Clinical Biochemistry of Domestic Animals (Sixth Edition) - UMK ...

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Chapter | 5 Proteins, Proteomics, and the Dysproteinemias
1 . Principle of Electrophoresis
The principle for all protein electrophoresis is based on
the movement of charged protein molecules in an electric
field. In original studies in the early 20th century, electrophoresis
was carried out in solution. This “ free electrophoresis
” was subsequently replaced by methods in which the
proteins are separated in the matrix of a support medium in
which the charged proteins and buffer ions are still able to
move. The use of support medium has the benefit of reducing
interfering problems and allows greater reproducibility.
The choice of support can have direct consequences on
the separation obtained during electrophoresis. As well as
the nature of the support media, the migration of proteins
depends on their size and charge, the pH and ionic composition
of the buffer, and the strength of the electric field.
The charge on the protein is dependent on the balance of
acidic and basic amino acids in its primary structure and
varies with pH. Thus, at a neutral pH, protein with a high
proportion of acidic amino acids will have an overall negative
charge, whereas a protein with a preponderance of
basic amino acids will have an overall positive charge.
SPE is usually performed at a basic pH (pH 8.6) so that
most protein will have a negative charge. Molecules with a
negative charge move toward the anode (positive electrode)
when an electric current is passed through the solution.
A side effect of agarose electrophoresis is that electroendosmosis
occurs because of impurities in the agarose, causing
the migration of the more basic ( γ -globulins) proteins
to the cathode (negative electrode).
Interestingly, one of the most recent innovations in
electrophoretic separation has been to revert to electrophoresis
in the absence of support media, but in this case
using a very narrow bore capillary column. This “ capillary
zone electrophoresis ” has the benefit of providing more
rapid and reproducible analyses for individual samples,
but the equipment is more specialized than that for SPE.
These methods have been applied to canine serum protein
fractionation ( Martinez-Subiela et al. , 2002b ) with results
analogous to conventional SPE, though hemolysis and
lipemia were found to cause interference in the β and α
regions, respectively. Specific proteins such as haptoglobin
can also be measured using capillary zone electrophoresis
( Pirlot et al. , 1999 ).
can move relatively freely. The introduction of plastic
backed previously prepared gels meant that the handling,
staining, and quantification of results could be easily
undertaken.
Typically in an SPE run, serum samples are diluted 1:5
in buffer, and 5 μ l are placed close to the center of the gel,
slightly on the cathodal side; after the sample has diffused
into the agarose, a voltage is applied across the agarose and
the proteins are allowed to separate. The mobility of the
proteins is based on a mix of their charge at the pH of the
buffer (usually pH 8.6) and the size of the protein. Albumin
has a high negative charge under these conditions and is
also relatively small, so it is one of the most mobile proteins
in moving toward the anode. At the other extreme the
immunoglobulins (especially γ -globulins) have the least
negative charge and are affected by electroendosmosis and
migrate toward the cathode. The α - and β -globulins have
intermediate mobility between albumin and the γ -globulins.
Agarose SPE allows the subdivision of these groups,
and α 1 , α 2 , β 1 , β 2 , γ 1 , and γ 2 fractions of proteins may
be observed ( Trumel et al. , 1996 ), though this can vary
between samples and between species. Following electrophoresis
the proteins are fixed in the gel and visualized
by staining with a stain such as amido black. Usually 10
samples will be run on one agarose gel with each sample
in a different “ track. ” After staining and clearing the gel,
the proportion of proteins in each fraction can be estimated
by densitometry. In most instruments a computer-generated
printout will provide a graphical representation of the
absorbance readings from the densitometer and will also
calculate the percentage of protein per fraction. This allows
the calculation of the protein content of each fraction based
on the total serum protein concentration. Examples of agarose
SPE and densitometer scans of the major domestic
animal species are illustrated in Figures 5-2 and 5-3 .
2 . Cellulose Acetate and Agarose Electrophoresis
For several decades, cellulose acetate was the method
used in diagnostic laboratories for SPE, but the easier use,
greater reproducibility, and commercial availability of agarose
gels specifically produced for SPE have meant that
use of the latter method is now more common. Agarose
is a polysaccharide-based material derived from seaweed.
When used for electrophoresis at a concentration of 1%
(w/v), the agarose forms a gel through which serum protein
a b c
d e f
FIGURE 5-2 Agarose gel serum protein electrophoresis showing the
separation of normal serum protein from healthy animals. Samples are
from (a) sheep, (b) cow, (c) pig, (d) dog, (e) cat, and (f) horse.