Summary
Chromatofocusing has many potential applications in the field of proteomics, such as for the isolation and removal of major sample components to facilitate the analysis of low-abundance components, and for sample prefractionation prior to a subsequent separation using SDS-PAGE, narrow-pI-range 2D-PAGE, or additional chromatography steps. However, the chromatofocusing techniques that are most commonly used employ propriety polyampholyte elution buffers and highly specialized column packings, both of which limit the use of chromatofocusing in practice. To expand the range of application for this technique, this chapter considers chromatofocusing methods which employ common ion-exchange column packings and elution buffers which are simple mixtures of readily available buffering species. Of particular interest is the use of chromatofocusing with a multistep pH gradient for the fractionation of protein mixtures into narrow-pI-range fractions. The cross-contamination characteristics of these fractions using SDS-PAGE are also assessed.
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Acknowledgements
This work has been supported by grant CTS-0442072 from the National Science Foundation. The authors also thank Remco van Soest and Chris Pohl (Dionex/LC Packings, Sunnyvalle, CA) for providing technical support and for donating several of the columns used in this study.
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Shen, H., Li, X., Bieberich, C.J., Frey, D.D. (2008). Reducing Sample Complexity in Proteomics by Chromatofocusing with Simple Buffer Mixtures. In: Posch, A. (eds) 2D PAGE: Sample Preparation and Fractionation. Methods in Molecular Biology™, vol 424. Humana Press. https://doi.org/10.1007/978-1-60327-064-9_16
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