Cell Signaling Protease Assays Protease-Glo™ Assay Product Protease-Glo™ Assay Available Separately pGloSensor™-10F Linear Vector For Research Use Only. Not for Use in Diagnostic Procedures. Description: The Protease-Glo™ Assay is a novel method to detect and measure protease activities using a genetically engineered firefly (Photinus pyralis) luciferase and represents one example of the GloSensor™ platform technology. The assay uses a circularly permuted firefly luciferase, the GloSensor™-10F protein, with a protease recognition site as the protease substrate. This assay system allows rapid generation of protease substrates through molecular cloning and coupled transcription/translation cell-free expression, thus enabling the facile evaluation of protease function. Oligonucleotides encoding a protease recognition sequence are designed and cloned into the GloSensor™-10F gene located on a linearized vector. The GloSensor™ protein containing the protease site of interest is then synthesized in a cell-free protein expression system and subsequently used as a protease substrate. Cleavage of the protease recognition sequence leads to activation of the GloSensor™ protein and light emission. The level of luminescence correlates to protease activity. The Protease-Glo™ Assay has the advantage of a bioluminescent readout, which provides easy quantitation, high sensitivity and wide dynamic range. Visit the Protease-Glo™ Assay Design Tool to see how to generate your protease recognition site of interest in the pGloSensor™-10F Linear Vector and express the protein using cell-free translation. Features: • Flexible: Use with P´ requiring proteases. • Avoid Fluorescent Background Problems: Physical and chemical features of luminescence overcome problems due to fluorescence interference. • Greater Sensitivity: Ease and dynamic range of luminescence. • Open Platform System: Create your own recognition substrates. • Interrogate Sequences: Excellent tool to determine optimal protease recognition sequences or effects of amino acid substitutions. • Web Application: Makes proper oligo design fast and easy; simply enter your amino acid sequence of interest. See the Protease-Glo™ Assay Design Tool. Storage Conditions: Store all components at –20°C, except the TNT® SP6 High-Yield Wheat Germ Master Mix, which must be stored at –70°C. Protocol Protease-Glo™ Assay Technical Manual Luminometer Plates Product Luminometer Plates For Research Use Only. Not for Use in Diagnostic Procedures. Description: These plates are White 96-Well Cliniplate, Universal Binding, Flat Bottom, and are multiwell plates recommended for use with the Protease-Glo™ Assay. The plates offer excellent optical, binding precision and are compatible with all common instruments (manufactured by Thermo Fisher Scientific). Features: • Compatible with All Common Instruments: Excellent optical and binding properties. Storage Conditions: Store at room temperature in a cool and dry location. For complete and up-to-date product information visit: www.promega.com/catalog 141 Size Cat.# Price (Fr) 50 plates Z3291 Pls. Enq. Part# TM303 Size Cat.# Price (Fr) 1 each G9451 1385.00 1 µg G9461 796.00 DUB-Glo™ Protease Assay Product DUB-Glo™ Protease Assay (DUB/SENP/NEDP) For Research Use Only. Not for Use in Diagnostic Procedures. Description: The DUB-Glo™ Protease Assay (DUB/SENP/NEDP) is a homogeneous, bioluminescent assay that measures the activity of numerous deconjugating enzymes including deubiquitinating (DUB), deSUMOylating (SENP) and deneddylating (NEDP) proteases. These proteases reverse the protein modification by ubiquitin and ubiquitin-like proteins (Ubl proteins) and thus are integral components in the complex mechanisms of posttranslational protein regulation in eukaryotes. Features: • Greater Sensitivity: The luminescent format provides enough sensitivity to enable use of a simple peptide-based substrate, Z-RLRGGaminoluciferin, for assaying deconjugating proteases. Fluorescence generally requires the use of full-length substrates. • Broad Dynamic Range: The assays are linear over 2–3 logs of deconjugating protease concentrations. • Signal Stability: The coupled-enzyme format results in very stable signal with a half-life >3 hours. Substrate depletion is not a concern as it is when using the full-length substrates, Ub-AMC, SUMO-AMC or Nedd8-AMC. • Fast: Maximum sensitivity is reached in 10–30 minutes after reagent addition because the signal is not dependent on accumulation of cleaved product for sensitivity in the coupled-enzyme format. • Accurate and Robust: The broad linear range and excellent sensitivity readily translate to accurate kinetic analysis of inhibitors. Assays can be scaled to 384-well with suitable Z´ factors. • Greater Flexibility: The Km values for the peptide substrates are much higher than they are for full-length substrates, yet the sensitivity of the luminescent assay allows the assay to be run significantly below Km while still achieving good signal-to-background ratios for extended time periods. A single luminescent substrate concentration can be used for a wide variety of DUB/SENP/NEDP proteases without worrying about substrate depletion or substrate inhibition. • Batch-Processing Capability: The homogeneous coupled-enzyme format results in a continuous signal, providing excellent stability and allowing plates to be read over an extended period of time. Storage Conditions: Store components at –20°C protected from light. Protocol DUB-Glo™ Protease Assay (DUB/SENP/NEDP) Technical Manual Part# TM319 Size Cat.# Price (Fr) 10 ml G6260 535.00 50 ml G6261 1873.00 6 Drug Discovery Pagina 144
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