P450-Glo? CYP3A4 Screening System with Luciferin-IPA

• 产品编号：V9920      品牌：promega       原厂货号：V9920
• 产品分类：生化和细胞分析 > 生化分析试剂盒 > CyP450生化筛查试剂盒
• 应用分类：暂定分类 > Cytochrome P450 Activity

其它组分：

描述：

原厂资料：

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 (<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.

Features - Benefits

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/

Applications

Screen drugs and new chemical entities for their capacity to modulate CYP450 activities.

注意事项：

CYP3A4 膜制备物储存于-70℃。反复的冻融循环可使细胞色素P450 活性丧失。将 CYP3A4 膜制备物分装成单次用量的小份( 如：50μl 用于96 个反应)，避免多次冻融。将这些小份的膜制备物存于-70℃。将其它所有组分储存在-20℃或-70℃，避光。重组的萤光素检测试剂可在–20℃最长储存三个月。为了方便实验，重组的萤光素检测试剂可在室温( 约23℃ ) 放置24 个小时，或在4℃放置1 个星期，不会造成活性丧失。所有组分均需避免多次冻融。