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

互联网2013-12-31

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Abstract
Table of Contents
Materials
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Literature Cited

Abstract

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 protein?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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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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Materials

Basic Protocol 1: 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 ( 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% 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; 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

Materials

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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Figures

Figure 17.1.1 (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.

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Literature Cited

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

Abstract

Table of Contents

Materials

Basic Protocol 1: FRET Microscopy of Fixed Cells

Support Protocol 1: Nuclear and Cytosolic Microinjection

Support Protocol 2: Protein Labeling with Cy3

Figures

Videos

Literature Cited