Universal, HTS Kinase Screening Assays
Use higher ATP concentrations: Linear response up to 500μM ATP.
Use any kinase and kinase-substrate combination, including peptide, protein, lipid, and sugar substrates.
Perform the assay without substrate modifications.
Obtain reliable results: Z´-factor values routinely >0.7.
Reduce false hits: Luminescence is much less susceptible to interference from library compounds than fluorescence-based assays.
How It Works
The kinase reaction is conducted under the appropriate conditions. ATP remaining at the time that the Kinase-Glo® 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 500μM ATP
The Kinase-Glo® Platform consists of three assay formats: the Kinase-Glo® Assay, which is used to monitor kinase activity using up to 10μM ATP; the Kinase-Glo® Plus Assay, which is used for assays requiring higher ATP concentrations (up to 100μM); and the Kinase-Glo® Max Assay, which is used for assays requiring up to 500μM ATP.
Luminescence correlates with amount of ATP. A direct relationship exists between the luminescence measured with the Kinase-Glo® Reagent and the amount of ATP. Reactions were performed as described in Technical Bulletin TB372. Values represent the mean of two replicates. There is a linear relationship between the luminescent signal and the amount of ATP in the kinase reaction buffer from 0–10μM using the Kinase-Glo® Assay (Panel A, r2 = 0.9903); 0–100μM using the Kinase-Glo® Plus Assay (Panel B, r2= 0.9993); and 0–500μM using the Kinase-Glo® Max Assay (Panel C, r2 = 0.9971).
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.
Results of Z´-factor analysis in a solid white 384-well, flat-bottom plate. Panel A. (Kinase-Glo® Plus Assay) Reactions were performed in solid white, 96-well plates as described in Technical Bulletin TB372. Panel B. (Kinase-Glo® Max Assay) PKA was diluted in 50μl kinase reaction buffer (40mM Tris [pH 7.5], 20mM MgCl2, 0.1mg/ml BSA) containing 500μM Kemptide Substrate using 0.2 units/well PKA and 100μM ATP for 30 minutes at room temperature (solid symbols) or without PKA (open symbols). Final volumes for the 384-well plate assays were 20μl. Solid lines indicate the mean, and the dotted lines indicate ± 3 S.D. Z´-factor values were ~0.8 for 10μM ATP and 100μM ATP plates.
Uncover non-ATP binding site inhibitors
Use the Kinase-Glo Assays to distinguish ATP competitive inhibitors from noncompetitive inhibitors. Use PKA inhibitor (noncompetitive, PKI, and competitive, H89) titrations were performed using the Kinase-Glo Plus Assay. 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.