Promega Corporation

Homogeneous Bioluminescent Proteolysis Assay for Therapeutic Targets Scientific Poster

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Abstract

Martha O'Brien, Mike Scurria*, William Daily*, Rich Moravec, Terry Riss, Dieter Klaubert*, Keith V. Wood and Robert F. Bulleit
Promega Corporation, 2800 Woods Hollow Rd. Madison, WI 53711
*Promega Biosciences Inc. 277 Granada Dr., San Luis Obispo, CA 93401

Rapid and sensitive assays of proteolytic activity are necessary general characterization of proteases and high-throughput screening for protease inhibitors. Fluorescence has been widely used for monitoring protease activity using peptide-conjugated fluorophores as substrates, but fluorogenic substrates can exhibit significant background. As a means for improving assay sensitivity, we developed a new homogeneous, bioluminescent method that combines peptide-conjugated aminoluciferin substrates and a stabilized luciferase. In comparison to the analogous fluorescent assays, the luminescent assays for several protease targets, including caspases, DPPIV, calpain, and proteasome, showed lower background resulting in increased dynamic range and improved detection sensitivity. In addition, the luminescent assays rapidly attained maximum signal and held stable light emission, allowing for assay flexibility. We also demonstrated a lower occurrence of false hits compared to an analogous fluorescent assay in a test screen using a LOPAC library. The reproducible improvements in sensitivity seen for a variety of proteases indicates that, in general, bioluminescent substrates in a homogeneous assay format provide sensitive and rapid protease assays ideal for high-throughput screening.

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  • Part# PS057
  • Printed in USA.

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