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Abstract for Homogeneous High Throughput Luminescent Assay
Technologies to Monitor Protein Kinase Activity at Significantly
High ATP Concentrations
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.
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