Visualization of protein interactions in living Caenorhabditis elegans using bimolecular fluorescence complementation analysis.
The bimolecular fluorescence complementation (BiFC) assay is a powerful tool for visualizing and identifying protein interactions in living cells. This assay is based on the principle of protein-fragment complementation, using two nonfluorescent fragments derived from fluorescent proteins. When two fragments are brought together in living cells by tethering each to one of a pair of interacting proteins, fluorescence is restored. Here, we provide a protocol for a Venus-based BiFC assay to visualize protein interactions in the living nematode, Caenorhabditis elegans. We discuss how to design appropriate C. elegans BiFC cloning vectors to enable visualization of protein interactions using either inducible heat shock promoters or native promoters; transform the constructs into worms by microinjection; and analyze and interpret the resulting data. When expression of BiFC fusion proteins is induced by heat shock, the fluorescent signals can be visualized as early as 30 min after induction and last for 24 h in transgenic animals. The entire procedure takes 2-3 weeks to complete.
Shyu
Y John
YJ
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907-2091, USA.
Hiatt
Susan M
SM
Duren
Holli M
HM
Ellis
Ronald E
RE
Kerppola
Tom K
TK
Hu
Chang-Deng
CD
D000818:Animals; D017173:Caenorhabditis elegans / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000378:metabolism*; D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D005816:Genetic Complementation Test / Q000379:methods*; D049452:Green Fluorescent Proteins; D008856:Microscopy, Fluorescence / Q000379:methods*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011485:Protein Binding
D029742:Caenorhabditis elegans Proteins; D049452:Green Fluorescent Proteins