Agriculture Reference
In-Depth Information
Protein electroPhoresis
The DNA marker techniques described above use gel electrophoresis as a tool to visualize DNA fragments
subsequent to digestion and ampliication. In that respect, electrophoresis is only the last of many steps in
DNA-based variety testing. Assessing the degree of variation in protein composition is another molecular
approach to variety testing. This approach relies on electrophoresis as the basic method of protein analysis.
Electrophoresis is a simple, fast, sensitive and broadly applicable method. It separates a mixture of
proteins into distinct bands in a gel as a result of differential migration in an electric ield. The rate of separa-
tion (migration) depends on the size, net charge and shape of the molecules, as well as the ionic strength and
viscosity of the gel. The presence of a particular protein in an organism is under genetic control. Therefore,
samples characterized by different electrophoretic protein bands are considered to differ genetically while
those having the same protein bands may be the same. Protein bands are visualized after electrophoresis by
staining with a general protein stain or a stain for a speciic enzyme. When a protein band reacts positively
with an enzyme stain, the band is characterized as to function as well as size and charge.
Two main electrophoretic techniques have been used in protein-based analysis of varietal differences.
The irst and more common technique is starch gel electrophoresis commonly associated with isozyme
analysis (Fig. 9.2). The second is polyacrylamide gel electrophoresis usually associated with seed protein
analysis. Although these techniques have been superseded by DNA-marker techniques, they still present
rapid, fairly sensitive and inexpensive methods of variety testing. In addition, the “isoelectric focusing”
(IEF) technology is used to differentiate between cultivars that produce different proteins. It is an elec-
trophoresis system which utilizes a pH gradient to separate proteins of different cultivars based on their
isoelectric points, i.e., the pH point at which a particular protein molecule carries no net electrical charge.
At a pH below their isoelectric point (IEP), proteins carry a net positive charge; above their IEP, they carry
a net negative charge. Proteins can accordingly be separated on a gel based on their isoelectric points (Fig.
9.3).
Figure 9.2. An example of an electrophoresis banding pattern obtained on a starch gel for hybrid seeds (cour-
tesy of Carol Betzel, BioDiagnostics Inc., and Pam Marasko, Syngenta Seeds Inc.).
isozymes Analysis-starch Gel electrophoresis
Isozymes (isoenzymes) are variants of enzymes catalyzing the same reaction. The terms isozymes and allo-
zymes are usually used interchangeably, although, strictly speaking, allozymes represent different alleles of
the same gene, while isozymes represent different genes.
For this type of analysis, the presence of isozymes when protein extracts from two populations are
compared is indicative of varietal differences. Protein extracts from seeds, seedlings or other plant tissue
are subjected to electrophoresis on starch gels. Following electrophoresis, different slices of the starch
gel are stained with enzyme speciic stains. Different staining solutions make it possible to visualize dif-
ferent enzyme systems, and study the variability within each of those systems (Hamrick and Godt, 1989;
Manchenko, 1994). Variations in the banding patterns observed after staining are used to establish varietal
Search WWH ::




Custom Search