Technical Reference #692

692.

Measurement of FRET Efficiency and Ratio of Donor to Acceptor Concentration in Living Cells Huanmian Chen; Henry L. Puhl; 3rd; Srinagesh V. Koushik; Steven S. Vogel; and Stephen R. Ikeda, National Institutes of Health, Biophysical Journal: Biophysical Letters, 91(692), (2006)
Link To Paper

Abstract:
Measurement of fluorescence resonance energy transfer (FRET) efficiency and the relative concentration ofdonor and acceptor fluorophores in living cells using the three-filter cube approach requires the determination of two constants:1) the ratio of

Keywords:
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Materials & Methods:
In the cDNA plasmid constructs used in this study transcription was driven by the human CMV promoter and the start codon was under the optimal context for translational initiation (Kozak 1999). The preparation of constructs encoding monomeric Cerulean and Venus has been previously described (Thaler et al. 2005). Cerulean and Venus are variants of CFP and YFP respectively (Nagai et al. 2002; Rizzo et al. 2004) with photophysical properties superior to CFP and YFP for FRET studies. All Venus and Cerulean constructs used in this study had the A206K mutation to prevent formation of homo– or heterodimers (Zacharias et al. 2002). The constructs pC5V pCVC pVCV and pCTV have been previously described (Thaler et al. 2005). pC5V encodes a Cerulean–Venus fusion protein in which the first methionine in Venus was replaced by the linker SGLRS and the C-terminus flanked by the peptide EFCSRRYRGPGIHRI. pCVC encodes a Cerulean–Venus–Cerulean fusion protein in which the first methionine of Venus was replaced with the linker SGLRS and the first methionine in the second Cerulean replaced with the peptide linker EFCSRR. The second Cerulean was flanked at the C-terminus by the peptide GSTGSR. pVCV was identical to pCVC except that Cerulean was replaced with Venus and vice versa. pCTV encodes a Cerulean- TRAF domain-Venus fusion protein. The linker between Cerulean and the TRAF domain was a dipeptide SG the linker between the TRAF domain and Venus was the peptide SGLRS. The TRAF domain consists of residues 273–501 of the human tumor necrosis factor associating factor 2 (GenBank accession NM_021138; He et al. 2003). Venus was flanked at its C-terminus by the peptide EFCSRRYRGPGIHRI. Note there are a total of 236 residues separating Cerulean and Venus in the CTV. pC32V encodes a Cerulean–Venus fusion protein with the first methionine in Venus replaced with the peptide TSGLETRDIRSENLYFQGPREFPGGTAGPVAT. pC40V and pC50V encode Cerulean–Venus fusion proteins with the first methionine in Venus replace by a 40-residue peptide TSGLETR(P)10RSENLYFQGPREFPGGTAGPVAT and a 50-residue peptide TSGLETR(P)20RSENLYFQGPREFPGGTAGPVAT respectively. Cell culture and transfection HeLa cells (purchased from ATCC Manassas VA) suspended in Minimal Essential Medium containing 10% bovine calf serum were plated onto 35 mm polystyrene tissue culture dishes and stored in a humidified atmosphere containing 5% CO2 in air at 37 °C. HeLa cells were transfected with appropriate cDNA plasmids using Lipofectamine 2000 (Invitrogen Carlsbad CA). Cells were re-plated onto glass-bottom 2 dishes (MatTek Ashland MA) about four hours after transfection. One day after transfection culture medium was replaced with PBS containing 1 mM CaCl2 and MgCl2 before image acquisition. In some experiments as indicated cells were fixed with 4% paraformaldehyde (PFA) in PBS (pH 7.3) before or during the process of image acquisition.

Microscopic Technique
Fluorescence Microscopy, Wide-field

Cell Type(s)
HeLa