How it works
The kinase reaction is conducted under the appropriate conditions. ATP remaining at the time that Kinase-Glo Plus Reagent is added is used as a substrate by the Ultra-Glo™ Luciferase to catalyze the mono-oxygenation of luciferin and the generation of light. Luminescence is inversely related to kinase activity.
Linear out to 100µM ATP
A direct relationship exists between the luminescence measured with the Kinase-Glo Plus Reagent and the amount of ATP remaining in a completed kinase reaction. Serial dilutions of ATP were made in 50µl kinase reaction buffer. Following the addition of an equal volume of Kinase-Glo Plus the luminescence was recorded. There is a linear relationship between the luminescent signal and the amount of ATP in the kinase reaction from 0.100µM to 100µM (r2 = 0.9993).
Perfect for HTS applications
Z´-factor is a statistical value that compares the assay dynamic range to data variation in order to assess assay quality. Z´-factors greater than 0.5 indicate excellent assay quality. Z’-factor analysis was performed in a 384-well plate using PKA at 10µM ATP (Panel A) and 100µM ATP (Panel B). In both cases, the Z’-factor was calculated to be > 0.8.
Uncover non-ATP binding site inhibitors
Use Kinase-Glo Plus to distinguish ATP competitive inhibitors from noncompetitive inhibitors. PKA inhibitor (noncompetitive, PKI, and competitive, H89) titrations were performed. IC50 results determined using Kinase-Glo Plus varied just 2-fold for the noncompetitive inhibitor PKI (3.5nM vs. 7.9nM) at 10 and 100µM ATP, respectively. The IC50 results varied 7-fold for the competitive inhibitor H89 (0.06µM vs. 0.4µM) at 10 and 100µM ATP, respectively.
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