Biology Reference
In-Depth Information
30 nm in diameter) coated
with non-dialyzable polyvinylpyrrolidone (PVP). Percoll is non-toxic, chemically inert, easily
adaptable to physiological ionic strength and pH, is of low viscosity and can be adapted to
discontinuous or continuous gradients. Importantly it does not stick to membranes. It is its
role in continuous gradient centrifugation that makes Percoll so attractive for membrane
isolations (for example see [27] ). A continuous Percoll gradient can be simply and quickly
generated by moderate speed centrifugation (200
Percoll
(GE Healthcare [29] ) consists of silica particles (15
e
1,000g for a few minutes).
e
(GE Healthcare) is an uncharged, highly branched polymer formed by the co-
polymerization of sucrose and epichlorohydrin. Due to its many (
Ficoll
OH) groups, it readily
dissolves in water. Like Percoll, Ficoll can be adapted to discontinuous or self-generating
continuous gradients [9] .
More recently a new self-generating density gradient medium, OptiPrep
e
(Axis Shield
[30] ), has significantly impacted the field of membrane purification. OptiPrep is a solution
of 60% iodixanol in water.
Iodixanol ( Figure 12.11 ) is non-toxic to cells, is metabolically inert and has low viscosity
and osmolality. OptiPrep avoids the high viscosity problems associated with sucrose and
Ficoll gradients and the inconvenience of removing Percoll from membrane fractions after
centrifugation. OptiPrep has been successfully applied in the isolation of plasma membranes
and their domains, including lipid rafts.
Continuous density gradient centrifugations are of two basic types, isopycnic (or equilib-
rium) and rate zonal. In isopycnic, centrifugation is stopped when the particle no longer
moves down the density gradient. At this point density of the particle equals density of
the medium and s
0. Sometimes two different particles may reach the same or very similar
final location in the gradient and so cannot be separated after completion of the centrifuga-
tion. However, the particles may have different physical properties (e.g. size, shape) and so
reach the same final location at different rates. In rate zonal, centrifugation is stopped before
the particles reach final equilibrium. At this point, where density of the particles being sepa-
rated is greater than density of the medium, the particles may be separated from one another.
ΒΌ
D. MEMBRANE FRACTIONATION: NON-CENTRIFUGATION
METHODS
Isolation of membrane fractions remains very much an art form and so there appears to be
almost as many specific procedures as there are membranes to be isolated. While most isola-
tion procedures are based on centrifugation, an almost limitless number of clever adaptations
or 'tricks' have been employed for specific applications. Often these 'tricks' are combined
with standard centrifugation methods to enhance final membrane purification. A brief
sample of these techniques is discussed below.
Affinity Chromatography
Affinity chromatography is the most used of the non-centrifugation methods [31,32] . The
most commonly employed applications for the isolation of membranes use lectins or anti-
bodies. Fortunately, lectins and antibodies are among the most commercially available
Search WWH ::




Custom Search