This Application Bulletin describes a protocol for the Promega P450-Glo kit using the Agilent Bravo Platform for the measurement of Cytochrome P450 activity. The protocol involves a two-step reaction, in which the P450 enzyme metabolizes a luminogenic substrate (step 1) into a luciferin product in the presence of an NADPH regenerating system. The addition of a luciferin detection reagent (step 2) stops the enzyme reaction and converts the luciferin product into a luminescent signal that is directly proportional to the enzyme activity.
Cytochrome P450 enzymes are critical in drug discovery, and this document outlines a high-throughput assay protocol. The method utilizes the Promega P450-Glo kit with the Agilent Bravo Automated Liquid Handling Platform to measure enzyme activity via a luminescent signal.
The protocol details a two-step reaction process involving substrate metabolism and detection reagent addition. It specifies the required materials, including the Bravo Platform with specific gripper and pipette head configurations, as well as the necessary labware and reagents for processing up to five microplates simultaneously.
The microplate processing time is approximately one hour for a run of five plates.
You must dispense 6.25 pL of the P450/Substrate mixture from reservoir A.
The protocol requires you to incubate the microplate for 10 minutes.