Written by Gerard Rozing
At the 37th issue of the Microscale Separations and Bioanalysis symposium, I had the privilege to present a tutorial lecture on Capillary Iso-Electric Focusing.
Abstract. Since the inception of iso-electric focusing (IEF) as a high-resolution electrophoretic technique for the separation of amphoteric substances by Tiselius and Vestenberg, it has become a prime methodology for the separation of protein mixtures.
Initially, IEF was executed on a flat surface coated with a gel mixed with ampholytes to establish the pH gradient. The sample is applied to the center of the plate. This so-called slab gel IEF became more sophisticated by using flat surfaces with an immobilized pH gradient (IPG). With the introduction of instruments for Capillary Electrophoresis, the technique was adapted to allow in-capillary execution of iso-electric focusing (CIEF) followed by dedicated whole-column-image-detection electrophoresis systems (iCIEF).
With the commercialization of IEF, the technology has evolved into a recipe-driven, a kind of black-box approach. The users strictly follow the protocols provided by the system supplier. In this situation, the basic understanding of the separation mechanism on IEF fades. Diagnosis and troubleshooting when “it does not work” become an insurmountable hurdle, and the costly help of the system supplier is called.
Therefore there is a continuing need for teaching the basic principles of isoelectric focusing, mainly when applied in capillaries (CIEF)
In this tutorial lecture, it is intended to present:
- Basics of capillary iso-electric focusing.
- Instrumentation for CIEF and iCIEF.
- New developments
- Preparative CIEF.
- Coupling of CIEF with Mass Spectrometric Detection.
The presentation was recorded as a streaming video (MP4). You can view the talk via this link.
If you want a copy of the slides, please contact me via mail form.