in a total volume of 100 μl for 1 h. Samples with PAK4 protein in reaction buffer alone without the presence of PRP4 kinase and PAK4 protein with PRP4 kinase-dead mutant were used as negative controls. For measuring Compound A potency against PRP4 using PAK4 as substrate, Compound A ranging from 0.5 nM to 1 μM was preincubated with PRP4 for 30 min at room temperature before the reaction was initiated with PAK4 protein substrate. Compound dilution (3-fold) was prepared in 100% DMSO and diluted 1:100 into the final reaction. After a 60-min incubation at room temperature, the reaction was terminated with formic acid (1% final). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |