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Imaging Protein‐Protein Interactions by Fluorescence Resonance Energy Transfer (FRET) Microscopy

Fred S. Wouters¹, Philippe I.H. Bastiaens²

¹Imperial Cancer Research Fund, London, United Kingdom
²European Molecular Biology Laboratory, Heidelberg, Germany

Publication Name:

Current Protocols in Neuroscience

Unit Number:

Unit 5.22

DOI:

10.1002/0471142301.ns0522s34

Online Posting Date:

February, 2006

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Abstract

Detection of specific protein-protein interactions has long been restricted to bulk biochemical methods such as immunoprecipitation and immunoblotting. Even more sensitive methods using general immunofluorescence are limited, and it is difficult to infer protein-protein interactions from the results of these tests. Fluoresence Resonance Energy Transfer (FRET) is a photophysical process that can be exploited to obtain highly sensitive information about such interactions. It can sense the presence of acceptor fluorophores in the vicinity of a donor fluorophore within a separation distance that is the size of a single protein molecule. This unit details FRET microscopy based on release of quenched donor fluorescence after acceptor photobleaching, microinjection of reagents into the nucleus or cytosol, and labeling of antibodies for these procedures.

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Unit Introduction
Basic Protocol: FRET Microscopy of Fixed Cells
Support Protocol 1: Nuclear and Cytosolic Microinjection
Support Protocol 2: Protein Labeling with Cy3
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol: FRET Microscopy of Fixed Cells

Materials

Cells of interest
Plasmid for GFP-tagged protein
Transfection reagent (e.g., Fugene 5 from Boehringer Mannheim, Lipofectin from Life Technologies, Effectene from Qiagen, or Superfect from Qiagen)
Serum-free medium
Low-background fluorescence CO₂-independent medium (Life Technologies, or see recipe)
Phosphate-buffered saline (PBS; see recipe), pH 7.4
4% (w/v) formaldehyde fixative solution (see recipe)
Quench solution: 50 mM Tris×Cl (pH 8.0)/100 mM NaCl
0.1% (v/v) Triton X-100 in PBS
Antibody (e.g., PY72 monoclonal anti-phosphotyrosine antibody) labeled with Cy3 (see Support Protocol 2)
1% (w/v) bovine serum albumin (BSA, fraction V) in PBS
Mowiol mounting medium (see recipe)
6- and 12-well tissue culture plates
Coverslips
Microscope slides
Confocal laser scanning microscope (e.g., Zeiss LSM 510), equipped with argon (488 nm) and He/Ne (543 nm) lasers selected by the HFT 488/543 double dichroic filter, GFP fluorescence selected by the NFT 545 dichroic and BP 505-530 emission filter, and Cy3 fluorescence selected by the LP560 emission filter
Imaging software package (e.g., NIH-image or IPLab Spectrum from Scanalytics)
Additional reagents and equipment for transfection of cells by calcium phosphate (appendix 1C) or lipofection (appendix 1F)

NOTE: All solutions and equipment coming into contact with cells must be sterile, and aseptic technique should be used accordingly.

NOTE: All incubations are performed in a humidified 37°C, 5% CO₂ incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO₂ to maintain pH 7.4.

Support Protocol 1: Nuclear and Cytosolic Microinjection

Materials

Cells of interest, cultured in MatTek glass-bottom 35-mm dishes (MatTek)
DNA (e.g., human EGFR cDNA in the Clontech pEGFP-N1 expression vector)
HPLC-grade water
Millex-GV4 0.22-µm filtration unit
GELloader tips (Eppendorf)
Needles for microinjection (e.g., Femtotip from Eppendorf)
Microinjector (e.g., Eppendorf model 5244)
Micromanipulator (e.g., Eppendorf model 5170)
Inverted microscope with 10× and 40× air objectives
Additional reagents and equipment for preparation of DNA (appendix 1J)

