Biology Reference
In-Depth Information
are anionic, they are amenable to this technique. Membrane phospholipids are either neutral
(they are zwitterions like PC) or they are anions (full anions like PA, PS, PI, PG, CL or a partial
anion like PE). In addition, gangliosides by definition contain the anionic sugar sialic acid and
so provide negative membrane surface charge. As a result, all membranes have varying
degrees of negative charge density and will bind to anion exchange columns. The higher the
negative charge density, the tighter the membrane binding. As with affinity chromatography,
a cell homogenate is passed over the anion exchange column and washed with water.
Membrane fractions are released from the column (stationary phase) with either increasing
retention time in the presence of a constant anion concentration in the mobile phase (normally
Cl
) or by adding solutions of increasing anion concentrations. The poorest binding membrane
fraction has the lowest negative charge density and is the first to be released. The most negative
membrane fraction is the tightest bound and so is last to be released.
There are now many examples of commercially available anion exchange resins. The
leader in the field is Dow Chemical Company's division Dionex Corporation (Dow-Ion
Exchange). Membrane separations by anion exchange chromatography have serious limita-
tions and do not have the specificity and precision of affinity chromatography.
Derivitized Beads
Magnetic Beads
Since the 1980s magnetic micro- and nano-beads have been used for a wide array of bio-
logical applications
[41]
, including membrane separations. Unmodified magnetic beads
would be of little or no value for these studies. However, magnetic beads have proved to
be readily modified by several of the same molecules known to work so well with affinity
chromatography (e.g. lectins and antibodies). In addition, magnetic beads have assumed
a major role in molecular biology by being modified to bind to specific nucleic acid
sequences, thus allowing separation of various nucleic acids
[42]
and isolation of DNA-
binding proteins.
Separations using magnetic beads have proven to be not only versatile but also extremely
gentle. The rougher techniques of column chromatography and centrifugation can often be
completely avoided. The technique is quick, easy, sensitive, inexpensive, and amenable to
automation.
The leader in the field of magnetic beads is Dynabeads
(Invitogen, Life Technologies
[43]
). Dynabeads are exceptionally uniform spheres (
[43]
) of highly defined composition.
Importantly, they are superparamagnetic, meaning they have magnetic properties when
placed in a magnetic field, but lose these properties completely when removed from the field.
A uniform polymer shell surrounding the superparamagnetic core can be modified through
a variety of bioreactive conjugates (e.g. lectins, antibodies) and separates the inert internal
core from active surface components that protrude into the external aqueous environment.
An appropriately modified Dynabead will attach specifically to a target membrane in
a complex cell homogenate. A magnet placed on the outside of the tube containing the
homogenate will attract the Dynabeads to the tube wall. The remaining, non-attached
membrane fragments can then be simply washed away and discarded. After adding new
media, the external magnet is removed and the desired membrane attached to the Dynabeads
is left free in solution where it can be readily removed from the beads.
