Homogeneous, luminescent assays that individually measure the chymotrypsin-like, trypsin-like and caspase-like protease activities associated with the proteasome complex in cultured cells for more biologically relevant results. These assays can also be used to screen inhibitor libraries and monitor proteasome-regulated protein degradation in 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. The 26S proteasome is a 2.5MDa multiprotein complex found in all eukaryotic cells.
The Proteasome-Glo™ Cell-Based 3-Substrate System consists 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 simplified method minimizes handling steps and makes the assays amenable to automation. The luminescent assay format avoids inherent fluorescent background signals, providing excellent signal-to-background readings. The assays are miniaturizable to 384-well format.