Isoelectric-Focusing

Isoelectric focusing (IEF) is an electrophoretic technique for the separation of proteins based on their isoelectric point (pI). The pI is the pH at which a protein has no net charge and thus, does not migrate further in an electric field. IEF gels are used to determine the pI of a protein and to detect minor changes in the protein due to post-translational modifications such as phosphorylation and glycosylation.

How isoelectric focusing (IEF) works

In isoelectric focusing (IEF), proteins are applied to polyacrylamide gels (IEF gels) or immobilized pH gradient (IPG) strips containing a fixed pH gradient. An electrical field is applied and the protein sample containing a mixture of proteins migrates through the pH gradient. Individual proteins are immobilized in the pH gradient as they approach their specific pI. After staining the gel and documenting the results, proteins separated by pI can be separated by mass using 2D gel electrophoresis.

We offer comprehensive solutions for isoelectric focusing including Novex® IEF gels containing 5% polyacrylamide that are optimized for native, nondenaturing applications. The following reagents and instruments are typically used to get the best performance with Novex® IEF gels.

  • Novex®  IEF gels—excellent for native applications using soluble proteins
  • Optimized, premixed IEF buffers—reduce variability and enable consistent results
  • Novex® IEF protein markers—ready to use, accurate results
  • ZOOM® IEF fractionation system—a fast, reliable method to reduce sample complexity, enrich low-abundance proteins, and increase the dynamic range of detection

The ZOOM® IEF Fractionator offers a fast, reliable method to reduce sample complexity, enrich low-abundance proteins, and increase the dynamic range of detection. Solution-phase isoelectric focusing with the ZOOM® IEF Fractionator provides reproducible separations typically in three hours. Fractionated samples are ready for further analysis by two-dimensional gel electrophoresis (2DE), one-dimensional gel electrophoresis (1DE), or two-dimensional liquid chromatography/mass spectrometry (2D LC/MS).