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].

    Allozyme 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).. These phenotypic patterns indicate that the individuals have homozygous genotypes for one of three alleles: s, m, or f, respectively. These genotypes may be designated ADH_ss, ADH_mm, and ADH_ff, respectively. The next three individuals show the patterns expected from the three possible heterozygous genotypes: ADH_sm, ADH_sf, and ADH_mf, each of which has two bands, in lanes 4, 5, & 6, respectively.

      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 : TRN_ss, TRN_mm, and TRN_ff, 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 TRN_sm, TRN_sf, and TRN_mf  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.