Document Detail


Investigating protein-protein interactions in living cells using fluorescence lifetime imaging microscopy.
MedLine Citation:
PMID:  21886099     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Fluorescence lifetime imaging microscopy (FLIM) is now routinely used for dynamic measurements of signaling events inside living cells, including detection of protein-protein interactions. An understanding of the basic physics of fluorescence lifetime measurements is required to use this technique. In this protocol, we describe both the time-correlated single photon counting and the frequency-domain methods for FLIM data acquisition and analysis. We describe calibration of both FLIM systems, and demonstrate how they are used to measure the quenched donor fluorescence lifetime that results from Förster resonance energy transfer (FRET). We then show how the FLIM-FRET methods are used to detect the dimerization of the transcription factor CCAAT/enhancer binding protein-α in live mouse pituitary cell nuclei. Notably, the factors required for accurate determination and reproducibility of lifetime measurements are described. With either method, the entire protocol including specimen preparation, imaging and data analysis takes ∼2 d.
Authors:
Yuansheng Sun; Richard N Day; Ammasi Periasamy
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2011-08-11
Journal Detail:
Title:  Nature protocols     Volume:  6     ISSN:  1750-2799     ISO Abbreviation:  Nat Protoc     Publication Date:  2011 Sep 
Date Detail:
Created Date:  2011-09-02     Completed Date:  2012-02-09     Revised Date:  2014-09-14    
Medline Journal Info:
Nlm Unique ID:  101284307     Medline TA:  Nat Protoc     Country:  England    
Other Details:
Languages:  eng     Pagination:  1324-40     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Animals
CCAAT-Enhancer-Binding Protein-alpha / chemistry*,  metabolism
Calibration
Dimerization
Fluorescence Resonance Energy Transfer
Mice
Microscopy, Fluorescence / methods*
Pituitary Gland / cytology,  metabolism
Protein Interaction Mapping / methods*
Signal Transduction
Grant Support
ID/Acronym/Agency:
2R01 DK43701/DK/NIDDK NIH HHS; 3R01 DK43701-15S1/DK/NIDDK NIH HHS; P01HL101871/HL/NHLBI NIH HHS; R01 DK043701/DK/NIDDK NIH HHS; R01 DK043701-17/DK/NIDDK NIH HHS; R01 DK043701-18/DK/NIDDK NIH HHS; RR027409/RR/NCRR NIH HHS
Chemical
Reg. No./Substance:
0/CCAAT-Enhancer-Binding Protein-alpha
Comments/Corrections

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