The P450-Glo Screening Systems provide a complete set of reagents for performing luminescent cytochrome P450 assays. 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. The membranes are prepared from baculovirus-infected insect cells and contain human cytochrome P450 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 cytochrome P450 substrate with a cytochrome P450 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 cytochrome P450 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), a common solvent used to solubilize chemical compounds, can significantly inhibit the activity of the 3A4 isoform of cytochrome P450, even at low concentrations (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/