Summary
Protein fractionation is essential to uncovering low-abundance proteins in complex protein mixtures. Many common methods and techniques are used to fractionate proteins, including chromatography (size exclusion, affinity, ion exchange, etc.), electrophoresis, and solution chemistry. Regardless of the method employed, the ultimate goal of protein fractionation is to enable more protein analysis by today’s current proteomics technologies, such as one- (1-DGE) or two-dimensional gel electrophoresis (2-DGE) and liquid-chromatography and tandem mass spectrometry (LC-MS/MS).
The MicroRotofor\(^{\rm TM}\) isoelectric focusing (IEF) cell fractionates proteins in free solution according to their isoelectric point (pI). We demonstrate the ability of the MicroRotofor to enrich low-abundance proteins in mouse brain tissue, thus enabling further identification of potential biomarker candidates.
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
Bier, F. F., Egen, N. B., Allgyer, T. T., Twitty, G. E., and Mosher, R. A. (1979) New developments in isoelectric focusing, inStructure and biological function (Gross, E., and Meierhofer, J., Eds.), pp. 79–89, Pierce Chemical Company.
Bier, F. F., and Egen, N. B. (1979) Large-scale recycling electrofocusing inElectrofocus/78 (Haglund, H., and Westerfield, J. G., Eds.), Elsevier, New York.
Righetti, P. G., Castagna, A., Herbert, B., and Candiano, G. (2005) How to bring the “unseen” proteome to the limelight via electrophoretic pre-fractionation techniques.Biosci Rep 25, 3–17.
Davidsson, P., Folkesson, S., Christiansson, M., Lindbjer, M., Dellheden, B., Blennow, K., and Westman-Brinkmalm, A. (2002) Identification of proteins in human cerebrospinal fluid using liquid-phase isoelectric focusing as a prefractionation step followed by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionisation mass spectrometry.Rapid Commun Mass Spectrom 16, 2083–8.
Davidsson, P., Paulson, L., Hesse, C., Blennow, K., and Nilsson, C. L. (2001) Proteome studies of human cerebrospinal fluid and brain tissue using a preparative two-dimensional electrophoresis approach prior to mass spectrometry.Proteomics 1, 444–52.
Davidsson, P., and Nilsson, C. L. (1999) Peptide mapping of proteins in cerebrospinal fluid utilizing a rapid preparative two-dimensional electrophoretic procedure and matrix-assisted laser desorption/ionization mass spectrometry.Biochim Biophys Acta 1473, 391–9.
Davidsson, P., Puchades, M., and Blennow, K. (1999) Identification of synaptic vesicle, pre- and postsynaptic proteins in human cerebrospinal fluid using liquid-phase isoelectric focusing.Electrophoresis 20, 431–7.
Peirce M.J., Wait R., Begum S., Saklatvala J. and Cope A.P. (2004) Expression profiling of lymphocyte plasma membrane proteins.Mol.Cell. Proteomics 3: 56–65.
Hamler R.L., Zhu K., Buchanan N.S., Kreunin P., Kachman M.T., Miller F.R. and Lubman D.M. (2004) A two-dimensional liquid-phase separation method coupled with mass spectrometry for proteomic studies of breast cancer and biomarker identification. Proteomics 4: 562–577.
Wang M.Z., Howard B., Campa M.J., Patz, E.F. and Fitzgerald M.C. (2003) Analysis of human serum proteins by liquid-phase isoelectric focusing and matrix-assisted laser desorption/ionization-mass spectrometry.Proteomics 3: 1661–1666.
Harper R.G., Workman S.R., Schuetzner S., Timperman A.T. and Sutton J.N. (2004) Low-molecular-weight human serum proteome using ultrafiltration, isoelectric focusing, and mass spectrometry.Electrophoresis 25: 128–133.
Xiao Z., Conrads T.P., Lucas D.A., Janini G.M., Schaefer C.F., Buetow K.H., Issaq H.J. and Veenstra T.D. (2004) Direct ampholyte-free liquid-phase isoelectric peptide focusing: application to the human serum proteome.Electrophoresis 25: 128–133.
Janini G.M., Conrads T.P., Veenstra T.D. and Issaq H.J. (2003) Development of a two-dimensional protein-peptide separation protocol for comprehensive proteome measurements.J Chromatography B 787:43–51.
Bindschedler L.V., Palmblad M. and Cramer R. (2006) First-dimension separation with the MicroRotofor cell prior to SDS-PAGE and LC-MS/MS analysis. Bulletin 5451, Bio-Rad Laboratories, Inc.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Hey, J., Posch, A., Cohen, A., Liu, N., Harbers, A. (2008). Fractionation of Complex Protein Mixtures by Liquid-Phase Isoelectric Focusing. 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_19
Download citation
DOI: https://doi.org/10.1007/978-1-60327-064-9_19
Publisher Name: Humana Press
Print ISBN: 978-1-58829-722-8
Online ISBN: 978-1-60327-064-9
eBook Packages: Springer Protocols