The amount of genetic variation by examining phenotypic variation was difficult because many traits, a complex interaction of many genes and environmental factors determines the phenotype

#Id: 2501

#Unit 13. Methods in Biology

The higher no. of band will be observed in gradient gel electrophoresis comparison to uniform gel SDS and Native gel electrophoresis.

#Id: 2502

#Unit 13. Methods in Biology

Isoelectric Focusing Gels is based on the separation of molecules according to their different isoelectric points at which the show zero electrophoretic mobility.

#Id: 2503

#Unit 13. Methods in Biology

In the Isoelectric Focusing pH gradient is formed by the introduction into the gel of compounds known as ampholytes, which are complex mixtures of synthetic polyamino-polycarboxylic acids Ampholytes can be purchased in different pH ranges covering a wide band (e.g. pH 3–10).

#Id: 2504

#Unit 13. Methods in Biology

IEF is a highly sensitive analytical technique and is particularly useful for studying  microheterogeneity in a protein. For example, a protein may show a single band on an SDS gel but may show three bands on an IEF gel. This may occur, for example, when a protein exists in mono-, di- and tri-phosphorylated forms. 

#Id: 2505

#Unit 13. Methods in Biology

IEF method is particularly useful for separating isoenzymes, which are different forms of the same enzyme, often differing by only one or two amino-acid residues.

#Id: 2506

#Unit 13. Methods in Biology

In the Isoelectric Focusing due to the pH gradient (e.g. pH 3–10) positive charged protein are separated near pH 10 while negatively amino acid are separated near pH 3.