Journal
NATURE PROTOCOLS
Volume 3, Issue 11, Pages 1693-1702Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2008.157
Keywords
-
Categories
Funding
- Purdue Cancer Center [NCI-P30CA23168]
- Indiana Elks Inc.
- Walther Cancer Institute
- National Science Foundation [0420634-MCB]
- American Heart Association [0655570Z]
- NATIONAL CANCER INSTITUTE [P30CA023168] Funding Source: NIH RePORTER
Ask authors/readers for more resources
Studies of protein interactions have increased our understanding and knowledge of biological processes. Assays that utilize fluorescent proteins, such as fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC), have enabled direct visualization of protein interactions in living cells. However, these assays are primarily suitable for a pair of interacting proteins, and methods to visualize and identify multiple protein complexes in vivo are very limited. This protocol describes the recently developed BiFC-FRET assay, which allows visualization of ternary complexes in living cells. We discuss how to design the BiFC-FRET assay on the basis of the validation of BiFC and FRET assays and how to perform transfection experiments for acquisition of fluorescent images for net FRET calculation. We also provide three methods for normalization of the FRET efficiency. The assay employs a two-chromophore and three-filter FRET setup and is applicable to epifluorescence microscopes. The entire protocol takes about 2-3 weeks to complete.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available