Cytochrome P450 Assay on
the Agilent Bravo Platform
Application Bulletin

Summary• Small footprint liquid handler provides 5
microplate throughput per run• Microplate processing time is approximately 1 hour per 5 microplate run• Throughput can be easily scaled with theaddition of an Agilent BenchCel
Microplate Handling Workstation
The Agilent Bravo Automated Liquid Handling Platform

Introduction
Cytochrome P450 enzymes are thelargest group of drug-metabolizing
enzymes, and are therefore of criticalimportance in the drug discovery
process. Their potential inhibition bydrug candidates is an important part ofADME/Tox compound profiling. The
demand for this type of assay is highbecause of the increasing output fromhigh-throughput screening (HTS) andthe pressure to frontload as muchmetabolic testing as possible during
drug discovery. There are a number ofdifferent approaches for Cytochrome
P450 testing. Here we outline a protocol for the Promega P450-Glo kit
using the Agilent Bravo Platform. TheCytochrome 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 stops
the enzyme reaction and converts theluciferin product into a luminescentsignal (step 2), directly proportional tothe enzyme activity.

System Description
The Promega P450-Glo kit is easily adapted
for automation on a Bravo Platform that is
equipped with a microplate gripper and a384-channel pipette head. The microplatestacking feature allows multiple plates to belocated at one deck position. The plates canquickly be re-stacked to allow for first in –first out processes. The reagents are added

without tip touching, therefore only one tip
box is needed per reagent. Efficient mixing
is generated by the Orbital Shaking Station,
which doubles as a pipetting position to optimize plate throughput. The plates arestacked on the Bravo Platform deck forroom-temperature incubation. Agilent
VWorks Automation Control software manages all process and incubation times, toguarantee reliable and repeatable results.

P-450 CYPXXX
P-450 substrate(enzyme specific)

Luciferin(step 1)

Oxyluciferin(step 2)Detection Reagent

Luciferin

Overview of the two-step reaction involved in measuring Cytochrome P450 activity using a luminescent signal.

Materials

Instrument Layout

Component List
• Agilent Bravo Platform with gripper,
384ST disposable-tip head
• Three reservoirs• One Orbital Shaking Station• Agilent VWorks Automation
Control software

The Agilent Bravo deck layout with one
Orbital Shaking Station (location 8). One
stack of five microplates (location 5),
three reservoirs (locations 1-3), andthree tipboxes (locations 4-6) are placed
manually on the deck before theprotocol is started.

Labware List
• Microplate A (1-5): Greiner 384PS, white,flat-bottom• Tipbox A, B, C: Agilent Tips 384 ST 70 µL
Reagent List
• Reservoir A: Cyp P450/Substrate mix• Reservoir B: NADPH regenerating
system solution• Reservoir C: Luciferin detection reagent

Protocol WorkflowConclusions

1. Attach tips at location 4.

14. Incubate microplate A-1 for 10 min.

2. Move microplate A-1 from location 7 to
8 (Shaker).

Loop: Repeat steps 10 to 14 formicroplates A-2 to A-5.

3. Dispense 6.25 µL P450/Substrate fromreservoir A to microplate A-1.

15. Remove tips at location 5.

4. Shake for 20 s.

16. Restack microplates A-1 to A-5 fromlocation 9 to 7.

5. Move microplate A-1 from location 8 to 9.

17. Press on tips at location 6.

6. Incubate microplate A-1 for 10 min.

18. Move microplate A-1 from location 7 to8 (Shaker).

Loop: Repeat step 2 to 6 formicroplates A-2 to A-5.
7. Remove tips at location 4.

19. Dispense 25 µL Luciferin fromreservoir C to microplate A-1.

8. Restack microplates A-1 through A-5
from location 9 to 7.

20. Shake for 10 s.

9. Press on tips at location 5.

22. Incubate microplate A-1 for 20 min.

10. Move microplate A-1 from location 7 to8 (Shaker).

Loop: Repeat step 18 to 22 formicroplates A-2 to A-5.

11. Dispense 12.5 µL NADPH fromreservoir B to microplate A-1.

23. Remove tips at location 5.

12. Shake for 20 s.
13. Move microplate A-1 from location 8 to 9.

21. Move microplate A-1 from location 8 to 9.

24. Restack microplates A-1 to A-5 from 9to 7.

The platform provides a reliable and efficient solution for quantifying Cytochrome
P450 activity. Utilizing the plate stacking
capabilities of the Bravo Platform, up to fiveplates can be run without user intervention.
The typical throughput for this setup is
about 1 hour for five plates, depending on
exact protocol and liquid handling steps.
This instrument can easily scale to meetincreasing capacity demands with theintegration of an Agilent BenchCel
Microplate Handling Workstation.

www.agilent.com/lifesciences/automation
This item is intended for Research Use Only.
Not for use in diagnostic procedures. Information,
descriptions, and specifications in this publication
are subject to change without notice.
Agilent Technologies shall not be liable for errorscontained herein or for incidental or consequential
damages in connection with the furnishing,performance, or use of this material.
Promega and P450-Glo are registered trademarksof Promega Corporation.
© Agilent Technologies, Inc., 2009
Published in the U.S.A., February 26, 2009
Publication Number 5990-3550EN