section 3.4
Capillary Electrophoresis
41
FIG U R E 3-5
Separation of proteins by isoelectric focusing, (a) Proteins with different
isoelectric points are mixed with an appropriate ampholyte solution, (b)
When an electric potential is applied, the ampholytes migrate to their pi
values establishing a pH gradient, while the proteins migrate to the
positions of their respective isoelectric points.
intrinsic charge of the amino acid residues. SDS-protein
complexes, all of which are rod-shaped, migrate toward the
anode rapidly if they are small, but slowly if they are large.
The polyacrylamide medium retards migration according
to the size of the rigid rods. The molecular weight of a
protein is established by comparing its rate of migration
with those of a number of proteins of known molecular
weight.
3.4 Capillary Electrophoresis
Capillary electrophoresis is a technique that can be used
to analyze and separate proteins. It has a high resolving
power that exceeds other electrophoretic techniques and
is capable of distinguishing between proteins that differ
only slightly in amino acid composition or glycosylation.
Capillary electrophoresis is similar to high-performance
liquid chromatography (HPLC) in high level of resolution,
speed, on-line detection, and automation. However, it
functions like slab gel electrophoresis in which proteins
are separated based on mobility differences in an electric
field.
In capillary electrophoresis, protein samples are first in-
jected onto a fused silica microcolumn. The diameter of the
fused silica microcolumns ranges between 50 and 100 /xm.
The free silanol groups of the fused silica become ionized
at pH values greater than 2, causing the inside surface of
the column to be negatively charged; the charge density is
pH dependent. The micro-column sustains electric fields
that are at least 50-fold more powerful than those used in
slab gel electrophoresis. As a result of the high-intensity
electric field, separation of proteins is more readily accom-
plished and the increased sensitivity enables microcharac-
terization of proteins. Due to the small size of the column,
nanoliter quantities can be analyzed and separation occurs
within minutes, whereas in slab gels it may take several
hours. As the samples come off the column, proteins are
measured by a specialized detector.
Separation by Solubility
Proteins may also be purified by selective precipitation,
which is usually accomplished by changing the salt con-
centration (ionic strength) of the solution. Many proteins
that are readily soluble in aqueous solutions at low salt
concentration exhibit decreased solubility with increased
salt concentration. Protein precipitation by increasing salt
concentration is known as the
salting-out
phenomenon.
In this process, the salt ions compete with the protein
molecules for interaction with solvent (water) molecules,
such that the affinity between protein molecules increases
as water molecules are “removed” from the surface func-
tional groups of the proteins. Eventually, the decreased
polymer-solvent association results in precipitation of the
protein. Since different proteins have different surface
functional groups, proteins can be differentially precipi-
tated at different salt concentrations. The related
salting-in
phenomenon, which increases the solubility of some pro-
teins by addition of dilute salt solution, is believed to be due
to interactions between salt ions and the charged groups
on protein molecules, which minimize protein-protein
(precipitation) interaction and maximize protein-solvent
(dissolution) interaction.
Ammonium sulfate, (NH
4
)
2
S
0 4
, is the most commonly
used compound for salting out of proteins because it is very
soluble (706 g/L) and has four ionic charges per molecule.
Precipitations are generally performed slowly with cold
solutions to minimize protein denaturation due to the heat
