Focus on Isoelectric Focusing

Isoelectric focusing electrophoresis (IEF) of proteins is nowhere near as popular as its cousin – sodium dodecyl sulphate-polyacrylamide gel electrophoresis aka SDS-PAGE. While in both methods the proteins are denatured, IEF is a gel-based electrophoretic separation of proteins using difference in their overall charges. The sodium dodecyl sulphate – SDS part of the usual gel – neutralises proteins electric charge, so that only length of the polypeptide chain determines the speed of protein migration in the gel. IEF takes advantage of proteins different amino acid compositions. Some of the amino acids in a protein are charged negatively, others – positively, and the sum of the individual charges of the molecule is called an isoelectric point (pI).  It varies between 3 and 12, but most of the proteins have a pI value between 4 and 7. The porous gel for IEF contains an immobilised pH gradient, for example created by negatively and positively charged molecules, covalently attached to the acrylamide.  The pH range of the gel can vary to ensure a good separation, depending on the predicted isoelectric point of the protein in question in the same way as SDS-PAGE percentage of polyacrylamide determines separation of proteins with different molecular mass.  Therefore, the IFF gradient should be centred on the predicted isoelectric point of your protein, e.g. if the pI is 7, the IEF gradient should be between 4 and 10.  Fine tuning of the gradient will allow good separation of similar proteins. During IEF a protein (the net charge of which depends of the pH of its environment) migrates in the pH gradient to the oppositely charged electrode, until it reaches its isoelectric point. Different proteins will migrate into different positions within the gel (see Fig.1) until the charge on the molecules are completely compensated.

Fig.1 Schematic representation of isoelectric focusing (Picture: Wikimedia Commons).

If a protein molecule acquires charge, it starts migrating back until it is equilibrated (focused), which – at least in some instances – allows very precise focusing within 0.01 difference. This property of IEF allows using it to separate closely related molecules, such as enzyme isoforms, including phosphorylated and glycosylated forms, because these posttranslational modifications change charge of the resulting proteins. Making a good  IEF gel is much trickier that a SDS PAGE gel,  but because  IEF is  used as a first step in 2D electrophoresis  it is easy to buy a precast IEF, where  the gel is immobilized on a plastic  strip and use it in an 2D gel apparatus. I wish you a happy focusing.