Summary
Proteomics often involves systematic analyses of proteomes that are constantly changing in response to changes in the environment of the cell, tissue, or organism being analyzed. Due to limitations of all current protein profiling methods, powerful, reliable proteome prefractionation methods prior to two-dimensional electrophoresis (2DE) gels or alternative non-2DE gel methods are needed for in-depth quantitative comparisons of the complex proteomes typically encountered with samples from higher eukaryotes. The microscale solution isoelectrofocusing (MicroSol IEF) fractionation method is capable of reproducibly dividing complex proteomes into as many as seven well-resolved fractions based on the proteins’ pIs on a small volume scale (∼0.65mL/fraction). When MicroSol IEF is combined with narrow pH range 2DE gels or with alternative downstream analysis methods, it can substantially increase the detection dynamic range and the total number of proteins that can be quantitatively compared. Although MicroSol IEF is reasonably reproducible, subtle variations can occur in different separations similar to the minor variations often seen in most separations of proteins. Therefore, for reliable quantitative comparisons the samples to be compared should be differentially labeled with either Cy dyes or stable isotope labels prior to mixing and separation in a single MicroSol IEF run. Larger numbers of samples can be compared across many MicroSol IEF separations by using a differentially labeled internal standard composed of equal aliquots of all samples to be compared.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
O’Farrell, P.H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007–4021.
Bjellqvist, B., Ek, K.J., Righetti, P.G., Gianazza, E., Gorga, A., Westermeier, R., and Postel, W. (1982) Isoelectric focusing in immobilized pH gradients; principle, methodology and some applications. J. Biochem. Biophys. Methods 6, 317–339.
Gorg, A., Weiss, W., and Dunn M.J. (2004) Current two-dimensional electrophoresis technology for proteomics. Proteomics 4, 3665–3685.
Williams, K.L. (1999) Genomics and proteomes: towards a multidimensional view of biology. Electrophoresis 20, 678–688.
Dreger, M. (2003) Subcellular proteomics. Mass Spectrom. Rev. 22, 27–56.
Elortza, F., Nuhse, T.S., Foster, L.J., Stensballe, A., Peck, S.C., and Jensen, O.N. (2003) Proteomic analysis of glycosylphosphatidylinositol-anchored membrane proteins. Mol. Cell. Proteomics 2, 1261–1270.
Zuo, X., Lee, K., and Speicher, D.W. (2004) Electrophoresis prefractionation for comprehensive analysis of proteome. In Proteome Analysis: Interpreting the Genome. Speicher D.W. (Ed.), Elsevier, New York, pp. 93–117.
Bier, M. (1988) Recycling isoelectric focusing and isotachophoresis, Electrophoresis 19, 1057–1063.
Righetti, P.G., Castagna, A., Herbert, B., Reymond, F., and Rossier, J.S. (2003) Prefractionation techniques in proteome analysis. Proteomics 3, 1397–1407.
Sang, T.Q., Ginter, J.M., Johnston, M.V., Larsen, B.S., and McEwen, C.N. (2003) Carrier ampholyte-free solution isoelectric focusing as a prefractionation method for the proteomic analysis of complex protein mixtures. Electrophoresis 24, 2359–2368.
Righetti, P.G., Wenisch, E., and Faupel, M. (1989) Preparative protein purification in a multi-compartment electrolyser with immobiline membranes. J. Chromatogr. 475, 293–309.
Herbert, B. and Righetti, P.G. (2000) A turning point in proteome analysis: sample prefractionation via multicompartment electrolyzers with isoelectric membranes. Electrophoresis 21, 3639–3648.
Righetti, P.G., Wenisch, E., Jungbauer, A., Katinger, H., and Faupel, M. (1990) Preparative purification of human monoclonal antibody isoforms in a multi-compartment electrolyser with immobiline membranes. J. Chromatogr. 500, 681–696.
Zuo, X. and Speicher, D.W. (2000) A method for global analysis of complex proteomes using sample prefractionation by solution isoelectrofocusing prior to two-dimensional electrophoresis, Anal. Biochem. 284, 266–278.
Zuo, X. and Speicher, D.W. (2002) Comprehensive analysis of complex proteomes using microscale solution isoelectrofocusing prior to narrow pH range two-dimensional electrophoresis. Proteomics 2, 58–68.
Echan, L.A., Tang, H-Y., Ali-Khan, N., Lee, K., and Speicher, D.W. (2005) Depletion of multiple high-abundance proteins improves protein profiling capacities of human serum and plasma. Proteomics 5, 3292–3303.
Tang, H-Y, Ali-Khan, N., Echan, L.A., Levenkova, N., Rux, J.J., and Speicher, D.W. (2005) A novel four-dimensional strategy combining protein and peptide separation methods enables detection of low-abundance proteins in human plasma and serum proteomes. Proteomics 5, 3329–3342.
Han, M.J. and Speicher, D.W. (2008) Microscale isoelectric focusing in solution; A method for comprehensive and quantitativet proteome analysis using 1-D and 2-D DIGE combined with MicroSol IEF prefractionation. In: Methods in Molecular Biology. Posch A. (Ed.), Humana Press, Totowa, NJ, pp. 241–256.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Joo, WA., Speicher, D. (2009). Prefractionation Using Microscale Solution IEF. In: Tyther, R., Sheehan, D. (eds) Two-Dimensional Electrophoresis Protocols. Methods in Molecular Biology, vol 519. Humana Press. https://doi.org/10.1007/978-1-59745-281-6_18
Download citation
DOI: https://doi.org/10.1007/978-1-59745-281-6_18
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-58829-937-6
Online ISBN: 978-1-59745-281-6
eBook Packages: Springer Protocols