TECHNICAL DATA SHEET
BIOLUMINESCENCE RESONANCE ENERGY TRANSFER
GFP2--ARRESTIN2 FUSION VECTOR
Following this technical data sheet is a suggested protocol for the V2 Vasopressin/
β-arrestin assay (Appendix A) and a section outlining general requirements to perform
BRET2 assays (Appendix B).
Product: pGFP2--arrestin2 Vector
Catalog number:
6310176
Lot number:
6310176-1F1
Description:
The pGFP2--arrestin2 vector, which expresses the GFP2--arrestin2 fusion
protein, has been designed to serve as the Acceptor moiety in a BRET2 GPCR/arrestin assay. The fusion gene is placed under the control of the CMV promoter
thus assuring a high level of expression in mammalian cells. When HEK-293T cells
co-transfected with V2 Vasopressin Receptor-Rluc(h) (cat. # 6310193) and GFP2-βarrestin2 are stimulated with an agonist such as [Arg8]-Vasopressin (8-AVP), dosedependent BRET2 signals are obtained in the presence of the Rluc coelenterazine
substrate, DeepBlueCTM. The GFP2-β-arrestin2 fusion vector can be used for other
agonist-dependent GPCR stimulation assays.
Amount:
10 g lyophilized plasmid DNA (store lyophilized plasmid at –20°C)
Reconstitution Protocol
Reconstitution:
 Centrifuge briefly to recover contents
 Reconstitute to 1.0 g/l with 10 l of 10 mM Tris-HCl pH 8.0, 1 mM EDTA
Storage conditions:
 Store reconstituted plasmid at -20°C
 After thawing, centrifuge briefly to recover contents
Shelf life:
 1 year from date of receipt under recommended storage conditions
Renilla Luciferase Substrate - DeepBlueCTM
BRET2 requires a modified form of the Rluc coelenterazine substrate, called DeepBlueC (cat.# 6310100F,
6310101M). DeepBlueC has been selected for its ability to confer superior spectral properties to the reaction,
resulting in excellent discrimination of the Rluc and GFP2 signals. Important Note: the use of other
coelenterazine derivatives may seriously hamper BRET2 results.
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pGFP2--arrestin2 Vector Map
Plasmid size:
7482 bp
Antibiotic resistance:
Prokaryotic: Ampicillin (100 g/ml)
Eukaryotic: Hygromycin (concentration is cell type dependent)
Quality Control Data





The identity of the pGFP2--arrestin2 vector has been confirmed by sequence analysis.
Incubation in standard restriction enzyme buffer at 37°C for 16 hours showed no evidence of nuclease
activity as detected by agarose gel electrophoresis.
No RNA and chromosomal DNA were detected in a 1 g sample of plasmid DNA following agarose gel
electrophoresis.
Percent DNA in Superhelical form: > 80%
Purity (A260/A280) at pH 8.0: 1.88
Functional QC Result
HEK-293T cells were co-transfected with either the pGFP2--arrestin2 (pGFP2-β-arr2, lot # 6310176-1F1) and the pV2
Vasopressin Receptor-Rluc(h) (pV2-Rluc(h), cat. # 6310193) vectors or reference plasmids (R&D batches of these same
vectors) and treated with 8-AVP for 20 minutes. DeepBlueC was added and the BRET2 signal was measured using a
FusionTM Universal Microplate Analyzer (Packard BioScience Co.). Results were shown to be within 15% of the
corresponding reference plasmids.
Numbers above the bar graphs represent Signal to Background (S/B) ratio =
BRET2 Signal (+) AVP
BRET2 Signal (-) AVP
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BRET2TM – BIOLUMINESCENCE RESONANCE ENERGY TRANSFER
BRET2 is a proprietary technology from BioSignal Packard. Patents pending.
BRET2, DeepBlueC, and Fusion are trademarks or registered trademarks of Packard BioScience Company or its subsidiaries in the United
States and/or other countries.
Renilla Luciferase is produced and sold under license, U.S. Patent Nos. 5,292,658 & 5,418,155; CA Patent No. 2,105,984; EPO Patent No.
91 908378.2; JP Patent No. 506544/91 exclusively licensed from Chemicon International, Inc.
Cytomegalovirus (CMV) is produced and sold under license, U.S. Patent Nos. 5,168,062 and 5,385,839.
Green Fluorescent Protein (GFP2) is produced and sold under license, U.S. Patent Nos. 6,020,192; 5,968,750 and 5,874,304.
