Use of protein electrophoresis to detect allozyme variation
Substitution mutations that
result in the replacement of one amino acid by another with a
different electrical charge can result in slight changes to the
overall charges of the protein. These protein variants are
called allozymes because
they are encoded by different alleles at an enzyme
gene locus. [Allozymes should not be confused with isozymes,
which are different forms of the same enzyme encoded at
different gene loci].
variation is detected by means of protein electrophoresis.
Tissue extracts are introduced into a solid support medium (a gel, often starch or
cellulose acetate) at the Origin.
An electrical field is applied: many proteins have a net
negative charge and will thus migrate away from the negative (cathode) end at the top of the gel towards the positive (anode) at the bottom end of the
field. After several hours, the positions of the allozymes may
be detected either directly by staining, or by a coupled
enzymatic reaction that links the enzyme substrate to a coloured
dye. The result is an electropherogram.
In the example
below, an analysis of
the monomeric (single-subunit) enzyme Alcohol
Dehydrogenase (ADH), three distinct bands are seen
in lanes 1, 2, & 3 with slow,
medium, or fast mobilities (measured
from the upper cathodal end)..
patterns indicate that the individuals have homozygous
genotypes for one of three alleles: s,
m, or f, respectively. These genotypes
may be designated ADHss,
ADHmm, and ADHff,
respectively. The next three individuals show the patterns
expected from the three possible heterozygous genotypes:
ADHsf, and ADHmf,
each of which has two bands, in lanes 4, 5, & 6,
In the second example, analysis of the dimeric
(two-subunit) allozyme Transcendentalase (TRN),
three bands are again seen in lanes 1, 2, & 3 with slow, medium,
or fast mobilities,
corresponding to homozygotes
of the alleles s, m, or
f : TRNss,
respectively. As before, the two alleles in each of the
heterozygous genotypes encode alternative protein
subunits, but in forming the dimeric protein
these subunits combine in the ratio 1 : 2 :1
. The heterozygous TRNsm,
therefore produce three-banded phenotypes,
recognizable by the mobilities of the products of the
homozygotes, with an intermediate band with twice the
protein product in the heterozygote.
Be sure to distinguish between the observed allozyme
phenotypes on the gel, and the inferred allele
genotypes in the DNA.