I am making preformed lipid vesicles for use in protein reconstitution and after 5-7 x freeze-thaw cycles with N2l to ~ 10-15C, extrusion through a 0.2 um filter x 10, and extrusion through 2 x 80 nm filters, I get quite a number of Poly's by Cryo-EM imaging. Has anyone found a way to reduce the number of Poly's since this is absolutely critical to my experiments?
Buffer is 20 mM HEPES, pH 7.5, 75 mM NaCl
Extrusion temperature is RT
Extruder is a Lipex 10 ml extruder : 10/1.5mL Thermobarrel and Regular Barrel Extruder from Transferra Nanosciences
Depending on your formulation and the payload properties, excess lipids will form additional layers on loaded vesicles, instead of forming independent empty vesicles. So I would suggest conducting a study where you study different Lipid:payload ratios and observing the size over DLS or ideally, cryo-EM.
The usual route would be to lower the concentration by a factor of 10 or 100.
David W Chester
You can experiment with an aqueous solution of ethyl alcohol instead of water to obtain vesicles. With an increase in the alcohol content in water to 0.1 mole fraction, the hydrophobic interaction is enhanced. The addition of urea weakens the hydrophobic interaction.
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