BRET2 vector kits are designed for laboratory use only and should not be made available for resale either in their original form or in any
variation thereof without explicit approval from BioSignal Packard.
LIMITED WARRANTY BioSignal Packard warrants that, at the time of shipment, the products sold by it are free from defects in material and workmanship and
conform to specifications which accompany the product. BioSignal Packard makes no other warranty, express or implied, with respect to the products, including
without limitation any warranty of merchantability or fitness for any particular purpose. Notification of any breach of warranty must be made within 30 days of
receipt unless provided in writing by BioSignal Packard. No claim shall be honored if the customer fails to notify BioSignal Packard within the period specified.
The sole and exclusive remedy of the customer for any liability of BioSignal Packard of any kind, including liability based upon warranty (express or implied
whether contained herein or elsewhere), strict liability, breach of contract or otherwise, is limited, at BioSignal Packard's sole election, to replacement of the goods
or a refund of the invoice price of the goods. BioSignal Packard shall not in any case be liable for special, incidental or consequential damages of any kind.
6310176
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APPENDIX A
Suggested* Protocol for pV2 Vasopressin-Rluc(h) / pGFP2-β-arrestin2 Assay
*Note : This protocol is an example only and should be optimized to study any other GPCR (see
Appendix B). pV2 Vasopressin-Rluc(h) used in this protocol is available from BioSignal
Packard (cat. # 6310193).
Introduction
-arrestin contributes to receptor internalization upon agonist stimulation. It is known to interact with most
agonist-activated G-protein coupled receptors. In this BRET 2 TM assay the agonist-stimulated interaction between
the GFP2-β-arrestin2 (GFP2-β-arr2) and the V2 Vasopressin Receptor-Rluc(h) (V2-Rluc(h)) fusion proteins brings
Rluc and GFP2 into close proximity to give a BRET2 signal in the presence of DeepBlueCTM.
Material
1234567891011121314151617-
100 mm Petri dishes
Sterile culture tubes (13 mL)
Microfuge tubes
Packard 96-well OptiPlateTM microplates (cat. # 6005290)
Packard 96-well black OptiPlateTM microplates (cat. # 6005270)
Dulbecco's Modified Eagle Medium (D-MEM)
Minimum Essential Medium (MEM)
LipofectAMINETM 2000 (Invitrogen Life Technologies cat. # 11668-027)
Fetal Bovine Serum
Glutamine
Trypsin 0.05%
BRET2 buffer: Dulbecco's Phosphate-Buffered Saline (D-PBS, Invitrogen Life Technologies
cat. #14287-080) supplemented with 2 g/mL Aprotinin (which is a protease inhibitor)
AVP (vasopressin-8-L-arginine) agonist
DeepBlueCTM (cat. # 6310100F, 6310101M)
coelenterazine h (note: this is used strictly to observe expression levels of Rluc-fusion constructs. It
should not be used as a substitute for DeepBlueC in BRET2 assays).
pcDNA3.1 (zeo)+ (Invitrogen Life Technologies cat. # V860-20)
FusionTM universal microplate analyzer (Packard BioScience)
Transfection of HEK-293T cells using LipofectAMINE 2000 (3 X 100 mm petri dishes)
1- The day before transfection, trypsinize and plate 3 dishes of HEK-293T cells at 3 X 106 cells per
100-mm dish so that they are 40-50% confluent on the day of transfection. Cells are plated in 10 mL of
normal D-MEM growth medium supplemented with 10% serum, and 2mM glutamine without antibiotics.
2- For each dish to be transfected, mix 40 µL of LipofectAMINE 2000 reagent with 3 mL of MEM medium
without serum and glutamine by vortexing and incubate 5 minutes at room temperature.
3- Add the following preparation of various transfection solutions to each tube in step 2, as per Table 1
below. Vortex thoroughly and incubate at room temperature for 20 minutes.
Tube 1
Tube 2
Tube 3
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Table 1 : Transfection Conditions
1 μg of pV2-Rluc(h) + 40 μg pGFP2-β-arr2
1 μg pV2-Rluc(h) + 40 μg pcDNA3.1
Non-transfected cells
Positive Control
Negative Control
Background
4
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4- Add 7 mL of D-MEM growth medium supplemented with 14.3% serum (10% final), and 2mM glutamine
without antibiotics to the DNA-LipofectAMINE 2000 complexes prepared in step 3 for a total of 10 ml per
tube.
5- Remove the growth medium from the three dishes (step 1) and replace with the DNA-LipofectAMINE
2000 reagent complexes (10 mL) (step 4). Mix gently by rocking each dish back and forth.
