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Abstract for Cell-Based Bioluminescent Assays for Proteasome
Activity
Richard Moravec, Martha O'Brien, Tracy Worzella, Bill Daily*,
Mike Scurria*, Laurent Bernad*, Kay Rashka, Jeri Culp, Sandra Hagen,
Brian McNamara, Alyssa TenHarmsel and Terry Riss
Promega Corporation, 2800 Woods Hollow Rd., Madison, WI 53711
*Promega Biosciences Inc., 277 Granada Dr., San Luis Obispo, CA
93401
We have developed a series of cell-based assays that individually
measure the chymotrypsin-like, trypsin-like or caspase-like
activities of the proteasome. Each assay utilizes a specific
luminogenic proteasome peptide substrate in a buffer optimized for
cell permeabilization, proteasome activity and luciferase activity.
Addition of the reagent directly to cultured cells gently
permeabilizes cellular plasma membranes, allowing substrate and
reagent access to the cytosolic proteasome. Luminescence is
generated in a coupled enzyme format, as proteasome cleavage of the
peptide substrate generates aminoluciferin, which is a substrate for
luciferase. A stable glow-type luminescent signal is obtained 10-15
minutes after addition of reagent. Luminogenic proteasome substrates
enabled development of the "add-mix-measure" assays with adequate
sensitivity to be miniaturized into 96 and 384-well plate formats. |
