Science Catalog 2012 Life Cell Signaling P450-Glo™ CYP450 Screening Systems Product P450-Glo™ CYP3A4 Screening System with Luciferin-IPA P450-Glo™ CYP3A4 Screening System (Luciferin-PPXE) DMSO-Tolerant Assay P450-Glo™ CYP1A2 Screening System P450-Glo™ CYP2C9 Screening System P450-Glo™ CYP3A4 Screening System P450-Glo™ CYP2C19 Screening System P450-Glo™ CYP2D6 Screening System Available Separately NADPH Regeneration System 1,000 assays V9910 1422.00 1,000 assays V9770 1422.00 1,000 assays V9790 1422.00 1,000 assays V9800 1422.00 1,000 assays V9880 1422.00 1,000 assays V9890 1422.00 1,000 assays V9510 183.00 For Research Use Only. Not for Use in Diagnostic Procedures. Description: The P450-Glo™ Screening Systems provide a complete set of reagents for performing luminescent cytochrome P450 (CYP450) assays. The systems include a membrane preparation containing recombinant human CYP450 enzyme, a luminogenic CYP450 substrate appropriate for the enzyme, an NADPH Regeneration System, reaction buffer, Luciferin Detection Reagent and Luciferin-Free Water. The membranes are prepared from baculovirusinfected insect cells and contain human CYP450 and P450 reductase (and cytochrome b5 for CYP2C9 and CYP3A4). The P450-Glo™ Screening Systems also contain a membrane fraction devoid of cytochrome P450 activity as a negative control. The assays are ideal for testing the effects of drugs and new chemical entities on cytochrome P450 enzyme activities. The cytochrome P450 reaction is performed by incubating a luminogenic CYP450 substrate with a CYP450 enzyme and the NADPH Regeneration System. The luminogenic P450-Glo™ Substrates are derivatives of beetle luciferin ((4S)-4,5-dihydro-2-(6-hydroxybenzothiazolyl)-4-thiazolecarboxylic acid or D-luciferin), a substrate of firefly luciferase. The P450-Glo™ Substrates do not react with luciferase but are converted by cytochrome P450 to luciferin, which in turn reacts with luciferase to produce light. Light is used to monitor CYP450 activity because the amount of light produced is directly proportional to the amount of D-luciferin produced by cytochrome P450. The new P450-Glo™ CYP3A4 Screening System with Luciferin-IPA contains our latest 3A4 substrate and is the most sensitive P450-Glo™ 3A4 substrate available. Luciferin-IPA displays the widest range of inhibition profiles of all the P450-Glo™ CYP3A4 substrates. Dimethyl sulfoxide (DMSO) can inhibit the activity of the 3A4 isoform of CYP450, even at low concentrations (<0.1%). The P450-Glo™ CYP3A4 Screening System (Luciferin-PPXE) DMSO-Tolerant Assay is specifically designed to tolerate DMSO in the 3A4 reaction. The assay exhibits little to no change in the signal-to-background ratio in the presence of 0.2% DMSO as compared to a no-DMSO control. After the cytochrome P450 reaction has been performed, the reconstituted Luciferin Detection Reagent is added. This reagent simultaneously stops the cytochrome P450 reaction and initiates a stable glow-type luminescent signal. The glow-type reaction produces a stable signal and eliminates the need for strictly timed luminescence detection. Protocols are configured for multiwell plate formats but can be easily adapted for single-tube applications. Size Cat.# Price (Fr) 1,000 assays V9920 1422.00 Features: • Complete Systems: The systems include a membrane preparation containing recombinant human cytochrome P450 enzyme, a luminogenic cytochrome P450 substrate appropriate for the enzyme, an NADPH regeneration system, reaction buffer, Luciferin Detection Reagent and Luciferin-Free Water. • Speed: The luminescent format eliminates the need for time-consuming analyses such as HPLC. • Robust: Z´ values greater than 0.8 in either 96- or 384-well plate formats. Highly predictive results. • Luminescent Output: No interference by fluorescent compounds. • Broad Dynamic Range and Low Background: Excellent sensitivity. • Low False-Positive Rate: Use of a proprietary stabilized firefly luciferase and a proprietary luciferase assay formulation minimizes the incidence of false positives due to inhibition of luciferase by analytes when screening for cytochrome P450 inhibitors. • Scalable: Easily scalable to 384-well plate format. • Automate This Assay: Validated automated methods available at: www.promega.com/automethods/ Storage Conditions: Store the CYP1A2, CYP2C9 and CYP3A4 membranes at –70°C. Cytochrome P450 may lose activity with repeated freeze-thaw cycles. Avoid multiple freeze-thaw cycles by dispensing the CYP1A2, CYP2C9 and CYP3A4 membranes into single-use aliquots (e.g., 50μl for 96 reactions). Store aliquots at –70°C. All other components can be stored at –20°C or –70°C and protected from light. The reconstituted Luciferin Detection Reagent can be stored at –20°C for up to 3 months. For convenience, the reconstituted Luciferin Detection Reagent can be stored at room temperature (approximately 23°C) without loss of activity for 24 hours or at 4°C for 1 week. Avoid multiple freeze-thaw cycles of all components. Protocol P450-Glo™ Screening Systems Technical Bulletin Part# TB340 22 For complete and up-to-date product information visit: www.promega.com/catalog Pagina 25
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