P-450 substrate
(enzyme specific)
P-450 CYPXXX
Luciferin
Luciferin
Detection Reagent
Oxyluciferin
(step 1)
(step 2)
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 approxi-
mately 1 hour per 5 microplate run
• Throughput can be easily scaled with the
addition of an Agilent BenchCel
Microplate Handling Workstation
Introduction
Cytochrome P450 enzymes are the
largest group of drug-metabolizing
enzymes, and are therefore of critical
importance in the drug discovery
process. Their potential inhibition by
drug candidates is an important part of
ADME/Tox compound profiling. The
demand for this type of assay is high
because of the increasing output from
high-throughput screening (HTS) and
the pressure to frontload as much
metabolic testing as possible during
drug discovery. There are a number of
different approaches for Cytochrome
P450 testing. Here we outline a pro-
tocol for the Promega P450-Glo kit
using the Agilent Bravo Platform. The
Cytochrome P450 enzyme metabolizes
a luminogenic substrate (step 1) into a
luciferin product in the presence of an
NADPH regenerating system. The addi-
tion of a luciferin detection reagent stops
the enzyme reaction and converts the
luciferin product into a luminescent
signal (step 2), directly proportional to
the enzyme activity.
Overview of the two-step reaction involved in measuring Cytochrome P450 activity using a luminescent signal.
The Agilent Bravo Automated Liquid Handling Platform
System Description
The Promega P450-Glo kit is easily adapted
for automation on a Bravo Platform that is
equipped with a microplate gripper and a
384-channel pipette head. The microplate
stacking feature allows multiple plates to be
located at one deck position. The plates can
quickly 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 opti-
mize plate throughput. The plates are
stacked on the Bravo Platform deck for
room-temperature incubation. Agilent
VWorks Automation Control software man-
ages all process and incubation times, to
guarantee reliable and repeatable results.
The Agilent Bravo deck layout with one
Orbital Shaking Station (location 8). One
stack of five microplates (location 5),
three reservoirs (locations 1-3), and
three tipboxes (locations 4-6) are placed
manually on the deck before the
protocol is started.
Materials
Component List
• Agilent Bravo Platform with gripper,
384ST disposable-tip head
• Three reservoirs
• One Orbital Shaking Station
• Agilent VWorks Automation
Control software
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 Workflow
1. Attach tips at location 4.
2. Move microplate A-1 from location 7 to
8 (Shaker).
3. Dispense 6.25 µL P450/Substrate from
reservoir A to microplate A-1.
4. Shake for 20 s.
5. Move microplate A-1 from location 8 to 9.
6. Incubate microplate A-1 for 10 min.
Loop: Repeat step 2 to 6 for
microplates A-2 to A-5.
7. Remove tips at location 4.
8. Restack microplates A-1 through A-5
from location 9 to 7.
9. Press on tips at location 5.
10. Move microplate A-1 from location 7 to
8 (Shaker).
11. Dispense 12.5 µL NADPH from
reservoir B to microplate A-1.
12. Shake for 20 s.
13. Move microplate A-1 from location 8 to 9.
Instrument Layout
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 errors
contained herein or for incidental or consequential
damages in connection with the furnishing,
performance, or use of this material.
Promega and P450-Glo are registered trademarks
of Promega Corporation.
© Agilent Technologies, Inc., 2009
Published in the U.S.A., February 26, 2009
Publication Number 5990-3550EN
14. Incubate microplate A-1 for 10 min.
Loop: Repeat steps 10 to 14 for
microplates A-2 to A-5.
15. Remove tips at location 5.
16. Restack microplates A-1 to A-5 from
location 9 to 7.
17. Press on tips at location 6.
18. Move microplate A-1 from location 7 to
8 (Shaker).
19. Dispense 25 µL Luciferin from
reservoir C to microplate A-1.
20. Shake for 10 s.
21. Move microplate A-1 from location 8 to 9.
22. Incubate microplate A-1 for 20 min.
Loop: Repeat step 18 to 22 for
microplates A-2 to A-5.
23. Remove tips at location 5.
24. Restack microplates A-1 to A-5 from 9
to 7.
Conclusions
The platform provides a reliable and effi-
cient solution for quantifying Cytochrome
P450 activity. Utilizing the plate stacking
capabilities of the Bravo Platform, up to five
plates 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 meet
increasing capacity demands with the
integration of an Agilent BenchCel
Microplate Handling Workstation.