Download BRET (Bioluminescence Resonance Energy Transfer) Method

BRET (Bioluminescence Resonance Energy Transfer) Method
By SW Gersting, et al. (2012)
The following protocol focuses on the use of the first generation of BRET (BRET), using Rluc as energy
donor, YFP as energy acceptor, and Coelenterazine 400A as the Rluc-specific substrate. Hence, to study
the interaction of two proteins by BRET, one protein of interest is genetically fused to Rluc (energy
donor) and the other to YFP (energy acceptor). Both proteins are then coexpressed in cells, and the
BRET signal is detected after the addition of coelenterazine 400A. To enable large-scale transfection of
cells at high efficiency, an electroporation system in 96-well format is applied.
Cell Culture
1. To gain a sufficient number of cells, COS7 cells are cultured under monolayer conditions in a
HYPERFlask cell culture vessel. For this, ten million COS7 cells are seeded into one vessel and
cultured in RPMI 1640 medium at 37°C and 5% CO2 for 1 week.
2. On the day of transfection, cells are detached by trypsinization and the cell number is
determined in a conventional Neubauer counting chamber. Usually, a total of 240 million COS7
cells can be harvested at this juncture.
Transfection (Electroporation)
1. For electroporation, the use of a 96-well nucleofection device is described resulting in sufficient
transfection efficiency and providing transfection at adequate throughput.
2. For nucleofection in 96-well format, 2 × 105 COS7 cells, 1 µg of plasmid DNA and 20 µl of 96-well
Nucleofector Solution SE are needed per well.
3. To analyze binary PPI by BRET, cotransfection of BRET expression vectors coding for the two
proteins of interest is performed at a ratio of 3:1 of acceptor (YFP) to donor (Rluc) constructs.
4. Each protein pair is tested in duplicate and two independent experiments are performed.
5. Several control experiments are performed (in triplicate) for every plate. A plasmid coding for an
YFP–Rluc fusion protein serves as a positive control and always gives similar intraassay results
(~1.0). As a device-specific negative control, a construct expressing the Rluc-tagged protein of
interest with an YFP construct in the absence of the protein of interest is cotransfected. The
BRET ratio measured will be used as correction factor (cf) and subtracted from every BRET pair.
Here, the light emission detected in the acceptor channel (535 nm) predominantly results from a
bleed-through of donor emission that is specific for the filter set used. In addition, a background
control with nontransfected cells is included to ensure stable assay conditions.
6. DNA is prepared in a 96-well V-bottom plate (sterile) or conventional PCR-stripes (sterile) with
0.65 µg for the donor construct and 1.95 µg for the acceptor construct (0.25 and 0.75 µg,
respectively, multiplied by 2 for duplicates and an additional dead volume factor of 1.3).
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7. The required total number of cells (2 × 105 COS7 cells multiplied by the respective number of
wells and the dead volume factor of 1.3) is pelleted at 200 × g for 5 minutes at 37°C and
resuspended in the corresponding volume of prewarmed (37°C) electroporation buffer (20 µl
solution multiplied by the respective number of wells and the dead volume factor of 1.3).
8. A volume of 52 µl of the cell suspension (20 µl multiplied by 2 for duplicates and the dead
volume factor of 1.3) is then added to the prepared DNA and mixed by pipetting up and down.
9. The DNA-cell solution mix (20 µl) is then transferred to the wells of the 96-well nucleofection
plate in duplicates for each sample.
10. For electroporation using the nucleofection system, the appropriate program optimized for
COS7 cells (DSMZ ACC 60) is FP-100.
11. After transfection, prewarmed RPMI 1640 without phenol red (80 µl) is added to each well and
mixed thoroughly. A volume of 50 µl of each well is then transferred accordingly into a 96-well
white plate with clear bottom, prepared in advance with prewarmed 150 µl of RPMI media
without phenol red per well. [RPMI 1640 Medium with L-glutamine, with phenol red
(Phenol Red, GoldBio Catalog # P-117), supplemented with 10% fetal bovine serum and 1%
antibiotic–antimycotic solution (corresponding to 100 U/ml penicillin (Penicillin G
Potassium, GoldBio Catalog # P-303), 0.1 mg/ml streptomycin (Streptomycin Sulfate,
GoldBio Catalog # S-150), and 0.25 g/ml amphotericin B (Amphotericin B, GoldBio
Catalog # A-560)].
Detection of BRET Signal
1. BRET signals can be detected 24 hours after transfection.
2. To prepare plates for BRET measurement, aspirate the culture medium (170 µl) and place the
plate into the luminescence plate reader.
3. Coelenterazine 400A solution has to be prepared at least 15 minutes before the measurement.
To prepare coelenterazine 400A solution for the measurement of one plate, 127 µl of
Coelenterazine 400A [Coelenterazine 400A, GoldBio Catalog # C-320] stock (1 mg/ml suspended
in methanol) are added to 1 ml of Renilla luciferase assay buffer to obtain a 300µM solution.
Immediately prior to the start of measurement, a volume of 1.1 ml of the 300µM solution is
diluted with 6.6 ml PBS (equivalent to the total volume for one plate: 70 µl/well × 96 wells + 1
ml dead volume for priming of the injection pump).
4. After washing the injection pump with pure water, the pump is primed with the coelenterazine
400A solution.
5. For BRET measurement, a protocol has to be set up, starting with a sequential injection of 70 ml
of the coelenterazine 400A solution to each well (resulting in a concentration of 30µM),
followed by an incubation time of 2 minutes. Signals are then detected using the dual emission
option at 485 nm (Rluc-signal) and 535 nm (BRET-signal) over 60 seconds with a total of 60
intervals.
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St. Louis, MO
Ph: (314) 890-8778
email: [email protected]
Calculation of BRET Ratios
1. To allow data evaluation, Rluc signals at 485 nm for transfected cells should exceed the interval
of the mean value and the nine-fold standard error of the nontransfected background control.
2. The BRET-ratio is calculated based on the equation: R = (IA/ID) - cf, where R is the BRET ratio, ID is
the intensity of acceptor luminescence emission at 535 nm, IA is the intensity of donor
luminescence emission at 485 nm, and cf is a correction factor (BRETcontrol/Rluccontrol D) with the
control being the cotransfection of donor fusion-proteins with YFP in the absence of the second
protein of interest.
3. As a positive control, the YFP–Rluc fusion protein should result in BRET ratios around 1.0.
4. A positive interaction of two investigated protein pairs is assumed, if at least one out of eight
tested tag combinations results in a BRET ratio above a method-specific threshold of 0.1
Reference
Gersting, Søren W., Amelie S. Lotz-Havla, and Ania C. Muntau. (2012). Bioluminescence Resonance
Energy Transfer: An Emerging Tool for the Detection of Protein–Protein Interaction in Living Cells.
Methods in molecular biology (Clifton, NJ) 815: 253.
Gold Biotechnology
Web: www.goldbio.com
St. Louis, MO
Ph: (314) 890-8778
email: [email protected]