| Assay Method Information | |
| | FRET-Based Enzyme-Coupled Assay |
| Description: | A robust FRET-based, enzyme-coupled assay format, Z-Lyte® Kinase Assay Ser/Thr 13 Peptide Kit (Invitrogen, cat. PV3793), was employed in this study to monitor inhibition of ROCK1 (Invitrogen, cat. PV3691) and ROCK2 (Invitrogen, cat. PV3759) enzyme activities. Compounds were tested on three separate days with 8 point dilutions done in duplicate to determine the average IC50 values. The assay conditions were optimized to 15 μl of kinase reaction volume with 5 ng of enzyme in 50 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM EGTA, and 0.01% Brij-35. The reaction was incubated 1 hr. at room temperature in the presence of 1.5 μM of substrate with 12.5 μM of ATP (ROCK1 assay) or 2 μM of substrate with 50 μM of ATP (ROCK2 assay) in the presence of various concentrations of the compounds. The reaction was then stopped and the ratio of phosphorylated to unphosphorylated peptide substrate was determined by selective cleavage of only the unphosphorylated peptide as described by the manufacturer (Invitrogen, cat. PV3793). This was followed by excitation of coumarin at 400 nm resulting in emission at 445 nm and energy transfer to fluorescein and final emission at 520 nm. The substrate contains both coumarin and fluorescein and only uncleaved phosphorylated substrate will undergo FRET. |
| Affinity data for this assay | |
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