Said Goueli, Marina Zdanovskaia and Hicham Zegzouti
Promega Corporation, Madison, WI
Because of its versatility (all types of substrates), robustness (Z' > 0.8), rapid performance and its ease of use, the luminescence-based Kinase-Glo® Assay platform has gained wide acceptance in many drug screening programs for protein kinase inhibitors. The new ADP-Glo™ Assay maintains all the positive features of Kinase-Glo®. It is universal, applicable to all kinds of kinase substrates regardless of their nature with no prior modification (peptides, protein, polymer, lipid and sugars). Instead of measuring ATP depletion, ADP-Glo™ Assay monitors ADP production. It detects ADP at early stages of enzyme reactions with very high signal-to-background (SB) ratio. It can be run at a wide range of ATP concentrations (low micromolar to millimolar), is sensitive to low ADP concentrations, and it can be carried out in high density plate formats. The assay is robust as is indicated by the high Z' values (>0.8), is homogeneous, does not require antibodies or custom synthesized substrates, and is ideal to search for optimal substrates in a collection of peptides, proteins, lipids in one easy assay format and to screen for ATP competitive and noncompetitive inhibitors. Because of the high dynamic range and more importantly, the high SB values at low % ATP to ADP conversion, the ADP-Glo™ Assay allows the use of low amount of enzyme during high-throughput screenings. ADP-Glo™ Assay is applicable to all types of kinases, ATPases and many other ADP-producing enzyme reactions.