6- Incubate the cells at 37°C in a CO2 incubator for 48 h. It is not necessary to remove the medium
containing the DNA-LipofectAMINE 2000 complexes.
BRET2 assay
1- Forty-eight hours post-transfection, estimate transfection efficiency by examining the cells with a
fluorescence microscope using the following filters: excitation 425 nm with 20 nm bandpass, emission
515 nm with
30 nm bandpass. Transfection efficiency should range from 70-90%. Cell confluence should be 100%.
2- Harvest the cells by rinsing with D-PBS followed by the addition of 1mL trypsin (0.05%). Incubate with
trypsin for 5 minutes followed by addition of 10 mL growth medium supplemented with 10% serum, and
2mM glutamine without antibiotics to inactivate the trypsin. Centrifuge at 800 rpm for 5 minutes,
resuspend in 10 mL D-PBS and determine the cell density. Recentrifuge the cells at 800 rpm and
resuspend to a density of 2 x 106 cells/mL in BRET2 buffer.
3- Take a sample to read fluorescence and luminescence in order to estimate expression levels of the
GFP2 and Rluc fusion proteins (as described in the following section), respectively. Incubate the cells at
room temperature in the BRET2 buffer for at least one hour prior to starting the experiment.
4- Transfer 30 µL of suspension (60,000 cells) into a 96-well OptiPlate for each transfection according to
the plate map (Table 2 below).
5- Prepare a 4 M (1 M final) solution of AVP agonist in BRET2 buffer.
a) In a microtube, dilute 4 µL of AVP stock solution (1000 M in H2O) in 996 L of BRET2 buffer to
give a 4 µM solution.
b) Vortex thoroughly.
Add 10 µL of 4 µM AVP agonist or BRET2 buffer alone to the wells according to the plate map (Table 2)
and incubate for 20 minutes at room temperature.
Table 2: Sample Map for BRET2 V2/β-arrestin Assay (not all 96 wells shown)
1
untransfected
pV2-Rluc(h) + pGFP2-arrestin2
pV2-Rluc(h) + pcDNA 3.1
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+ AVP
2
3
4
-AVP
5
6
7
8
9
10
11
12
A
B
C
D
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BRET2TM – BIOLUMINESCENCE RESONANCE ENERGY TRANSFER
6- The luminescence reaction is started by the addition of 10 µL of 25 µM (5 µM final) DeepBlueC substrate
to all wells, prepared as follows:
a) In a microtube, dilute 7.5 µL of DeepBlueC stock solution (1000 M in ethanol stored at –200C)
in 292.5 L of BRET2 buffer to give a 25 µM solution.
b) Vortex thoroughly.
Note: Diluted DeepBlueC is stable for approximately 1 hour at room temperature.
7- Immediately following addition of DeepBlueC, read the plate on a FusionTM universal microplate analyzer
(Packard BioScience Co.) with the following settings: one second per well, PMT: 1100 volts and gain:
100, using the following 410/515 nm filter pairs:
a) Rluc emission: 410 nm bandpass 80 nm (cat. # 3400410)
b) GFP2 emission: 515 nm bandpass 30 nm (cat. # 3401515)
The average counts obtained with non-transfected cells (treated or untreated with agonist) are
subtracted from the reading of transfected cells in each well (with or without agonist, respectively) to
obtain the corrected counts at both wavelengths. Using the corrected counts, the BRET 2 signal is then
determined as the ratio between GFP2 emission (515 nm) and Rluc/DeepBlueC emission (410 nm).
When subtraction of the background counts yields negative values, a BRET 2 ratio of zero is assigned.
BRET2 signal = GFP2 emission (515nm) – GFP2 emission of non-transfected cells (515nm)
Rluc emission (410 nm) – Rluc emission of non-transfected cells (410 nm)
Determination of fluorescence and luminescence levels of cells transfected with GFP2
and Rluc fusion constructs
These experiments are performed independently from the BRET 2 assay in order to estimate expression levels of
the GFP2-β-arrestin2 and V2 Vasopressin Receptor-Rluc(h) fusion proteins. Coelenterazine h is used instead of
DeepBlueC to determine Rluc expression levels since it generates higher counts and a stable signal for more
than 20 minutes after addition. This method provides consistent values that can be used to determine Rluc
expression levels.
1- Fluorescence: Into a black 96-well OptiPlate add 175 L BRET2 buffer + 25 L cells (50 000 cells) in
triplicate. Read on the Fusion at excitation 425/20 nm and emission 515/30 nm for 0.5 s per well at
voltage 1000.
