Kinase glo luminescent kinase assay

The ROCK kinase assay is a luminescent kinase assay that measures IOP formed from a kinase reaction. ADP is converted into ATP and is converted into light by Kinase-Glo Luciferase. The Kinase-Glo Assay can be used to monitor the activity of virtually any ADP-generating enzyme. The relative light unit (RLU) without test compound was set as 100% (blank value), and that without enzyme and compound was set as 0% (normal value). The reaction rate (% of black) was then calculated from the RLU.
The following reagents were used in this assay: ROCK1 (750 ug/mL, Carna Biosciences, Cat.01-109); long S6 kinase substrate peptide (Millipore, Cat#12-420); magnesium chloride solution (Sigma, 60142-100 mL); EGTA solution (0.5 M pH 8.0, Bioword, Cat. 4052008-1); Trizma^® hydrochloride solution (1 M, pH 7.5 Sigma, T2319-100 mL); bovine serum albumin solution (20 mg/mL in water, Sigma); Kinase-Glo luminescent kinase assay (Promega, RV6712); ripasudil (K115) hydrochloride dehydrate (Selleckchem, S7995-5 mg); ATP solution (100 mM, GE Healthcare, Cat.27-2056-010). A ROCK kinase inhibitor k-115 was used as the positive control.
$$\mathrm{Inhibition} (\%)=\frac{100 –\{\left(\mathrm{activity} \mathrm{of} \mathrm{Enzyme} \mathrm{with} \mathrm{Test} \mathrm{Cmpd} \& \mathrm{Sbustrate}-\mathrm{Min}\right)\}\times 100}{\mathrm{Max}-\mathrm{Min}}$$
Max = observed enzyme activity measured in the presence of enzyme, substrate(s), and cofactors utilized in the method.
Min = Normal value in the method

A total of 3000 exponential-phase cells were plated into each well of a 96-well plate (100 μL medium/well) and cultured overnight. Then, the cells were transfected with miR-338 mimics or control mimics, inhibitor RNA or control RNA for another 48 h. The assays were conducted with a Kinase-Glo® Luminescent Kinase Assay (Promega) according to the manufacturer's protocol. Data were expressed as fold-changes.

Enzyme kinetics was measured by Kinase-Glo Luminescent Kinase Assay and ADP-Glo Kinase Assay (Promega, WI, United States). The 5 μl of different concentrations of LM49 solution, 2.5 μl different concentrations of ATP solution, 2.5 μl of GSK3β solution, and 10 μl Kinase-Glo Reagent were mixed and incubated in a well of a solid white 96-well plate for 30 min at 30°C. Then, 20 μl of ADP-Glo Reagent was added to the well and incubated at room temperature for 40 min. Finally, 40 μl of Kinase Detection Reagent was added to the well and incubated at room temperature for 30 min. Luminescence was measured using a SpectraMax i3X multifunctional microplate reader (Molecular Devices, CA, United States). Lineweaver-Burker plot was generated by 1/V and 1/[ATP].