It's a basic summary of Electrophoresis to gain basic knowledge about Electrophoresis, how it is done etc.

1. ALLOZYME ELECTROPHORESIS
Introduction-
 Electrophoresis (Gk. Phorein, to bear) means migration of suspended particles
such as protein macromolecules under the influence of an electric field ie.
Separation of a mixture of electrically charged molecules in an electric field.
 Electrophoresis is a bio-chemical technique that enables geneticists to
determine protein phenotypes and their genotypes and to study DNA and
RNA.
 Protein electrophoresis, the migration of protein under the influence of an
electric field, is among the most cost-efficient method of investigating genetic
phenomena at the molecular level.
 Isozymes are functionally similar forms of enzymes including all polymers of
sub-units produced by different gene loci or by different alleles at the same
locus.
 Allozymes, a subset of isozymes which are variants of polypeptides,
representing different allelic alternative of the same gene locus.
or,
Allozymes refers to the electrophoreic expression of allelic protein of a
particular locus.
Application-
 Electrophoresis is used extensively by population geneticists and by those
who study evolution, because it enables them to compare gene frequencies
across both time and space.
 Electrophoresis can also be used to conduct taxonomic work. Species are
traditionally separated by the presence of, absence of, shapes of or counts of

2. phenotypes. Usually gross visible ones such as dorsal fin ray number, presence
or absence of an adipose fin etc. Species can now be differentiated
biochemically and biochemical systematics have revolutionized the study of
taxonomy.
 Electrophoresis can also be used to identify the species.
 Electrophoresis can even be used as a forensics tool to differentiate flesh that
comes from legally harvested animal from that which comes from endangered
species and is thus illegally harvested, marketed.
 Aquaculturists can also use this techniques to identify species.
 Another use for electrophoresis is to detect genetic drift and inbreeding,
out-crossing and dispersal.
 Electrophoresis can also be used to determine the result of polyploidy
experiment.
 Electrophoresis can be used to help manage natural populations and to
assess the survival or reproductive success of different group of fish or the
contribution of stocked fish to local fisheries or local gene pools.
 This technique can also be used to examine protein phenotypes produced by
mt DNA genes to provide independent assessment of evolution, mt DNA can
be used to identify species and sub-species.
 It can be used to differentiate hatchery produced and wild fish.
 It can also be used to study of species boundaries.
 This technique can also be used to study of ecological genetics.
BASIC PRINCIPLES OF ALLOZYME ELECTROPHORESIS:
The principle that govern this technology are rather simple.

3. 1. Proteins which are the phenotypes produced by genes are composed of
amino acids, joined together by covalent peptide bonds to form
polypeptides.
2. All amino acids have the same backbone.
NH2 C COOH
R
3. Radicals(R) is different for every amino acids and the radical is what
distinguishes one amino acid from another. The radical groups import
different charges to the amino acids.
 Five of the twenty common amino acids which make up proteins are charged.
The charges of Lysine, Arginine and Histidine (LAH) are positive, while those
of aspartic acid and glutamic acid are negative. Thus different proteins tend to
have different net electrical charges.
4. Charges size, chains are responsible for the movement of the proteins
through a matrix during electrophoresis.
5. The net charge of each protein varies with pH
. At a low pH
the amino group
become positively charged and at high pH
the carboxyl group become
negatively charged.
6. Electrophoresis uses physical, chemical, property of protein to separate
mixtures of proteins on the basis of charges.
However, the process of allozyme electrophoresis is described below-
A. Gel preparation-
The process of electrophoresis includes a gel (common starch /
polyacrylamide) preparation in which influenced solutions of protein were
separated by passage of a direct electrical current through the gel.
B. Sample collection, storage (-200C) and tissue preparation-
Procedures for the collection and storage of sample for electrophoresis are
extremely important because of the lability on enzyme in vitro. Proper cryogenic
storage will preserve enzyme activity and minimize breakdown. To separate and

4. isolate the protein initially mixtures of proteins were extracted with water (or
buffered aqueous solvents) from tissues such as skeletal muscle, heart, eye, liver
or blood.
Fig. - Schematic diagram of how protein phenotype and genotype are determined
electrophoretically. A. Protein is extracted from tissue; B. proteins from tissue are then placed
in a gel and the gel is placed in a chamber where an electric current is run through the gel to
move the protein; C. The gel is stained to reveal the protein phenotype; D. The phenotype are
expressed as based on the media.
C. Sample loading to the gel-
The water soluble protein mixtures were typically introduced to the gel on
a piece of filter paper that was saturated with the mixture.
D. Running the gel-
A direct current was usually applied for 3-5 hours through the gel. The
actual time was determined by such variables as composition of the
buffer solution used to make the gel, its ionic strength and the thickness of the
gel. Proteins with a positive net electrical charge move towards the negative (or
catodal) pole and negatively charged proteins move towards the positive (or
anodal) pole. The rate of migration was determined by the absolute charge of the

5. protein. A dye solution was added to the sample to mark the progress of
electrophoresis.
E. Gel slicing-
This gel was sliced horizontally into the multiple slab and each slab was
stained for the activity of a specific protein.
F. Staining gels for particular enzymes (eg- LDH, MDH, PGM)-
Specific staining for an enzymes activity, particular isozyme to be
distinguished. One at time in a mixture of hundreds of protein typically found in
a tissue extract, from an individual fish. The final results of the electrophoresis
procedure was band which identify the locations of various forms of a single type
of portion on a gel. The bonding pattern of individual contained information of
that individuals genotype with respect to the locus coding for the particular
protein.
In the diploid condition, each gene is composed of two alleles that help to
produce various protein phenotypes. These phenotypes were expressed as band
on the media. A fish that was homozygous was produced only one band while a
fish that was heterozygous was produced two or more bands depending on the
makeup of inheritance.
G. Gel preservation-
The gel was preserved for further study.
Types of gel-
 Starch gel
 Polyacrylamide gel
 Cellulose acetate gel
 Agarose gel
Selected Enzymes-
LDH MDH AAT PGM ME SOD
PGI ADH G-6 PDH

6. Limitations-
 Taxonomic limit;
 Sampling limitation;
 Null allele and isoloci;
 Other sources of phenotypic variation of isozyme
Conclusion-
Among different method, electrophoresis is one of the definite method by which
we know stock difference between one or more populations. Historically genetic
differentiation has usually been inferred from a comparison of morphological
characters. We hope to provide a basic understanding of the data and the ways,
this information can be used to address certain issues that concern fish biologists.