Science Catalog 2012 Life Cell Signaling Calpain-Glo™ Protease Assay Product Calpain-Glo™ Protease Assay For Research Use Only. Not for Use in Diagnostic Procedures. Description: The Calpain-Glo™ Protease Assay is a homogeneous, luminescent assay that measures calpain 1 (μ) and 2 (m) activities. Calpains are a family of calcium-activated cysteine proteases involved in cleaving a wide variety of proteins. Calpains modulate the biological activities of their substrates via limited proteolysis. The Calpain-Glo™ Protease Assay provides a succinyl, proluminescent calpain substrate, Suc-LLVY-aminoluciferin, in a buffer system optimized for calpain and luciferase activities. The addition of the calpain reagent in an “add-mix-measure” format results in calpain cleavage of the substrate and rapid development of a “glow-type” luminescent signal produced by the luciferase reaction. The signal is proportional to the amount of calpain activity present. The assay is designed for use with purified enzyme preparations. Features: • Faster Results: The homogeneous, enzyme-coupled format is especially well suited for rapidly autolysed enzymes like calpain; maximum sensitivity is reached in as little as 10 minutes, while the enzyme is fully active. • Simple Protocol: The homogeneous “add-mix-measure” protocol makes the assay easy to automate. • Greater Sensitivity: The assay is up to 1,000 times more sensitive than competitive fluorometric assays. The luminescent assay avoids inherent fluorescent background signals, providing excellent signal-to-background readings. The assay is linear over 4 logs of calpain concentration. Storage Conditions: Store components at –20°C protected from light. Protocol Calpain-Glo™ Protease Assay Technical Bulletin H Suc-LLVY – N Calpain I or II Suc-LLVY +H2N N S N S Luciferase S N COOH Part# TB344 Size Cat.# Price (Fr) 10 ml G8501 519.00 50 ml G8502 1817.00 100,000 10,000 1,000 100 10 0.001 0 1 Suc-LLVY-aminoluciferin; 12 minutes Suc-LLVY-aminoluciferin; 30 minutes Suc-LLVY-aminoluciferin; 60 minutes FRET substrate; 30 minutes Suc-LLVY-AMC; 60 minutes 0.01 0.1 1 10 Calpain I (nM) Sensitivity of the Calpain-Glo™ Protease Assay compared to fluorescent assays. 100 1,000 S N ATP, Mg++, O2 COOH Aminoluciferin Light The proluminescent substrate containing the Suc-LLVY sequence recognized by calpain. Following calpain cleavage, the substrate for luciferase (aminoluciferin) is released, allowing the luciferase reaction to occur and producing light. 144 For complete and up-to-date product information visit: www.promega.com/catalog 5105MA Signal-to-Noise Ratio 5108MA Pagina 147
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