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PMID: 17406412 Published · ppublish English Journal Article

Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells.

Nature protocols ·Vol. 1 ·No. 3 ·2006-00-00 ·Pages 1278-86

Kerppola TK

Abstract

Bimolecular fluorescence complementation (BiFC) analysis enables direct visualization of protein interactions in living cells. The BiFC assay is based on the discoveries that two non-fluorescent fragments of a fluorescent protein can form a fluorescent complex and that the association of the fragments can be facilitated when they are fused to two proteins that interact with each other. BiFC must be confirmed by parallel analysis of proteins in which the interaction interface has been mutated. It is not necessary for the interaction partners to juxtapose the fragments within a specific distance of each other because they can associate when they are tethered to a complex with flexible linkers. It is also not necessary for the interaction partners to form a complex with a long half-life or a high occupancy since the fragments can associate in a transient complex and un-associated fusion proteins do not interfere with detection of the complex. Many interactions can be visualized when the fusion proteins are expressed at levels comparable to their endogenous counterparts. The BiFC assay has been used for the visualization of interactions between many types of proteins in different subcellular locations and in different cell types and organisms. It is technically straightforward and can be performed using a regular fluorescence microscope and standard molecular biology and cell culture reagents.

MeSH Terms
Fluorescent Dyes/metabolism Genetic Complementation Test/methods Microscopy, Fluorescence Multiprotein Complexes/analysis,metabolism Spectrometry, Fluorescence/methods
Chemicals
Fluorescent Dyes Multiprotein Complexes
Authors & Affiliations
1 authors, click to expand affiliations / ORCID
Kerppola Tom K
Howard Hughes Medical Institute and Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA. kerppola@umich.edu
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Article Info
Journal
Nature protocols
Abbr.
Nat Protoc
ISSN
1750-2799
DOI
10.1038/nprot.2006.201
Email
kerppola@umich.edu
Published
2006-00-00
Pages
1278-86
Language
English
Region
England
NLM ID
101284307
PMCID
PMC2518326
Subset
IM
Grants
Howard Hughes Medical Institute · United States
External Links
PubMed source DOI: 10.1038/nprot.2006.201 PMC Full Text View journal details
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