When and where proteins associate is a central question in many biomolecular studies. Förster resonance energy transfer (FRET) measurements can be used to address this question when the interacting proteins are labeled with appropriate donor and acceptor fluorophores. We describe an improved method to determine FRET efficiency that uses a mode-locked laser, a confocal microscope and a streak camera. We applied this method to study the association of α and β1 subunits of the human cardiac sodium channel. The subunits were tagged with the cyan and yellow variants of the green fluorescent protein (GFP) and expressed in human embryonic kidney (HEK293) cells. Pronounced FRET between the channel subunits in the endoplasmic reticulum (ER) suggested that the subunits associate before they reach the plasma membrane. The described method allows simultaneous measurement of donor and acceptor fluorescence decays and provides an intrinsically validated estimate of FRET efficiency.
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We thank U. Meisel, G. Möhler, U. Simon, G. Watzinger, G. Weiss and R. Wolleschensky (Carl Zeiss Jena GmbH) for their help in adapting the LSM to our needs. We also appreciate the support of U. Denzer and G. Rousseau (Hamamatsu Photonics Germany) for the installation of the streak camera setup. We are grateful to K. Schoknecht, A. Kolchmeier, S. Bernhardt, G. Ditze and A. Hertel for excellent technical assistance.
The authors declare no competing financial interests.
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Biskup, C., Zimmer, T. & Benndorf, K. FRET between cardiac Na+ channel subunits measured with a confocal microscope and a streak camera. Nat Biotechnol 22, 220–224 (2004). https://doi.org/10.1038/nbt935
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