| Assay Method Information | |
| | In Vitro Effect Assay |
| Description: | Table 2: Inhibitory effect of each compound synthesized above on Rho-associated protein kinase (ROCK) was tested.Method 1. 10 mM test compound was diluted to 1 mM with DMSO, and then further diluted to 300 nM. Netarsudil (AR-13324) is a commercial drug for decreasing intraocular pressure, and AR-13503 is an active metabolite of AR-13324. 2. The 300 nM test compound mentioned above was serially diluted to obtain test compounds with concentrations of 100 nM, 33 nM, 11 nM, 3.7 nM, 1.2 nM and 0.4 nM, respectively. 3. 1 μL of the serially diluted test compound mentioned above was added to 49 μL of the modified ROCK reaction buffer (0.05 M Trizma hydrochloride buffer (pH 7.5) containing 0.1 M KCl, 0.01 M MgCl2, 0.1 mM EGTA and 2.25 μg/mL ROCK1) to obtain an experimental group sample. 1 μL of DMSO was taken to add to 49 μL of an adjusted ROCK reaction buffer to obtain a positive control group sample (maximum value=100%). 1 μL of DMSO was taken to add to 49 μL of a buffer (0.05 M Trizma hydrochloride buffer (pH 7.5) containing 0.1 M KCl, 0.01 M MgCl2 and 0.1 mM EGTA) to obtain a vehicle control group sample (minimum value=0%). 4. 20 μL of the above prepared sample was added to a flat-bottomed 96-well plate, and 20 μL ROCK ATP buffer was added to each well. 5. The 96-well plate mentioned above was placed on an orbital shaker and reacted at room temperature for 90 minutes.6. Next, 40 μL of Kinase-Glo luminescent kinase assay solution (Promega, RV6712) was added to the 96-well plate mentioned above, and the 96-well plate was placed on an orbital shaker and reacted at room temperature for 10 minutes. 7. The luminescence value of each well of the 96-well plate was determined by SpectraMax M5 microplate reader, and the ROCK inhibition rate (%) was calculated by the following formula. |
| Affinity data for this assay | |
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