Kinase RR Kit is based on a method of measuring the first order reaction rate constant of protein kinase reactions by monitoring the ATP degradation rate using bioluminescence. The assay starts by adding ATP to a reaction mixture containing all necessary assay components, including luciferase and luciferin. In HTS the light is measured at two points in time. In non-HTS the light can be measured repeatedly until a significan reduction of light is obtained. The assay is set up so that
a) the kinase reaction follows first order kinetics
b) the sample light emission, normalized against the blank (no kinase), is proportional to the remaining ATP concentration.
The first order rate constant of the kinase reaction is proportional to the kinase activity over several orders of magnitude, which makes it very easy to set up new assays. Furthermore variation in ATP and luciferase cancel out in calculations and do not affect assay results.
It is recommended to use a microplate luminometer with a dispenser for adding ATP. However, microplate luminometers without dispensers can be used. In such case please consult BioThema AB for technical support.
Detailed described procedures of Standard Curve Generation, Substrate Optimisation, HTS and IC50 Determination are found in the “Instructions for Use”, which is included with every kit.
Lundin, A., and Eriksson, J., Assay and Drug Development Technologies, Volume 6, Number 3, 2008 (in press)
No. determinations per kit: 1000 in 96-well plates and 2000 in 384-well plates. Storage conditions: +4 degrees celsius.
A Kinase RR Toolbox (Microsoft Excel) is available for calculation and presentation of results. This includes a detailed description on luminometer set up, instructions for data collection and analysis for the following applications:
HTS assays of compound libraries