Isoelectric focusing

Isoelectric focusing (IEF), also known as electrofocusing, is a high-resolution, stand-alone technique used in protein separation and characterization of proteins by their isoelectric point. The isoelectric point (pI) is the pH at which a particular molecule or surface carries no net electrical charge.

Principle

IEF works by changing the pH gradient, allowing proteins to migrate along this gradient to the point where their net charge is zero (their isoelectric point). This is achieved by applying an electric field across a pH gradient, which is established by small molecules known as ampholytes. These ampholytes are a mixture of polyamino-polycarboxylic acids of different pK values, which, when subjected to a pH gradient, migrate to the position in the gradient where the pH equals their pK value.

Method

The method of isoelectric focusing involves the following steps:

1. Preparation of a gel with a pH gradient.
2. Application of the protein sample.
3. Application of an electric field.
4. Focusing of the proteins at their isoelectric point.

Applications

Isoelectric focusing is used in various fields such as:

1. Proteomics: It is used in the first dimension of 2-D gel electrophoresis, where proteins are first separated by their pI and then by their molecular weight.
2. Biotechnology: It is used for the characterization and quality control of biopharmaceuticals.
3. Clinical chemistry: It is used for the detection of protein abnormalities.

See also

• Two-dimensional gel electrophoresis
• Capillary electrophoresis
• Zeta potential
• Isoelectric point

References

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