Biology Reference
In-Depth Information
diameter of 30-50 nm can be created by sonication. A water bath (above the phase
transition temperature) can be used and the lipid suspension sonicated for a pro-
longed period of time (typically on the order of 30-60 min depending on the lipid).
Alternatively, a probe sonicator can be used but care must be taken not to cause the
lipids to foam; this method is more rapid than using a water bath (typically 30 s to
1 min). Probe sonication should be performed in pulses to avoid excess heating of the
lipid sample, which can then lead to lipid hydrolysis. Clarification of the lipid mix-
ture is indicative of SUV formation although the size and distribution will depend on
a number of factors including lipid type, sonication power, and time. Alternatively,
SUVs can be created by extrusion using a filter with a pore size of
30 nm although
this can be challenging to perform for concentrated lipid stocks. However SUVs are
created, they should be used immediately as their high surface tension makes them
unstable leading to liposome fusion.
Our preferred method is to generate LUVs with a diameter of
100 nm via ex-
trusion using a filter with a corresponding pore size. Lipid can be directly extruded
to 100 nm or, more easily, using filters with a larger pore size, for example, 400 nm
first. Clarification of the lipid solution should occur as particles are created smaller
than the wavelength of visible light. Extruded liposomes can be stored at
80
C
for weeks with no apparent fusion. An excellent guide to extrusion is provided by
Avanti Polar Lipids Inc. (
http://www.avantilipids.com
)
. If significant losses of lipid
are observed at this stage, it is advisable to perform a phosphate assay to determine
the concentration of the lipid stock used for reconstitution. To ensure that the lipid
ratio is correct within the final mixture, thin layer chromatography can be used.
Additionally, if required, liposomes can be sized using dynamic light scattering
or a similar technique.
18.1.4
Reconstitution
Reconstitution essentially reinserts the purified GPCR of interest into a lipid bilayer
of defined composition at defined lipid-protein ratios. Ideally, receptor activity is
retained during the process. There are various methods of reconstitution in use;
the most common of which are the dilution and dialysis method or hydrophobic ad-
sorption using Bio-beads SM-2 (Bio-Rad) (
Fig. 18.1
). There are advantages to both
methods, and which one to be used will depend on the nature of the sample and of the
experiment for which the proteoliposomes are required.
Reconstitution by rapid dilution and dialysis can be an effective and reproducible
reconstitution strategy for a relatively robust membrane protein, but the main disadvan-
tage of this method is the time it takes for the process to be complete, as GPCRs are typ-
ically in detergent and will lose activity over time. Nevertheless, dilution and dialysis
can be a relatively gentle means of reconstitution compared to Bio-beads to which both
lipids and proteins can become adsorbed, changing the desired lipid-protein ratio.
Reconstitution using Bio-beads has been more reproducible in our hands (
Harding
et al., 2009
). This is partly because of the ability to control the amount of detergent
removed with each step, due to relatively well-characterized Bio-bead-to-detergent