Support Protocol 2: Protein Labeling with Cy3

Materials

Antibody (PY72 monoclonal anti-phosphotyrosine antibody; supplier )
1 M Tris×Cl, pH 8.0 (appendix 2A)
10 mM and 100 mM Bicine, pH 8.0 (adjusted with NaOH)
100 mM citric acid, pH 2.8 (adjusted with NaOH)
1 M Bicine, pH 9.0 (adjusted with NaOH)
1 M NaCl
Labeling buffer: 100 mM Bicine (pH 8.0)/100 mM NaCl
Cy3.29–OSu monofunctional sulfoindocyanine succinimide ester (Amersham Pharmacia Biotech)
Dimethylformamide (DMF) dried by addition of 10 to 20 mesh 3-Å pore diameter molecular sieve dehydrate (Fluka)
1-ml Protein G HiTrap columns (Amersham Pharmacia Biotech)
Centricon YM30 concentrators (Amicon)
Biogel P6DG Econopac prepacked size-exclusion columns (5.5 × 1.5–cm, ~10 ml; Bio-Rad)
1-ml and 10-ml syringes with HPLC Luer-Lok fitted tubing
Additional reagents and equipment for spectrophotometric protein determination (cpmb unit 10.1A) and SDS-PAGE (cpmb unit 10.2A)

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Figures

Figure 5.22.1

(A) Cy3-PY72 photobleaching releases FRET-quenched EGFR-GFP emission. The histogram shows the distribution of calculated FRET efficiencies in the cell. (B) FRET measured by fluorescence lifetime imaging microscopy. The histogram shows the distribution of measured lifetime (nsec) and corresponding calculated FRET efficiencies prior and after photobleaching of the acceptor.

