Said A Goueli1,2, Kevin Hsiao1, Marina Zdanovskaia1, and Jolanta Vidugiriene1
1Promega Corporation Madison, WI
2University of Wisconsin School of Medicine and Public Health, Madison, WI
Because of its versatility (all types of substrates), robustness (Z’>0.8), and rapid performance (10 minutes), and its ease of use, the luminescence based Kinase Glo®, Kinase Glo® Plus, and now Kinase Glo® Max assay platform have gained wide acceptance in many drug screening programs for protein kinase inhibitors. It is applicable to all kinds of kinase substrates regardless of their nature with no prior modification (peptides, protein, polymer, lipids, and sugars). It also detects additional phosphorylation sites of already existing phosphopeptide substrates by enzymes such as GSK-3 and CK1, and monitors the activity of kinases phosphorylating their substrates on multiple sites. Since the linear range of ATP is extended to 500 µM, it is feasible to screen libraries for compounds that are not only competitive with ATP but also for those that are non-competitive which broaden the selection of inhibitors of both serine/threonine protein kinases as well as tyrosine protein kinases. The assay is robust as indicated by the high Z’ values (more than 0.8), homogenous, can be completed in one step after completion of kinase reaction, 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 assay format and to screen for ATP and non-ATP competitive inhibitors.