Lipid mixing assays were performed as previously described with minor modifications (Lee et al., 2015 (link); Lee et al., 2019 (link)). Briefly, labeled donor proteoliposomes and unlabeled acceptor proteoliposomes were mixed at a molar ratio of 1:5, transferred to a black polystyrene 384-well plate, and incubated at 30°C for 10 min. The reaction was initiated by adding 1 mM GTP and 1 mM Mg2+. NBD fluorescence was measured every minute using a SpectraMax Gemini XPS plate reader (Molecular Devices). After 40 min, β-octygludoside (final concentration 90 mM) was added to determine the total NBD fluorescence in the sample. Fusion was expressed as the percentage of total fluorescence.
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Lipid Mixing Assay for Membrane Fusion
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Corresponding Organization : Gwangju Institute of Science and Technology
Other organizations : Ulsan National Institute of Science and Technology
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Variable analysis
independent variables
- Addition of 1 mM GTP and 1 mM Mg^2+
- NBD fluorescence measured every minute
- Incubation temperature at 30°C
- Incubation time of 10 min before reaction initiation
- Molar ratio of 1:5 for labeled donor proteoliposomes and unlabeled acceptor proteoliposomes
- Addition of β-octygludoside (final concentration 90 mM) to determine total NBD fluorescence in the sample
- Not explicitly mentioned
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