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

Research output: Contribution to journalArticle

23 Scopus citations

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

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)

Fingerprint Dive into the research topics of 'Measuring protein interactions using Förster resonance energy transfer and fluorescence lifetime imaging microscopy'. Together they form a unique fingerprint.

  • Cite this