Detection of Protein-Protein
Interactions in vivo Using Cyan and
Yellow Fluorescent Proteins
The phenomenon of fluorescence resonance energy
transfer (FRET) describes the transfer of energy from
one fluorophore to another through dipole-dipole
interaction (Forster, 1946, 1948). It is a sensitive
"molecular ruler" that can detect molecular associations
within a range of 100Å (Stryer, 1978). Early work
using FRET to measure biological associations relied
on either fluorescent analogs of biomolecules or fluorescent
antibodies as donors and acceptors. However,
the utility of this technique in detecting biological
associations was limited by the availability of monoclonal
antibodies that recognize epitopes of the receptor
where maximum energy transfer can occur without
disrupting the interactions under examination. This
limitation was overcome with the use of spectral variants
of the green fluorescent protein (GFP) (Tsien,
1998). The use of GFP variants such as CFP (cyan) and
YFP (yellow) in FRET analysis has the additional
advantage of allowing detection of intracellular associations
in living cells. While most of the applications
of FRET have employed fluorescence microscopic
imaging methods, it has recently become feasible to
perform FRET analysis using flow cytometry. For
example, it has been used successfully to determine
the ligand-independent association between different
Fas receptors (Siegel et al.
, 2000). More recently, flow
cytometric FRET analysis has been used to study the
interaction between histone acetylase PCAF and
histone deacetylase HDAC1 and other chromatinbinding
proteins (Kanno et al.
, 2004; Yamagoe et al.
2003), to detect the presence of Bacillus anthracis spores
in a test biological sample (Zahavy et al.
, 2003) and to
detect caspase activation during apoptosis induction
(He et al.
, 2003). Flow cytometry-based FRET analysis
permits the screening of a large number of interactions
within a short time and will be a useful technique in
the screening of molecular associations in the proteomics
era. We will focus our discussion to flow
cytometry-based FRET analysis. For a more detailed
description of fluorescence microscopic FRET analysis,
readers are referred to the following link: http://www.
stke. org/cgi / content / full / OC_sitrans;2000 / 38 / pl1.
II. MATERIALS AND
The FACS Vantage SE flow cytometer is from BD
Bioscience (San Jose, CA). RPMI 1640 without phenol
red (Cat. No. 11835055), penicillin and streptomycin
(Cat. No. 10378016), L
-glutamine (Cat. No. 25030081),
phosphate-bultered saline (PBS, Cat. No. 14190250),
and trypsin versene (Cat. No. 15040066) are from Invitrogen
(Carlsbad, CA). Fetal calf serum (FCS) is from
Biofluids (Cat. No. 200P-500). HEK 293T cells are from
ATCC (Cat. No. CRL-11268). Fugene 6 (Cat. No.
1814443) and propidium iodide (Cat. No. 1348639)
are from Roche. The CFP (Cat. No. 6900-1) and YFP
(Cat. No. 6006-1) parental plasmids are from BD Clontech
(CA). Flowjo software is from Treestar Inc. (San
Carlos, CA). β-Mercaptoethanol (Cat. No. M-6250) and
bovine serum albumin (BSA, Cat. No. A-7906) are from
A. Transfection of FRET Constructs
B. Collection of Data on FACS Vantage SE
1. Setup of Flow Cytometer
- Split HEK 293T cells by washing the cell monolayer
with 10ml PBS and incubating cells at 37°C for 5-10min in 1× trypsin versene (2-5 ml). Seed cells
at 2.5 × 105 cells per well in 1 ml volume of phenol
red-free RPMI 1640 (supplemented with 10% FCS,
100 units/ml of penicillin and streptomycin, 2 mM of L-glutamine, and 54µM β-mercaptoethanol) in a 12-
well culture plate.
- Incubate cells in a CO2 incubator for 16-20h.
Cells should be 70-90% confluent at the time of
- Prepare the DNA mixture by mixing an equal
molar ratio of CFP to YFP plasmids in an Eppendorf
tube. The optimal molar ratio of CFP to YFP plasmids
may be different for different FRET pairs and should
be determined empirically. The total amount of DNA
should be 1 µg per 3 µl of Fugene 6 for transfection in
- Add 100µl of serum-free Dulbecco's Modified
Eagles Medium to an Eppendorf tube. Add 3µl of
Fugene 6 to the serum-free medium. Gently tap the
tube to mix it. Incubate at room temperature for 5 min.
- Add the diluted Fugene 6 solution from step 4 to
the DNA mixture in step 3. Gently tap the tube to mix
well. Incubate the mixture at room temperature for
- Add the DNA/Fugene 6 mixture from step 5 to
the cells dropwise. Gently swirl the plate to distribute
the mixture evenly.
- Incubate the cells in a 37°C CO2 incubator for
24-48 h. It is not necessary to change the medium after
- After 24-48h, aspirate the medium from the
wells. Resuspend the cells in 1 ml PBS supplemented
with 2% BSA. Spin the cells down at 1500rpm in a
Beckman tabletop centrifuge for 5 min at 4°C.
