TY - JOUR
T1 - Rational design and evaluation of FRET experiments to measure protein proximities in cells.
AU - Snapp, Erik L.
AU - Hegde, Ramanujan S.
PY - 2006/10
Y1 - 2006/10
N2 - Fluorescence resonance energy transfer (FRET) refers to the nonradiative transfer of energy from one fluorescent molecule (the donor) to another fluorescent molecule (the acceptor). Measurement of FRET between two fluorophore-labeled proteins can be used to infer the subnanometer spatial and temporal characteristics of protein interactions in their native cellular environment. Multiple experimental methods exist for measuring FRET. The method that can be most widely and simply implemented, quantified, and interpreted is the acceptor-photobleaching FRET technique. In this method, the presence of FRET between a donor and acceptor is revealed upon destruction (by photobleaching) of the acceptor. Acceptor photobleaching can be exploited to detect changes in the composition and organization of subunit proteins within a multiprotein complex and to even gain insight into relative stoichiometries of proteins within the complex. In this unit, strategies, tools, and background for designing and interpreting acceptor-photobleaching FRET experiments in cells are described.
AB - Fluorescence resonance energy transfer (FRET) refers to the nonradiative transfer of energy from one fluorescent molecule (the donor) to another fluorescent molecule (the acceptor). Measurement of FRET between two fluorophore-labeled proteins can be used to infer the subnanometer spatial and temporal characteristics of protein interactions in their native cellular environment. Multiple experimental methods exist for measuring FRET. The method that can be most widely and simply implemented, quantified, and interpreted is the acceptor-photobleaching FRET technique. In this method, the presence of FRET between a donor and acceptor is revealed upon destruction (by photobleaching) of the acceptor. Acceptor photobleaching can be exploited to detect changes in the composition and organization of subunit proteins within a multiprotein complex and to even gain insight into relative stoichiometries of proteins within the complex. In this unit, strategies, tools, and background for designing and interpreting acceptor-photobleaching FRET experiments in cells are described.
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U2 - 10.1002/0471143030.cb1709s32
DO - 10.1002/0471143030.cb1709s32
M3 - Article
C2 - 18228480
AN - SCOPUS:43249119207
SN - 1934-2500
VL - Chapter 17
SP - Unit 17.9
JO - Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.]
JF - Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.]
ER -