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 isoelectric point (pI) of a protein and to detect minor changes in the protein due to post-translational modifications such as phosphorylation and glycosylation.
What is isoelectric focusing (IEF)?
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.
Novex® Isoelectric Focusing Electrophoresis System
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 hels —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
Novex® Isoelectric Focusing gels
Novex® IEF gels are excellent for native, nondenaturing applications using soluble proteins. They can be used to readily detect minor changes in a protein due to deamination, phosphorylation, or glycosylation, and can resolve different proteins of similar size which cannot be resolved on standard SDS-PAGE gels.
When used with our convenient, preoptimized buffers, solubilizers, and molecular weight markers, the Novex® IEF gels provide:
- Clear, sharp bands for easy identification of protein modifications
- Higher resolution of slight differences in size than SDS-PAGE
Prefocusing is not required and typical run time is 2.5 hours. Figure 1 demonstrates the migration pattern of typical proteins on Novex® isoelectric focusing gels. All gels are provided in 1.0 mm thickness at a 5% acrylamide concentration.
Novex® IEF Gel pH performance ranges
- pH 3–10 gels—pI performance range is 3.5–8
- pH 3–7 gels—pI performance range is 3.0–7.0
Figure 1. The migration patterns of protein markers on Novex® Tricine, IEF, and zymogram gels are shown. Optimal resolution is achieved when protein bands migrate within the shaded regions. The numbered bands on the Tricine gel patterns correspond to the migration of Mark12™ Unstained Standard under denaturing conditions. The numbered bands on the zymogram gel patterns refer to the following proteases: Band 1: collagenase type I (140 kDa); Band 2: thermolysin (37 kDa); Band 3: chymotrypsin (30 kDa); Band 4: trypsin (19 kDa).
Isoelectric focusing buffers
The following buffers are needed to perform isoelectric focusing with Novex® Isoelectric Focusing gels:
- Novex® IEF sample buffer—for sample preparations, samples for IEF gels are prepared without SDS to avoid affecting the pI of the protein
- Novex® IEF anode buffer—for the lower buffer chamber of the XCell SureLock™ Mini-Cell
- Novex® IEF cathode buffer—for the upper buffer chamber of the XCell SureLock™ Mini-Cell
Novex® IEF Protein Marker
The Novex® IEF Marker 3-10 is a ready-to-use protein marker especially developed for IEF applications. The marker works well on all horizontal and vertical one-dimensional IEF gels, and can be used for both native and denaturing conditions (e.g., 8 M urea). The marker provides:
- pI range of 3.5–10.7
- 13 purified isoforms—no need for extra "high pI range" or "low pI range" markers
- A ready-to-use format for IEF gels
- Ideal pI determination of unknown protein samples
- Monitoring of separation on IEF gels
- One standard applicable to all IEF gels (vertical/horizontal)
- Purified, salt-free protein components
- Marker compatible with silver staining
ZOOM® IEF Fractionation System
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 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).