Cell Signaling Cell-Based Proteasome-Glo™ Assays Product Proteasome-Glo™ Chymotrypsin-Like CellBased Assay Proteasome-Glo™ Trypsin-Like Cell-Based Assay Proteasome-Glo™ Caspase-Like Cell-Based Assay Proteasome-Glo™ 3-Substrate Cell-Based Assay System For Laboratory Use. Description: The Proteasome-Glo™ Cell-Based Assays are 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. 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 Proteasome-Glo™ 3-Substrate System consists of three homogeneous bioluminescent assays in an enzyme-based format (each of these three assays also is available separately). Features: • 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. Storage Conditions: Store the Proteasome-Glo™ Assay components at –20°C. Protocol Proteasome-Glo™ Chymotrypsin-Like, Trypsin-Like and CaspaseLike Cell-Based Assays Technical Bulletin Part# TB346 Size Cat.# Price (Fr) 10 ml G8660 696.00 5 × 10 ml G8661 2436.00 2 × 50 ml G8662 4142.00 10 ml G8760 696.00 5 × 10 ml G8761 2436.00 10 ml G8860 696.00 5 × 10 ml G8861 2436.00 10 ml G1180 1653.00 50 ml G1200 5786.00 Z-LRR – or or Z-nLPnLD – Proteasome Suc-LLVY + Z-LRR Z-nLPnLD or or Ultra-Glo™ rLuciferase H2 N N S S N ATP, Mg++, O2 COOH Aminoluciferin H Suc-LLVY – N N S S N COOH 6 Light The luminogenic substrates containing the Suc-LLVY, Z-LRR or ZnLPnLD sequence are recognized by the 20S proteasome. Following cleavage by the 20S proteasome, the substrate for luciferase (aminoluciferin) is released, allowing the luciferase reaction to produce light. 1,000 10,000 100,000 100 10 0.00001 0.0001 1 0.001 0.01 20S (µg/ml) Luminescent proteasome assays are more sensitive than fluorescent proteasome assays. 0.1 1 10 Suc-LLVY-Glo™ Substrate Z-LRR-Glo™ Substrate Z-nLPnLD-Glo™ Substrate Suc-LLVY-AMC Suc-LLVY-AMC+SDS Boc-LRR-AMC Ac-nLPnLD-AMC For complete and up-to-date product information visit: www.promega.com/catalog 143 Drug Discovery Signal-to-Noise Ratio 5994MB Pagina 146
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