2- Luminescence: Add 125 L of BRET2 buffer to 3 wells of a 96-well OptiPlate followed by 25 L cells
(50 000 cells). Start the reaction by adding 50 L 20 M coelenterazine h (5 M final) prepared as
follows.
a) In a microtube, dilute 20 µL of coelenterazine h stock solution (1000 M in ethanol stored at
-20°C) in 980 L of BRET2 buffer to give a 20 µM solution.
b) Vortex thoroughly.
Incubate the plate in the dark (cover with aluminium foil) at room temperature for 18 minutes and then
read on the Fusion at 410 nm for 1 s per well with a delay of 120 s at voltage setting of 900.
6310176
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Results
The following graphs show results typically obtained with this assay:
Legend
V2-Rluc(h) = V2 Vasopression Receptor-Rluc(h)
fusion protein
GFP2-β-arr2 = GFP2-β-arrestin2 fusion protein
Conclusion: Stimulation of the vasopressin receptor with its agonist can be detected with the BRET 2
-arrestin assay in HEK 293T cells expressing the pGFP2-β-arr2 and pV2-Rluc(h) constructs. A signal
to background ratio (S/B) of 2.5 is obtained between stimulated and unstimulated receptor. No
significant change occurs in cells transfected with the V2-Rluc(h) construct alone.
BRET2 is a proprietary technology from BioSignal Packard. Patents pending.
BRET2, DeepBlueC, OptiPlate and Fusion are trademarks or registered trademarks of Packard BioScience Company or its subsidiaries in
the United States and/or other countries.
LipofectAMINE 2000 and Zeocin are trademarks of Invitrogen Life Technologies.
Renilla Luciferase is produced and sold under license, U.S. Patent Nos. 5,292,658 & 5,418,155; CA Patent No. 2,105,984; EPO Patent No.
91 908378.2; JP Patent No. 506544/91 exclusively licensed from Chemicon International, Inc.
Green Fluorescent Protein (GFP2) is produced and sold under license, U.S. Patent Nos. 6,020,192; 5,968,750 and 5,874,304.
6310176
Rev. D 12/01
7
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Tel (514) 937-1010 • (800) 293-4501 (US/Canada) • Fax (514) 937-0777 • Email: bret2@biosignal.com
BRET2TM – BIOLUMINESCENCE RESONANCE ENERGY TRANSFER
APPENDIX B
BRET2 Assay Requirements
Some Simple Steps for Successful BRET2 Assays
As a first step in the development of a successful BRET2 assay, three questions must be addressed:
a. What fusion construct should be used?
b. Are the fusion constructs functional and are they efficiently expressed upon transfection?
c. What is the optimal DNA ratio of the two fusion constructs when co-transfected to
maximize the BRET2 signal?
1. Creation of expression constructs
Consider creating four vectors for each protein to test the orientation of N- vs. C- terminal fusions.
The orientation of the fusion linkage and the composition of the fusion protein can affect protein
expression and/or activity which can dramatically affect the signal. Both combinations should be
tested as shown in Table A below. Note : This step does not apply to the BRET2 β-arrestin
assay where Rluc is fused to the C-terminal end of the GPCR, or to the biosensor assay.
Table A : GFP2 and Rluc Fusion Construct Combinations
N-terminal GFP2
C-terminal GFP2
N-terminal Rluc
C-terminal Rluc
Fusion Protein
Fusion Protein
Fusion Protein
Fusion Protein
Protein A
Protein B
Protein A
Protein B
Protein A
Protein B
Protein A
Protein B
Protein B
Protein A
Protein B
Protein A
Protein B
Protein A
Protein B
Protein A
Interaction
1
2
3
4
5
6
7
8
2. Verify the functionality and expression of the fusion constructs
The functionality of each fusion construct can be confirmed by testing enzymatic activity (if
available) of the target protein, or by measuring GFP2 fluorescence or Rluc activity. In addition,
because expression of fusion proteins can vary depending on the construct used, it is a good
practice to carry out preliminary transfection experiments to determine how much DNA will be
needed for the BRET2 assays.

Transfect cells with various amounts of DNA encoding the Rluc fusion protein alone and
test for luminescence of this protein in the presence of DeepBlueC. This will indicate that
the fusion construct is expressed and that the Rluc is active in this construct.

