Membrane leakage experiments were performed by measuring the leakage of 6-carboxyfluorescein dye from LUVs. Dye-filled LUVs were prepared by hydrating the dry lipid film with the buffer solution containing 6-carboxyfluorescein (80 mM 6-carboxyfluorescein, pH 7.4) according to the procedure described above. To remove nonencapsulated 6-carboxyfluorescein, we placed the solution containing LUVs on a Sephadex G50 gel exclusion column (Sigma-Aldrich, St.Louis, MO) and eluted using the buffer solution. The final concentration of lipids was checked by using the Stewart assay as described elsewhere.64 (link) Membrane damage was quantified by the increase in fluorescence emission intensity of 6-carboxyfluorescein due to its dilution (dequenching) in buffer as a result of the membrane leakage. All dye leakage curves represent the average ± SD calculated from three independent experiments with each run in quadruplicate.
Sephadex g50 gel exclusion column
Manufactured by Merck Group
Sephadex G50 is a gel exclusion column used for size-based separation and purification of molecules. It is composed of cross-linked dextran beads that are porous, allowing smaller molecules to enter the pores while larger molecules are excluded. This size-based separation enables the isolation and fractionation of different molecular weight components from complex mixtures.
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Lab products found in correlation
3 protocols using sephadex g50 gel exclusion column
1
Membrane Leakage Measurement via Fluorescence
2
Measuring Membrane Leakage with Fluorescent Dye
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Membrane leakage experiments
were performed by measuring the leakage of 6-carboxyfluorescein dye
from LUVs. Dye-filled bTLE LUVs were prepared by hydrating the dry
lipid film with the buffer solution containing 6-carboxyfluorescein
(80 mM 6-carboxyfluorescein, pH 7.4) according to the procedure described
above. Non-encapsulated 6-carboxyfluorescein was removed by eluting
the solution containing LUVs through a Sephadex G50 gel exclusion
column (Sigma-Aldrich, St. Louis, MO) using the buffer solution. The
final concentration of lipids was checked by using the Stewart assay
as described elsewhere.87 (link) Membrane damage
was quantified by the increase in fluorescence emission intensity
of 6-carboxyfluorescein due to its dilution (de-quenching) in the
buffer as a result of the membrane leakage. Time traces were recorded
in Corning 96-well non-binding surface plates using a Varioskan (ThermoFisher,
Waltham, MA) plate reader using a λecc of 490 nm
and a λem of 510 nm at 37 °C, shaking the samples
for 10 s before each read. All curves represent the average of three
independent experiments.
were performed by measuring the leakage of 6-carboxyfluorescein dye
from LUVs. Dye-filled bTLE LUVs were prepared by hydrating the dry
lipid film with the buffer solution containing 6-carboxyfluorescein
(80 mM 6-carboxyfluorescein, pH 7.4) according to the procedure described
above. Non-encapsulated 6-carboxyfluorescein was removed by eluting
the solution containing LUVs through a Sephadex G50 gel exclusion
column (Sigma-Aldrich, St. Louis, MO) using the buffer solution. The
final concentration of lipids was checked by using the Stewart assay
as described elsewhere.87 (link) Membrane damage
was quantified by the increase in fluorescence emission intensity
of 6-carboxyfluorescein due to its dilution (de-quenching) in the
buffer as a result of the membrane leakage. Time traces were recorded
in Corning 96-well non-binding surface plates using a Varioskan (ThermoFisher,
Waltham, MA) plate reader using a λecc of 490 nm
and a λem of 510 nm at 37 °C, shaking the samples
for 10 s before each read. All curves represent the average of three
independent experiments.
3
Preparation and Purification of POPC/POPS LUVs
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