| Solution Information | help | |
| Enzyme: | Rho-associated protein kinase 2 | |
| inhibitor: | BDBM39188 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Assay Overview: Compounds identified from a previously described set of experiments entitled "Primary high-throughput assay for chemical inhibitors of Rho kinase 2 (Rhok2) activity" (PubChem AID = 604) were selected for testing in this assay. Out of 212 compounds identified during the primary screen, 206 compounds were assessed in this dose response experiment. Each compound was assayed in 10 point, 1:3 serial dilutions starting at a nominal test concentration of 60 micromolar. As with the primary screen, the assay is based on ability of Rhok2 to phosphorylate a specific peptide sequence derived from its substrate - ribosomal protein S6 (amino acid residues 229-239). Rhok2 uses ATP as a donor of phosphate for the phosphorylation of the substrate, which leads to the depletion of ATP in the reaction mix. An assay kit (#Kinase-Glo#, Promega) was used to quantify enzyme activity. Using this kit, residual amounts of ATP are measured by a secondary enzymatic reaction, through which lucif | |
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