TheProteasome-Glo™ Cell-Based Assaysare homogeneous, luminescent assays that individually measure the chymotrypsin-like, trypsin-like and caspase-like protease activities associated with the proteasome complex in cultured cells. The 26S proteasome is a 2.5MDa multiprotein complex found in all eukaryotic cells. Proteasome-Glo™ Cell-Based Assays provide luminogenic proteasome substrates in buffers optimized for cell permeabilization, proteasome activity and luciferase activity. Addition of the Proteasome-Glo™ Cell-Based Reagent in an "add-mix-measure" format results in proteasome cleavage of the substrate and rapid generation of a luminescent signal produced by the luciferase reaction.
The three luminogenic substrates used to monitor specific protease activities include: Suc-LLVY-aminoluciferin for chymotrypsin-like, Z-LRR-aminoluciferin for trypsin-like, and Z-nLPnLD-aminoluciferin for caspase-like activity. Each luminogenic substrate is added to a buffer system optimized for its specific proteasome activity and luciferase activity. The reagents are added to cells in culture, and the proteasome cleaves the substrates, releasing luciferin, which is consumed by luciferase, producing "glow-type" luminescence correlating to enzyme activity or inhibition.
TheProteasome-Glo™ Cell-Based 3-Substrate Systemconsists of three homogeneous bioluminescent assays that measure the three proteolytic activities associated with the proteasome in a cell-based format (each of these three assays also is available separately).
The Proteasome-Glo™ 3-Substrate Systemconsists of three homogeneous bioluminescent assays in a purified enzyme-based format (each of these three assays also is available separately).
Features - Benefits
More Biologically Relevant Results:Obtain activity data directly from a cellular environment with the Proteasome-Glo™ Cell-Based Assay.
Simplified Method:The "add-mix-measure" protocol minimizes handling steps and makes the assays amenable to automation.
Faster Results:Maximum sensitivity is reached 10–30 minutes after reagent addition.
Greater Sensitivity:The luminescent assay format avoids inherent fluorescent background signals, providing excellent signal-to-background readings. The assays are miniaturizable to 384-well format.
Applications
Measuring the chymotrypsin-like, trypsin-like and caspase-like activities of the proteasome in cultured cells.
Screening of inhibitor libraries and monitoring proteasome-regulated protein degradation in cells.
Notes
Cat.# G8660, G8760, G8860 and G1180 (for each of the 3 systems) are each sufficient for 100 assays at 100μl/assay in 96-well plates or 400 assays at 25μl/assay in 384-well plates. Cat.# G1200 (for each of the 3 systems) is sufficient for 500 assays at 100μl/assay in 96-well plates or 2,000 assays at 25μl/assay in 384-well plates.
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