View Image

Literature Cited

Literature Cited
	Adams, S.R., Harootunian, A.T., Buechler, J., Taylor, S.S., and Tsien, R.Y. 1991. Fluorescence ratio imaging of cyclic AMP in single cells. Nature. 349:694-697.
	Bastiaens, P.I.H. and Jovin, T.M. 1996. Microspectroscopic imaging tracks the intracellular processing of a signal-transduction protein: Fluorescent labeled protein kinase C beta I. Proc. Natl. Acad. Sci. U.S.A. 93:8407-8412.
	Bastiaens, P.I.H. and Jovin, T.M. 1998. Fluorescence resonance energy transfer microscopy. In Cell Biology a Laboratory Handbook, Vol. 3 (J.E. Celis, ed.), pp. 136-146. Academic Press, New York.
	Bastiaens, P.I.H. and Squire, A. 1999. Fluorescence lifetime imaging microscopy: Spatial resolution of biochemical processes in the cell. Trends Cell Biol. 9:48-52.
	Bastiaens, P.I.H., Majoul, I.V., Verveer, P.J., Soling, H.D., and Jovin, T.M. 1996. Imaging the intracellular trafficking and state of the AB(5) quaternary structure of cholera-toxin. EMBO J. 15:4246-4253.
	Bonifacino, J.S. and Dell'Angelica, E.S. 1998. Immunoprecipitation. In Current Protocols in Cell Biology (J.S. Bonifacino, M. Dasso, J.B. Hartfort, J. Lippincott-Schwartz, and K.M. Yamada, eds.) pp. 7.2.1-7.2.21. John Wiley & Sons, New York.
	Budelier, K. and Schorr, J. 1998. Purification of DNA by anion-exchange chromatography. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 2.1.11-2.1.18. John Wiley & Sons, New York.
	Clegg, R.M. 1996. Fluorescence resonance energy transfer spectroscopy and microscopy. In Fluorescence Imaging Spectroscopy and Microscopy (X.F. Wang and B. Herman eds.) pp. 179-251. John Wiley & Sons, New York.
	Day, R.N. 1998. Visualization of Pit-1 transcription factor interactions in the living cell nucleus by fluorescence resonance energy transfer microscopy. Mol. Endocrinol. 12:1410-1419.
	Förster, T. 1948. Zwischenmolekulare Energiewanderung und Fluoreszenz. Ann. Phys. 2:55-75.
	Gadella, T.W.J. and Jovin, T.M. 1995. Oligomerization of epidermal growth-factor receptors on A431 cells studied by time-resolved fluorescence imaging microscopy—a stereochemical model for tyrosine kinase receptor activation. J. Cell Biol. 129:1543-1558.
	Gadella, T.W.J., Jovin, T.M., and Clegg, R.M. 1993. Fluorescence lifetime imaging microscopy (FLIM)—spatial resolution of microstructures on the nanosecond time-scale. Biophys. Chem. 48:221-239.
	Gordon, G.W., Berry, G., Huan Liang, X., Levine, B., and Herman, B. 1998. Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys. J. 74:2702-2713.
	Haugland, R.P. 2000. Antibody conjugates for cell biology. In Current Protocols in Cell Biology (J.S. Bonifacino, M. Dasso, J.B. Hartford, J. Lippincott-Schwartz, and K.M. Yamada, eds.) pp. 16.5.1-16.5.22. John Wiley & Sons, New York.
	Lakowicz, J.R. and Berndt, K. 1991. Lifetime-selective fluorescence imaging using an rf phase-sensitive camera. Rev. Sci. Instrum. 62:1727-1734.
	Mahajan, N.P., Linder, K., Berry, G., Gordon, G.W., Heim, R., and Herman, B. 1998. Bcl-2 and bax interactions in mitochondria probed with green fluorescent protein and fluorescence energy transfer. Nature Biotechnol. 16:547-552.
	Matz, M.V., Fradkov, A.F., Labas, Y.A., Savitsky, A.P., Zaraisky, A.G., Markelov, M.L., and Lukyanov, S.A. 1999. Fluorescent proteins from nonbioluminescent Anthozoa species. Nature Biotechnol. 17:969-973.
	Mittman, S., Flaming, D.G., Copenhagen, D.R., and Belgum, J.H. 1987. Bubble pressure measurement of micropipette tip outer diameter. J. Neurosci. Methods 22:161-166.
	Miyawaki, A., Llopis, J., Heim, R., McCaffery, J.M., Adams, J.A., Ikura, M., and Tsien, R.Y. 1997. Fluorescent indicators for Ca²⁺ based on green fluorescent proteins and calmodulin. Nature 388:882-887.
	Ng, T., Squire, A., Hansra, G., Bornancin, F., Prevostel, C., Hanby, A., Harris, W., Barnes, D., Schmidt, S., Mellor, H., Bastiaens, P.I.H., and Parker, P.J. 1999. Imaging protein kinase C alpha activation in cells. Science 283:2085-2089.
	Sefton, B. 1995. Detection of phosphorylation by immunological techniques. In Current Protocols in Protein Science (J.E. Coligan, B.M. Dunn, D.W. Speicher, and P.T. Wingfield, eds.) pp. 13.4.1-13.4.5. John Wiley & Sons, Hoboken, N.J.
	Squire, A. and Bastiaens, P.I.H. 1999. Three dimensional image restoration in fluorescence lifetime imaging microscopy. J. Microsc. 193:36-49.
	Tsien, R.Y. 1998. The green fluorescent protein. Annu. Rev. Biochem. 76:509-538.
	Wilson, K. 1994. Preparation of genomic DNA from bacteria. In Current Protocols in Molecular Biology (F.A. Ausubel R. Brent R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds) pp. 2.4.1-2.4.5. John Wiley & Sons, New York.
	Wouters, F.S. and Bastiaens, P.I.H. 1999. Fluorescence lifetime imaging of receptor tyrosine kinase activity in cells. Curr. Biol. 9:1127-1130.
	Wouters, F.S., Bastiaens, P.I.H., Wirtz, K.W.A., and Jovin, T.M. 1998. FRET microscopy demonstrates molecular association of non-specific lipid transfer protein (nsL-TP) with fatty acid oxidation enzymes in peroxisomes. EMBO J. 17:7179-7189.