| Assay Method Information | |
| | kinetic assay |
| Description: | Purified protein is diluted in reaction buffer to 100 nM in an opaque 96-well plate. The protein is incubated with or without compound in DMSO at different concentrations for 15 minutes while shaking. The reaction is initiated by the addition of a 50 μM FRET substrate (Dabcyl-KTSAVLQ↓SGFRKM-E(Edans-NH2); GL Biochem) in reaction buffer. Cleavage of the substrate generates a free Edans group. Fluorescence is monitored at an excitation wavelength of 360nm and an emission wavelength of 460nm. Baseline subtraction was performed to control for intrinsic fluorescence of each compound. All experiments were performed in triplicate. |
| Affinity data for this assay | |
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