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

Fred S. Wouters1, Philippe I.H. Bastiaens2

1 Imperial Cancer Research Fund, London, United Kingdom, 2 European Molecular Biology Laboratory, Heidelberg
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 17.1
DOI:  10.1002/0471143030.cb1701s07
Online Posting Date:  May, 2001
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FRET microscopy enables the detection of different biochemical states of proteins in cells. The use of fluorescence in the detection of proteins, by chemical modification, by immunofluorescence, or by genetic encoding of a green fluorescent protein fusion protein, provides more information than just the location of the protein in the cell. The properties of the fluorophore can be exploited to extract information on proteinā€protein interactions. A straightforward, quantitative imaging approach is presented to measure FRET that is based on internal calibration by acceptor photobleaching.

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Table of Contents

  • Basic Protocol 1: 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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Basic Protocol 1: FRET Microscopy of Fixed Cells

  • 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 CO 2‐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 ( appendix 2A)
  • 0.1% (v/v) Triton X‐100 in recipePBS
  • Antibody (e.g., PY72 monoclonal anti‐phosphotyrosine antibody) labeled with Cy3 (see protocol 3)
  • 1% (w/v) bovine serum albumin (BSA, fraction V) in recipePBS
  • 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 mammalian cells ( appendix 3A)
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% CO 2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO 2 to maintain pH 7.4.

Support Protocol 1: Nuclear and Cytosolic Microinjection

  • 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; appendix 3A)
  • 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 3A)

Support Protocol 2: Protein Labeling with Cy3

  • Antibody (PY72 monoclonal anti‐phosphotyrosine antibody)
  • 1 M Tris⋅Cl, pH 8.0 ( appendix 2A)
  • 10 mM and 100 mM Bicine/NaOH, pH 8.0
  • 100 ml citric acid/NaOH, pH 2.8
  • 1 M Bicine/NaOH, pH 9.0
  • 1 M NaCl ( appendix 2A)
  • Labeling buffer: 100 mM Bicine/NaOH (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 ( appendix 3B)
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Literature Cited

Literature Cited
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   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.
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