Bimolecular Fluorescence Complementation (BiFC) Analysis of Protein-Protein Interactions and Assessment of Subcellular Localization in Live Cells

Methods Mol Biol. 2016;1474:153-70. doi: 10.1007/978-1-4939-6352-2_9.

Abstract

Bimolecular fluorescence complementation (BiFC) is a fluorescence imaging technique used to visualize protein-protein interactions (PPIs) in live cells and animals. One unique application of BiFC is to reveal subcellular localization of PPIs. The superior signal-to-noise ratio of BiFC in comparison with fluorescence resonance energy transfer or bioluminescence resonance energy transfer enables its wide applications. Here, we describe how confocal microscopy can be used to detect and quantify PPIs and their subcellular localization. We use basic leucine zipper transcription factor proteins as an example to provide a step-by-step BiFC protocol using a Nikon A1 confocal microscope and NIS-Elements imaging software. The protocol given below can be readily adapted for use with other confocal microscopes or imaging software.

Keywords: BiFC; Bimolecular fluorescence complementation; Confocal microscopy; PPI; Protein localization; Protein–protein interaction.

MeSH terms

  • Activating Transcription Factor 2 / genetics
  • Activating Transcription Factor 2 / metabolism
  • Animals
  • COS Cells
  • Chlorocebus aethiops
  • Fluorescence Resonance Energy Transfer
  • Gene Expression
  • Microscopy, Confocal / statistics & numerical data*
  • Nuclear Localization Signals / genetics
  • Nuclear Localization Signals / metabolism
  • Optical Imaging / methods*
  • Plasmids / chemistry
  • Plasmids / metabolism
  • Protein Binding
  • Protein Interaction Mapping / methods*
  • Proto-Oncogene Proteins c-fos / genetics
  • Proto-Oncogene Proteins c-fos / metabolism
  • Proto-Oncogene Proteins c-jun / genetics
  • Proto-Oncogene Proteins c-jun / metabolism
  • Proto-Oncogene Proteins c-myc / genetics
  • Proto-Oncogene Proteins c-myc / metabolism
  • Signal-To-Noise Ratio
  • Software

Substances

  • Activating Transcription Factor 2
  • Nuclear Localization Signals
  • Proto-Oncogene Proteins c-fos
  • Proto-Oncogene Proteins c-jun
  • Proto-Oncogene Proteins c-myc