Isoelectric focusing is used to separate proteins or peptides which are charged in a medium that is either above or below their isoelectric point. When an electric field is applied, the proteins or peptides respond by migrating to their isoelectric point. We use isoelectric focusing as the first dimension of a two-dimensional gel study or to simplify complex mixtures for shotgun proteomics workflows.
Our Bio-Rad IEF instrument is used to focus proteins for subsequent separation in a 2D gel. A pH gradient is immobilized in a dried piece of gel that is bound to a plastic backer. The gel is rehydrated with a protein solution, then an electric field is ramped up to drive the proteins to their isoelectric point. The strip is then placed on top of an SDS-PAGE gel and the proteins separated by molecular weight. Find more information about considerations for selecting strip size here.
Our Digital Proteome Fractionator from Cell Biosciences is used for high resolution and highly reproducible separation of proteins and peptides. Here, biomolecules in solution are placed into a chamber with a cassette containing gel plugs with differences of 0.05 pH units. An applied electric field allows the molecules to migrate to the plug containing the relevant pI, where they are captured. Proteins in the plug are digested using our in gel digestion protocol and peptides are eluted prior to analysis by mass spectrometry.
In both cases, samples must be thoroughly desalted prior to separation. Visit our Protocols section for various desalting techniques.