Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence Complementation (BiFC) Analysis

Chang‐Deng Hu1, Asya V. Grinberg1, Tom K. Kerppola1

1 Howard Hughes Medical Institute and University of Michigan Medical School, Ann Arbor, Michigan
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 19.10
DOI:  10.1002/0471140864.ps1910s41
Online Posting Date:  September, 2005
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Abstract

Protein interactions integrate stimuli from different signaling pathways and developmental programs. Bimolecular fluorescence complementation (BiFC) analysis has been developed for visualization of protein interactions in living cells. This approach is based on complementation between two fragments of a fluorescent protein when they are brought together by an interaction between proteins fused to the fragments, and it enables visualization of the subcellular locations of protein interactions in the normal cellular environment. It can be used for the analysis of many protein interactions and does not require information about the structures of the interaction partners. A multicolor BiFC approach has been developed for simultaneous visualization of interactions with multiple alternative partners in the same cell, based on complementation between fragments of engineered fluorescent proteins that produce bimolecular fluorescent complexes with distinct spectral characteristics. This enables comparison of subcellular distributions of different protein complexes in the same cell and allows analysis of competition between mutually exclusive interaction partners.

     
 
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Table of Contents

  • Basic Protocol 1: Visualization of Protein Interactions in Living Cells
  • Support Protocol 1: Quantification of Fluorescence Complementation Efficiency
  • Alternate Protocol 1: Simultaneous Visualization of Multiple Protein Interactions in the Same Cell
  • Alternate Protocol 2: Analysis of the Competition Between Alternative Interaction Partners in Living Cells
  • Quantitation of Multicolor BiFC Analysis
  • Support Protocol 2: Comparison of the Absolute Fluorescence Intensities of Bimolecular Fluorescent Complexes Produced When a Pair of Interaction Partners Is Expressed in the Presence and Absence of a Competitor in Separate Cells
  • Support Protocol 3: Comparison of the Relative Fluorescence Intensities of Bimolecular Fluorescent Complexes Produced When a Protein Is Expressed with Two Alternative Interaction Partners in the Same Cells
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Visualization of Protein Interactions in Living Cells

  Materials
  • Plasmid vectors for expression of proteins of interest (also see Critical Parameter): e.g., pFlag‐CMV2 (Sigma) or pHA‐CMV (Clontech)
  • DNA encoding amino acid residues 1 to 154 of yellow fluorescent protein (YN fragment; also see and Table 19.10.1)
  • DNA encoding amino acid residues 155 to 238 of yellow fluorescent protein (YC fragment; also see and Table 19.10.1)
  • Linkers (see )
  • DNA encoding proteins (interaction partners, wild‐type) of interest
  • DNA encoding mutated, single amino acid–substitution variants of the protein of interest that do not interact with each other (controls)
  • Cells that can be transfected using plasmid DNA (preferably adherent, monolayer cell line)
  • Appropriate culture medium
  • FuGENE 6 (Roche Diagnostics) or other transfection reagent
  • Tissue culture vessels appropriate for experiment: e.g., cluster plates, slide chambers, or glass coverslips
  • Inverted fluorescence microscope equipped with:Sensitive CCD camera20× to 100× objectivesFilters for visualization of YFP (excitation 500 ± 10 nm; emission 535 ± 15 nm)Software for instrument control and data analysis
  • Additional reagents and equipment for expressing proteins in mammalian cells (unit 5.10), mammalian cell culture ( appendix 3C), and immunoblotting (unit 10.10)
    Table 9.0.1   MaterialsSelection of Fluorescent Protein Fragments for BiFC and Multicolor BiFC Analyses

    Fluorescent protein fragments Applications Filters
    YN155+YC155 BiFC analysis YFP
    YN173+YC173 BiFC analysis YFP
    CN155+CC155 BiFC analysis CFP
    YN155+CN155+CC155 Multicolor BiFC analysis CFP YFP

Support Protocol 1: Quantification of Fluorescence Complementation Efficiency

  • Plasmid encoding full‐length CFP (assuming YFP fragments are used in the protocol 1) or other spectrally distinguishable fluorescent protein
  • Filters for visualization of YFP (excitation 500 ± 10 nm; emission 535 ± 15 nm) and CFP (excitation 436 ± 5 nm; emission 470 ± 15 nm) fluorescence

Alternate Protocol 1: Simultaneous Visualization of Multiple Protein Interactions in the Same Cell

  • DNA encoding amino acid residues 155 to 238 of cyan fluorescent protein (also see and Table 19.10.1)
  • DNA encoding several alternative interaction partners
  • DNA encoding amino acid residues 1 to 154 of the cyan and yellow fluorescent proteins (also see and Table 19.10.1)
  • Interference filters for fluorescence microscope designed to minimize cross‐talk between the fluorescence signals from bimolecular fluorescent complexes formed by fragments of different fluorescent proteins (see below)

Alternate Protocol 2: Analysis of the Competition Between Alternative Interaction Partners in Living Cells

  • Constructs prepared in protocol 3, step
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Figures

Videos

Literature Cited

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