Isoelectric focusing (IEF) is a high-resolution technique where proteins are separated according to their isoelectric points within a continuous pH gradient. IEF is an electrophoretic method for separating proteins based on their isoelectric point. The isoelectric point is the pH at which the net charge of the protein is zero. With the presence of a pH gradient in the IEF technique, the protein will migrate to the position in the gradient where its charge is zero. Proteins with a positive net charge will migrate toward the cathode until it meets its pI. Proteins with a negative net charge will migrate toward the anode until it meets its pI. If the protein diffuses away from its pI, it will regain its charge and migrate back. This focusing effect allows proteins to be separated based on very small charge differences. IEF is performed under high voltages (> 1000 V) until the proteins have reached their final position in the pH gradient. If IEF is performed under denaturing conditions very high resolution and cleanliness of sample can be obtained. IEF is “traditionally” used as first stage separation for 2D-PAGE, separating proteins by charge prior to second dimension separation by SDS-PAGE. This additional separation allows resolution of a couple of thousand proteins on a 2D-PAGE gel–enough for the entire proteome of an organelle or bacterium. It can also be used to examine post-translation modifications of proteins. Another use for IEF is for fractionation of proteins or peptides prior to mass spec. Previously, it was difficult to recover molecules separated by IEF. However, with the Agilent OFFGEL system, the proteins or peptides remain in solution rather than being trapped in the gel as with standard IEF. Electrofocusing of proteins in this way provides a convenient alternative to SDS-PAGE for sample fractionation prior to mass spec.
Using IEF to separate peptides also provides an alternative to strong cation exchange (SCX) fractionation, which is one of the most popular techniques for the separation of peptides prior to mass spectrometry. IEF separation of peptides has been shown to result in more peptide identifications from whole proteome samples than with SCX for some samples