Biology Reference
In-Depth Information
FIGURE 13.16
High pressure extruder (Lipex Extruder).
Courtesy of ATS Engineering Inc
Vesicles by Detergent Dilution
Detergents are essential for membrane studies. They are amphipathic molecules that in
some ways resemble membrane polar lipids but are orders of magnitude more water
soluble. If added to membranes at high enough concentration, they can replace the resi-
dent membrane lipids while maintaining normal protein conformation and activity. Deter-
gents make functional protein isolation and reconstitution into proteoliposomes possible,
but also provide another path to unilamellar vesicle construction. If membrane polar
lipids are hydrated in a buffer containing a large excess of detergent, they become solu-
bilized into detergent micelles. The water-soluble detergents can then be easily removed
by dialysis or by gel filtration. When the detergent/membrane lipid mixture is sealed
in a dialysis tube, the detergent diffuses down its concentration gradient, eventually
crossing the dialysis tubing and entering the external bathing solution. As the detergent
concentration decreases inside the dialysis tube, the membrane polar lipids fall out of
solution as unilamellar lipid vesicles that are too large to fit through the dialysis tubing
pores. These vesicles do not have sequestered lipids and so are unilamellar. Detergents
may also be eliminated rapidly by passing the detergent/membrane lipid mixture through
a gel filtration column. As the mixture passes down the column, the detergent is extracted
into the small spaces in the gel matrix, while the polar membrane lipids come out of solu-
tion as unilamellar vesicles that are excluded from the gel. The newly formed unilamellar
vesicles leave the column before the free detergent and so can be isolated largely devoid
of detergent. Vesicle size is dependent on the detergent used and can be partially altered
