title: Bimolecular Fluorescence Complementation (BiFC) Analysis of Protein-Protein Interactions and Assessment of Subcellular Localization in Live Cells.
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.
authors: Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN, 47907, USA.
authors: Evan P S
authors: EP
authors: Pratt
authors: Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN, 47907, USA.
authors: Jake L
authors: JL
authors: Owens
authors: Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN, 47907, USA.
authors: Gregory H
authors: GH
authors: Hockerman
authors: Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN, 47907, USA. hu1@purdue.edu.
authors: Chang-Deng
authors: CD
authors: Hu
mesh_terms: D051698:Activating Transcription Factor 2 / Q000235:genetics / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D031541:Fluorescence Resonance Energy Transfer; D015870:Gene Expression; D018613:Microscopy, Confocal / Q000706:statistics & numerical data*; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism; D061848:Optical Imaging / Q000379:methods*; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D059629:Signal-To-Noise Ratio; D012984:Software
chemical_list: D051698:Activating Transcription Factor 2; D019913:Nuclear Localization Signals; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D016271:Proto-Oncogene Proteins c-myc
keywords: BiFC; Bimolecular fluorescence complementation; Confocal microscopy; PPI; Protein localization; Protein–protein interaction