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Fret donor and acceptor dyes

Manufactured by Thermo Fisher Scientific
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FRET donor and acceptor dyes are fluorescent probes used to measure Förster Resonance Energy Transfer (FRET) in biological systems. These dyes are designed to function as an energy donor and acceptor pair, enabling the detection of molecular interactions and conformational changes.

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2 protocols using fret donor and acceptor dyes

1

Reconstitution of SNARE-Mediated Membrane Fusion

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His6×-tagged syntaxin HT (amino acids 168–288; two cysteines replaced with alanines) was cloned into pET28a, and GST-tagged synaptosomal-associated protein25 (SNAP-25) (amino acids 1–206; four cysteines replaced with alanines) and synaptobrevin-2 (amino acids 1–116; one cysteine was replaced with alanine) were cloned into pGEX-KG. Proteins were expressed in Escherichia coli BL21 Rosetta (DE3) pLysS (Novagene) cells. The detailed purification procedures have been described elsewhere61 (link).
To form liposomes, 1-palmitoyl-2-oleoylsn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero3-(phospho-l-serine) (DOPS), and cholesterol (Chol) were used (Avanti Polar Lipids). DiI and DiD were used as FRET donor and acceptor dyes, respectively (Invitrogen). The molar ratio POPC:DOPS:Chol:DiI (or DiD) was 71:7:20:2 for both t-vesicles (DiI) and v-vesicles (DiD). SNARE proteins were incorporated into the vesicles at a protein/lipid molar ratio of 1:200. The detailed procedures for preparing proteoliposomes were described elsewhere32 (link).
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2

Preparation of Lipid Vesicles for In Vitro Assay

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Synthetic lipid components, including 1-palmitoyl-2-oleoylsn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero3-(phospho-L-serine) (DOPS), cholesterol (Chol), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (PEG-PE), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rhod-PE), were purchased from Avanti Polar Lipids (USA). As indicators in the lipid mixing assay, the lipid analog fluorescent dyes DiI and DiD were used as FRET donor and acceptor dyes, respectively (Invitrogen, USA). Chloroform stock solutions were mixed, and the chloroform was removed under vacuum in a desiccator for 16 h. The molar ratio of POPC:DOPS:PEG-PE was 92:7:1, and 10‐5 mol % Rhod-PE was added for the formation of the supported lipid bilayer (SLB). For an in vitro lipid mixing assay, the molar ratio of POPC:DOPS:Chol:DiI (or DiD) was 71:7:20:2. The lipid mixtures were then rehydrated in 25 mM HEPES buffer with 100 mM KCl (pH 7.4), and ten freeze-and-thaw cycles were performed using liquid nitrogen and a water bath at 37°C. The solution was passed through a mini extruder (Avanti Polar Lipids) ten times with a 100 nm polycarbonate filter (Whatman, UK). Next, extrusion was performed to obtain monodisperse unilamellar vesicles using a mini extruder (Avanti Polar Lipids) with a 100 nm polycarbonate filter (Whatman).
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