Isoelectric-Focusing

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 help obtain 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

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. All Novex® IEF gels are provided at1.0 mm thickness and a 5% acrylamide concentration. Figure 1 demonstrates the migration pattern of typical proteins on Novex® isoelectric focusing gels.

When used with our convenient, preoptimized buffers, solubilizers, and molecular weight markers, Novex® IEF gels provide:

  • Clear, sharp bands for easy identification of protein modifications
  • Higher resolution of slight differences in size than SDS-PAGE
  • Typical run times of 2.5 hours, with no refocusing required
Novex® IEF Gel ordering information and 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
Migration patterns of protein markers on Novex® Tricine, IEF, and Zymogram Gels
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

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
  • 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
  • Compatibility with silver staining