Transfect cells with various amounts of DNA encoding the GFP2 fusion protein alone and
test for fluorescence of this protein by exciting at 425 nm and examining the emission
signal at 515 nm. Note: A slightly right-shifted excitation wavelength of 425 nm
relative to the optimal excitation wavelength for GFP2 (410 nm) is used to minimize
the background due to absorption of light by the plastic, and to decrease
autofluorescence and photoisomerization.
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
GFP2 fusion proteins can be examined for appropriate intracellular localization via
microscopy.
3. Determine optimal DNA ratios
The optimal amount of each fusion construct to be used in co-transfection experiments will
depend on factors such as the affinity of the two interacting domains, the levels of endogenous
proteins and the levels at which each construct is expressed. For this reason, a simple 1 to 1
ratio (for example, 5 μg of each construct) may not give the best results. It is therefore a good
practice to carry out a transfection matrix experiment in which cells will be transfected with various
ratios of each construct and used for BRET2 signal measurements. It should be kept in mind that
the best BRET2 signal is obtained when each Rluc fusion protein interacts with a GFP2 fusion
protein. Excessive Rluc will contribute to a decrease in the BRET2 signal and therefore it is
generally more desirable to overexpress GFP2. Table B below is an example of a transfection
matrix. Note : This step does not apply to the biosensor assay.
Table B: Description of a transfection matrix for the BRET2 β-arrestin assay in 100-mm dishes using
LipofectAMINE 2000 as transfection reagent. Numbers in parenthesis show the ratio of
GFP2-β-arrestin2 fusion construct to GPCR-Rluc construct.
GPCR - Rluc
GFP2- arrestin2
(or -arrestin2-GFP2)
0 g
1 g
3 g
10 g
0 g
Condition 1
Condition 6
Condition 11
Condition 16
3 g
Condition 2
Condition 7 (3)
Condition 12 (1)
Condition 17 (0.3)
10 g
Condition 3
Condition 8 (10)
Condition 13 (3.3)
Condition 18 (1)
20 g
Condition 4
Condition 9 (20)
Condition 14 (6.7)
Condition 19 (2)
40 g
Condition 5
Condition 10 (40)
Condition 15 (13.3)
Condition 20 (4)
4. In parallel, confirm the transfection protocol using the GFP2-Rluc(h) positive control vector
(cat. # 6310030) transfected in cells of interest.
5. Consider assessing the use of various cell lines (if possible) to fine-tune the assay.
We recommend HEK293 or HEK293T cells transfected with LipofectAMINETM 2000 for the
-arrestin assay and CHO-K1, HeLa or BHK-21 cells transfected with LipofectAMINE for the
caspase (apoptosis) assay. However, other cell lines could be a better choice for expression of
specific proteins, provided that they can be transfected at high efficiency.
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Detection of BRET2 Chemistry
Instrument
The selection of an instrument capable of reading BRET2 is of great importance. The instrument
must feature sequential dual luminescence measurements: one for the donor (395 nm) and one for
the acceptor (510 nm) for each microplate well. Therefore, donor and acceptor light output must be
taken consecutively or simultaneously before moving to the next microplate well. We found that the
FusionTM universal microplate analyzer provides the right specifications for monitoring BRET 2 assays.
Filters
Packard BioScience Company provides BRET2 optimized filters with center wavelengths at 410 nm
with 80 nm bandpass and 515 nm with 30 nm bandpass for use with the Fusion universal microplate
analyzer.
BRET2 Demo Kit
It is recommended that the BRET2 Demo Kit (cat. # 6310556) be ordered and used to test the
feasibility of performing a BRET2 assay on alternate instruments that meet the above specifications.
BRET2 is a proprietary technology from BioSignal Packard. Patents pending.
BRET2, DeepBlueC, and Fusion are trademarks or registered trademarks of Packard BioScience Company or its subsidiaries in the United
States and/or other countries.
LipofectAMINE 2000 is a trademark of Invitrogen Life Technologies.
Renilla Luciferase is produced and sold under license, U.S. Patent Nos. 5,292,658 & 5,418,155; CA Patent No. 2,105,984; EPO Patent No.
91 908378.2; JP Patent No. 506544/91 exclusively licensed from Chemicon International, Inc.
Green Fluorescent Protein (GFP2) is produced and sold under license, U.S. Patent Nos. 6,020,192; 5,968,750 and 5,874,304.
6310176
Rev. D 12/01
10
BioSignal Packard, 1744 William, Suite 600, Montréal (Québec) Canada H3J 1R4 • http://www.biosignal.com
Tel (514) 937-1010 • (800) 293-4501 (US/Canada) • Fax (514) 937-0777 • Email: bret2@biosignal.com