Measuring protein interactions using Förster resonance energy transfer and fluorescence lifetime imaging microscopy

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

The method of fluorescence lifetime imaging microscopy (FLIM) is a quantitative approach that can be used to detect Förster resonance energy transfer (FRET). The use of FLIM to measure the FRET that results from the interactions between proteins labeled with fluorescent proteins (FPs) inside living cells provides a non-invasive method for mapping interactomes. Here, the use of the phasor plot method to analyze frequency domain (FD) FLIM measurements is described, and measurements obtained from cells producing the 'FRET standard' fusion proteins are used to validate the FLIM system for FRET measurements. The FLIM FRET approach is then used to measure both homologous and heterologous protein-protein interactions (PPI) involving the CCAAT/enhancer-binding protein alpha (C/EBPα). C/EBPα is a transcription factor that controls cell differentiation, and localizes to heterochromatin where it interacts with the heterochromatin protein 1 alpha (HP1α). The FLIM-FRET method is used to quantify the homologous interactions between the FP-labeled basic leucine zipper (BZip) domain of C/EBPα. Then the heterologous interactions between the C/EBPa BZip domain and HP1a are quantified using the FRET-FLIM method. The results demonstrate that the basic region and leucine zipper (BZip) domain of C/EBPα is sufficient for the interaction with HP1α in regions of heterochromatin.

Original languageEnglish
Pages (from-to)200-207
Number of pages8
JournalMethods
Volume66
Issue number2
DOIs
StatePublished - Mar 15 2014

Fingerprint

Optical Imaging
Energy Transfer
Energy transfer
Microscopy
CCAAT-Enhancer-Binding Protein-alpha
Microscopic examination
Fluorescence
Leucine Zippers
Imaging techniques
Proteins
Heterochromatin
Cell Differentiation
Transcription Factors
Fusion reactions
Cells

Keywords

  • Fluorescence lifetime imaging microscopy (FLIM)
  • Fluorescent proteins (FPs)
  • Förster resonance energy transfer (FRET) microscopy
  • Protein-protein interactions (PPI)

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Measuring protein interactions using Förster resonance energy transfer and fluorescence lifetime imaging microscopy. / Day, Richard.

In: Methods, Vol. 66, No. 2, 15.03.2014, p. 200-207.

Research output: Contribution to journalArticle

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