Quantifying macromolecular interactions in living cells using FRET two-hybrid assays.
Förster resonance energy transfer (FRET) is a versatile method for analyzing protein-protein interactions within living cells. This protocol describes a nondestructive live-cell FRET assay for robust quantification of relative binding affinities for protein-protein interactions. Unlike other approaches, our method correlates the measured FRET efficiencies to relative concentration of interacting proteins to determine binding isotherms while including collisional FRET corrections. We detail how to assemble and calibrate the equipment using experimental and theoretical procedures. A step-by-step protocol is given for sample preparation, data acquisition and analysis. The method uses relatively inexpensive and widely available equipment and can be performed with minimal training. Implementation of the imaging setup requires up to 1 week, and sample preparation takes ∼1-3 d. An individual FRET experiment, including control measurements, can be completed within 4-6 h, with data analysis requiring an additional 1-3 h.
Center for Integrated Protein Science CIPS-M and Zentrum für Pharmaforschung, Department Pharmazie; DZHK (German Center for Cardiovascular Research), partner site Munich Heart Alliance, Munich, Germany.
Elisabeth S
ES
Butz
Calcium Signals Laboratory, Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Manu
M
Ben-Johny
Calcium Signals Laboratory, Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Michael
M
Shen
Calcium Signals Laboratory, Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Philemon S
PS
Yang
Calcium Signals Laboratory, Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Lingjie
L
Sang
Center for Integrated Protein Science CIPS-M and Zentrum für Pharmaforschung, Department Pharmazie; DZHK (German Center for Cardiovascular Research), partner site Munich Heart Alliance, Munich, Germany.
Martin
M
Biel
Calcium Signals Laboratory, Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
David T
DT
Yue
Center for Integrated Protein Science CIPS-M and Zentrum für Pharmaforschung, Department Pharmazie; DZHK (German Center for Cardiovascular Research), partner site Munich Heart Alliance, Munich, Germany.
Christian
C
Wahl-Schott
D002470:Cell Survival; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D020798:Two-Hybrid System Techniques / Q000295:instrumentation