Isoelectric focusing orelectrofocusing is a well-established technique that is used for analyzing proteins, for example, hormones, enzymes and other biologically active proteins. With isoelectric focusing, a pH gradient is usually formed in a supporting medium by exposing the carrier ampholyte to an electric field. When the protein is introduced within the pHgradient, it will migrate to its specific isoelectric point and stay at that position, resulting in a typical banding pattern.
IEF differs from most electrophoresis techniques because the protein is actively focused or concentrated at its specific isoelectric point. Therefore, it is possible to separate different proteins with a pI difference of just 0.01pH units.
For a long time, electrophoretic techniques have been used as the main techniques for characterization and testing the purity of complex protein mixtures. Electrophoresis was first introduced in 1937 by researcher Tiselius. Over the years, a lotof effort has been devoted to refining this technique, resulting in the development of a wide range of electrophoretic techniques such as Isoelectric focusing.
Isoelectric focusing (IEF) is used in a variety of biochemical analysis. These include:
- Separation of proteins and peptides
- In research in cytology, immunology and taxonomy
- It can also be used in limit test when comparing the density of the band with the density of band of a standard preparation.
- Isoelectric focusing provides an effective alternative to the conventional electrophoresis in genetic marker typing.
- IEF gel can also be used in identity test when comparing the migration pattern on the gel with that of a standard preparation.
Conventional electrophoresis and electrofocusing
Electrofocusing is different from the conservative electrophoresis in that the pH is not maintained at a constant throughout the entire system. With IEF, the sample components will electrophoretically migrate in a motionless pH gradient. As a result, a steady state will be reached where all sample components will be focused or concentrated as sharp bands on their respective isoelectric points. Conventional electrophoresis also requires the sample to be applied as a narrow zone to guarantee optimal resolution. However, this is not the case with electro focusing where sample application is not crucial due to its concentrating characteristics.
The concentrating effect of isoelectric focusing has a lower detection limit when compared to conventional electrophoresis. This means that proteins that differ in isoelectric points by just a few hundredths of a pH unit can also be separated and analyzed. Check out mybiosource.com for more details.
Benefits of IEF
- IEF can be used to separate proteins that are as little as 0.001 pH units.
- High resolution can be achieved because the spreading of the bands is minimized because of application of the pH gradient and the applied field.
- The technique is simple to perform because the placement of the sample application is not crucial.
- Protein resolution is superb when you use very thin tube gels.
- Tube gels using carrier ampholytes are simple to prepare and do not need complicated gradient casting equipment.
- Ampholyte mixtures can be optimized or simply blended for more limited or wide pH ranges.
Over the years, the popularity of isoelectric focusing has been growing immensely in that fractionation and analysis of proteins. This is an indication that this technique will soon share an important place comparable to electrophoresis in shortlisted biochemical techniques. Electro focusing utilizes simple laboratory equipment and fractionation is complete within one hour or less, and the focusing protein zones are clearly seen during the entire experiment. Isoelectric focusing is a biochemical technique that researchers prefer because of its countless advantages and unmatched results.