Electrofocusing, also known as isoelectric focusing (IEF), is a scientific technique that is used to separate charged molecules, usually peptides or proteins based on their isoelectric point (pl). The pl or isoelectric point is basically the pH at which the specified molecules do not have a charge. IEF is a successful process because, in any electric field, molecules that are in a pH gradient will always migrate towards their respective isoelectric points.
In most instances, researchers use an immobilized pH gradient (IPG) stripthat is commercially available. This IPG strip is made up of an acrylamide gel that has wide pores to prevent the sieving effects that are based on protein mass, with a pH gradient. There are different gradients available in the market. Wider gradients like pH 3 to10 are suitable for wholesome proteome analysis, while the narrower ranges of pH 5 to 8 are suitable for more specialized applications.
To assist in migration, the protein sample is normally mixed with carrier ampholytes. Ampholytes are a combination of charged molecules that have a wide range of isoelectric points that matches with the pl ranges on the IPG strips. The migration of ampholytes supports the movement of sample molecules along the pH gradient. There are a variety of ampholyte mixtures with different pl ranges in the market today. After undergoing separation across the pH gradient, the protein mixture sample is separated and analyzed further. You can check out mybiosource.com for more details.
The greatest advantage of IEF is its high resolution that results in a better separation of the solutes. IEF of serum proteins gives many bands and the bands are sharper since every pH region tends to be very narrow.
It is also easier to perform IEF because the sample application is not that important. The ampholytes and the sample can be mixed prior to application. The ampholytes will first migrate creating a pH gradient, and the proteins will then separate and eventually migrate.
Some variant hemoglobins and isoenzymes in prenatal screening can be separated using IEF technique. IEF can also be used in detecting oligoclonal bands in the gamma-globulin
Uses of IEF
IEF is mainly used as the initial separation for the 2D-PAGE, involving separation of proteins by charge before they undergo second dimension separation by the SDS-PAGE technique. This extra separation process enables resolution of thousands of proteins on the 2D-PAGE gel that is enough for the entire bacterium, proteome or organelle. IEF can also be used in examining the post-translation modifications of various proteins.
IEF can also be used in separating peptides, providing an excellent alternative to the strong cation exchange (SCX) fractionation. SCX is one of the preferred methods of separating peptides before mass spectrometry. Separation of peptides using IEF technique brings about more peptide identifications from the entire proteome samples when compared to SCX.
IEF is also used for fractionation of peptides or proteins to mass spec. In the past, it was challenging to recover the molecules that were separated by IEF. However, with the current Agilent OFFGEL system, the peptides or proteins tend to remain in the solution instead of being trapped inside the gel. IEF of proteins offers a convenient method of sample fractionation before mass spec. It is without a doubt that isoelectric focusing is a technique that scientists should use.