- Decant the supernatant. Resuspend cells in 1 ml
of PBS supplemented with 2% BSA. Keep cells on ice
until they are ready for analysis. Alternatively, cells
can be fixed in 4% paraformaldehyde until they are
ready for analysis.
Analyze cells on a FACS Vantage SE flow cytometer
(equipped with an ILT air-cooled argon laser and a
Spectra Physics Model 2060 krypton laser). Tune the
argon to 514nm for direct excitation of YFP and the
krypton laser to 413 nm for excitation of CFP. Replace
the forward and side scatter filters with the 513/10
bandpass filter. Use a filter with 470/20-nm bandpass
for CFP detection in the P6 channel and filters with
546/10-nm bandpass for YFP and FRET detection in
P3 and P5 channels, respectively. Direct YFP fluorescence
for detection in the P7 channel for interlaser
compensation (see step 2). Use a 505LP dichroic mirror
to separate the CFP and FRET signals in P5 and P6
channels. Adjust the fluidics pressure to 29-30psi.
Perform electronic compensation to remove CFP
emission from the FRET channel (P5-P6). Perform
interlaser compensation between CFP and YFP using
Omnicomp circuitry to remove YFP excitation from the
413-nm laser. Alternatively, compensations can be
performed "off-line" using softwares such as Flowjo
during data analysis.
3. Run and Collect Samples
Collect 50,000 live events per sample. To facilitate
distinction of live and dead cells, 1µg/ml of propidium
iodide can be added to cells prior to collection of
events on the flow cytometer.
4. Analysis of Data
Analyze collected data using Flowjo software.
Control samples transfected with noninteracting FRET
pairs should be used as negative controls to determine
the baseline FRET signal. An example of step-bystep
analysis of the homotypic interaction of the
ectodomain of p60 TNFR-1 using flow cytometric
FRET analysis is given in Fig. 1. TNFR-1 forms ligandindependent
complexes through a domain at the N terminus
of the receptor called the preligand assembly
domain (PLAD) (Chan et al.
, 2000). The cytoplasmic
tail of TNFR-1 is replaced by CFP or YFP. The resulting
fusions are expressed in HEK 293T cells. A positive
interaction between the differentially tagged TNFR-1
ectodomains is observed as the FRET signal increases
over cells expressing only the CFP-tagged receptor
(Fig. 1a). Moreover, when TNF is added to the cells, a
ligand-dependent rearrangement of the complex
results in further increase of the FRET signal (Fig. 1a).
The interaction between different TNFR-1 chains is
specific, as replacing the fluorescence donor or acceptor
with another TNFR-like receptor DR4 abolishes the
FRET signal (Figs. 1b and 1d). However, cells expressing
DR4-CFP and DR4-YFP exhibit a positive FRET
signal (Fig. 1c), demonstrating that flow cytometric
FRET analysis can accurately recapitulate the known
biochemical interactions of these receptors (Chan et al.
|FIGURE 1 HEK 293T cells were transfected using Fugene 6 with (a) p60-CFP and p60-YFP, (b) p60-CFP
and DR4-YFP, (c) DR4-CFP and DR-YFP, or (d) DR4-CFP and p60-YFP. (Top) Live cells were analyzed for
their expression of CFP and YFP. Cells that express both CFP and YFP were gated (rectangular box) and analyzed
for FRET. (Bottom) Histogram overlays of control cells expressing only CFP (dotted lines) or cells
expressing CFP and YFP (solid lines) show that only (a) p60-CFP and p60-YFP or (c) DR4-CFP and DR4-YFP
exhibited FRET. (a) The heavy line shows the increased FRET when the ligand TNF was added to the cells.
In addition to the FACS Vantage SE flow cytometer,
other models of flow cytometers can be used for
flow cytometric FRET analysis if the proper lasers are
installed. For example, the LSRII flow cytometer from
BD Bioscience can be adapted for FRET analysis if
equipped with a violet laser for excitation at 405nm.
Other methods of detecting molecular interactions
using the concept of FRET include fluorescence lifetime
imaging microscopy and microscopic photobleaching
(Bastiaens and Squire, 1999; Miyawaki and
- FRET is sensitive to the distance and orientation
of the fluorescence donor and acceptor (Miyawaki and
Tsien, 2000; Pollok and Heim, 1999; Truong and Ikura,
2001). Moreover, the spacer length between the CFP or
YFP moieties and the protein of interest can also affect
the efficiency of FRET (Chan et al., 2001). Therefore, it
is sometimes necessary to test multiple fusion constructs
to find the optimal fusions for FRET analysis.
- Low transfection efficiency is detrimental in
FRET analysis. For 293T cells, Fugene 6 is the transfection
reagent of choice because of its consistency in
yielding high protein expression. Cell culture conditions
can also be significant in the success of transfections.
Typically, seeding 2.5 × 105 cells on 12-well plates
will yield a 70-80% confluent culture after a 16- to
20-h incubation, which is ideal for high transfection
- To optimize the FRET signal, it is necessary to
have the fluorescence acceptor molecule in slight
excess relative to the fluorescence donor molecule.
Therefore, different ratios of CFP to YFP plasmids
should be tested to determine the optimal ratio that
will yield maximal FRET signals.
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