Measurement of molecular interactions in living cells by fluorescence resonance energy transfer between variants of the green fluorescent protein.
Many signal transduction pathways operate through oligomerization of proteins into multi-subunit complexes. Although biochemical assays can identify potential protein-protein interactions, studying these interactions in living cells is more challenging. Fluorescence resonance energy transfer (FRET) has been used as a "spectroscopic ruler" to measure molecular proximity, but these methods have been limited by the need for chemical labeling of target proteins or labeled antibodies. We present methods for examining interactions between target proteins molecularly fused to cyan and yellow variants of the green fluorescent protein (GFP) by FRET in living cells. Flow cytometric and microscope-based methods are described that have been applied to a variety of interacting proteins.
Siegel
R M
RM
Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.
Chan
F K
FK
Zacharias
D A
DA
Swofford
R
R
Holmes
K L
KL
Tsien
R Y
RY
Lenardo
M J
MJ
D002460:Cell Line; D004735:Energy Transfer; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry; D049452:Green Fluorescent Proteins; D006801:Humans; D007668:Kidney / Q000737:chemistry / Q000166:cytology; D008164:Luminescent Proteins / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins