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Cobalt quenching of 1% Texas Red supported membranes formed by vesicle fusion shows that 70% of the Texas Red DHPE is in the top leaflet (data not shown).
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49
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2+ across the lipid bilayer.
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20444365227
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2+ quenching method provides a more direct method to determine lipid asymmetry, though it may not work for all lipids compositions and/or fluorophores. Although we measured the fluorophore distribution in supported membranes for a very limited set of cases, we find that different fluorophores can partition differently, and potentially asymmetrically, between the two leaflets of a supported membrane, which echoes the results of Crane et al. Likewise, we find that the structurally similar Rh-DPPE and Texas Red DHPE molecules seem to preferentially partition into the top (distal) leaflet. We find this asymmetry in a case where we would not necessarily expect asymmetry, i.e. supported membranes formed by vesicle fusion, suggesting that the asymmetry may be driven by interactions between surface charge on the glass support and charge in the fluorescently labeled lipids.
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46 This difference is likely a reflection of differences in the materials (DiI or NBD-PC in egg PC vs Rh-DPPE in POPC) or the exact methods used in preparation of the supported membranes. In our rather limited experience, we find that different fluorophores in different lipid mixtures partition differently.
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2+ across the membrane over the course of a single experiment.
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1 = 1.
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A fluorescence polarization experiment could be used to determine the average orientation of the Alexa 594 fluorophore relative to the surface normal. The surface coverage of fluorophores is on the order of 2 per square micrometer, so attaining sufficient signal-to-noise in such an experiment with our current setup would be extremely challenging.
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