The P450-Glo™ CYP1A2 Assay provides a homogeneous, luminescent method for measuring cytochrome P450 CYP1A2 activity. The assay employs luminogenic P450 substrates that are derivatives of beetle luciferin, a substrate for luciferase enzymes. The derivatives are not substrates for luciferase but are converted by P450s to luciferin, which in turn reacts with luciferase to produce light that is directly proportional to the activity of the P450.
This luminescent assay exhibits exquisite sensitivity, low background signals and broad dynamic range. It generates a "glow-type" luminescent signal, produced using derivatized luciferins as P450 substrates and a recombinant stabilized luciferase (Ultra-Glo Luciferase) coupled with a proprietary buffer system. The half-life of the luminescent output is greater than two hours, eliminating the need for luminometers with injectors and allowing batch plate processing. The luminescent format also eliminates the need for time-consuming analyses such as HPLC.
The luciferin-based substrates are readily taken up by cells and rapidly converted into luciferin inside the cell, which reduces the incubation time required (typically 30-60 minutes). Other benefits of the P450-Glo™ CYP2B6 Assay include a formulation that minimizes the incidence of false positives due to inhibition of luciferase by analytes when screening for cytochrome P450 inhibitors. Z´ values greater than 0.8 are achieved in either 96- or 384-well plate formats and confer highly predictive results.
Assays are complete systems that 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 P450-Glo™ CYP1A2 Induction/Inhibition Assay contains a new substrate for cytochrome 1A2 that is very well suited for all applications involving human CYP1A2 and is the best substrate available for cell-based applications. The substrate, Luciferin-1A2, is readily taken up by cells and converted into a luciferin precursor by CYP1A2. The luciferin precursor is rapidly converted into luciferin following the addition of D-cysteine. After addition of luciferin-1A2 to cells, the assay can be completed in approximately one hour. The low background and high signal-to-noise ratio produced using luciferin-1A2 means less starting material is required.
The P450-Glo™ CYP1A2 Screening System
The P450-Glo™ CYP1A2 Screening System provides a complete set of reagents for performing luminescent cytochrome P450 assays. The system includes 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, P450 reductase, and cytochrome b5. The P450-Glo™ Screening System also contains 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.