Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Liquid-based free-flow electrophoresis–reversed-phase HPLC: a proteomic tool

Nature Methods volume 2, pages 863–873 (2005)Cite this article

  • Carrier ampholytes: Servalytes pH 3–5, pH 7–9 and pH 3–10 (SERVA) or Sinulytes pH 2–4, pH 4–6, pH 5–7, pH 9–11 and pH 2–11 (SINUS GmbH)

  • FFE and IEF solutions and buffers (Table 1)

    Table 1 FFE-IEF solutions and buffers
    Full size table

  • Glycerol (AnalaR grade; BDH, Merck)

  • HPLC solvents A and B (Table 1)

  • Hydroxypropyl methyl cellulose (HPMC) stock solution, 2% (wt/vol; ∼86 K nominal molecular weight; 4,000-cP viscosity; Aldrich); prepare 24 h before use as an aqueous 2% (wt/vol) stock solution by adding 20 g of HPMC to 1 l of water; store at 4 °C for up to 5 d

  • Iodoacetic acid (IAA), 50 mM (Aldrich); prepare fresh stock solution by adding 9 mg IAA to 0.1 ml 1 M NaOH

  • Isopropanol (HPLC grade, ChromAR; Mallinkrodt)

  • Milli-Q water (deionized, HPLC grade, ≥18 MΩ from an A10-Synthesis water polishing system; Millipore)

  • Phosphoric acid (98%), sodium hydroxide and glacial acetic acid (all uniVAR grade; BDH, Merck)

  • Protein sample(s) to be analyzed

  • Protein standard mixture: prepare a 1 mg/ml mixture of the proteins listed in Table 2 by adding 1 mg of each protein to 1 ml Milli-Q water; store in small aliquots (0.5–1 ml) at −20 °C; do not freeze-thaw an aliquot more than twice

    Table 2 Protein standards to evaluate system performance
    Full size table

  • pI marker solution, to optimize IEF gradient (Table 1)

  • 2-(4-Sulfophenylazo)1,8-dihydroxy-3,6-naphthalenedisulfonic acid (SPADNS) trisodium salt or sulfanilic acid azochromotrop (this dye has maximum absorbance at 505–510 nm)

Carrier ampholytes: Servalytes pH 3–5, pH 7–9 and pH 3–10 (SERVA) or Sinulytes pH 2–4, pH 4–6, pH 5–7, pH 9–11 and pH 2–11 (SINUS GmbH)

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Schematic representation of the continuous FFE apparatus coupled off-line to RP-HPLC.
Figure 2: Separation of protein standards by nonreducing 2DE and 2D FFE/RP-HPLC.

References

  1. Simpson, R.J. Proteins and Proteomics: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2003).

    Google Scholar 

  2. Simpson, R.J. Purifying Proteins for Proteomics: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2004).

    Google Scholar 

  3. Hannig, K. New aspects in preparative and analytical continuous free-flow cell electrophoresis. Electrophoresis 3, 235–243 (1982).

    Article  CAS  Google Scholar 

  4. Moritz, R.L., Eddes, J. & Simpson, R.J. Methods in Protein Sequence Analysis (eds. Atassi, M.Z. & Appela, E.) 27–38 (Plenum Press, New York, 1995).

    Google Scholar 

  5. Moritz, R.L. et al. A proteome strategy for fractionating proteins and peptides using continuous free-flow electrophoresis coupled off-line to reversed-phase high-performance liquid chromatography. Anal. Chem. 76, 4811–4824 (2004).

    Article  CAS  Google Scholar 

  6. Smith, P.K. et al. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76–85 (1985).

    Article  CAS  Google Scholar 

  7. Vaidya, N.R., Gothoskar, B.P. & Banerji, A.P. Column isoelectric focusing in natural pH gradients generated by biological buffers. Electrophoresis 11, 156–161 (1990).

    Article  CAS  Google Scholar 

  8. Bjellqvist, B. et al. Isoelectric focusing in immobilized pH gradients: principle, methodology and some applications. J. Biochem. Biophys. Methods 6, 317–339 (1982).

    Article  CAS  Google Scholar 

  9. Egen, N.B. et al. Isolation of monoclonal antibodies to phencyclidine from ascites fluid by preparative isoelectric focusing in the Rotofor. Anal. Biochem. 172, 488–494 (1988).

    Article  CAS  Google Scholar 

  10. Faupel, M., Barzaghi, B., Gelfi, C. & Righetti, P.G. Isoelectric protein purification by orthogonally coupled hydraulic and electric transports in a segmented immobilized pH gradient. J. Biochem. Biophys. Methods 15, 147–161 (1987).

    Article  CAS  Google Scholar 

  11. Herbert, B. & Righetti, P.G. A turning point in proteome analysis: sample prefractionation via multicompartment electrolyzers with isoelectric membranes. Electrophoresis 21, 3639–3648 (2000).

    Article  CAS  Google Scholar 

  12. Zuo, X. & Speicher, D.W. Comprehensive analysis of complex proteomes using microscale solution isoelectrofocusing prior to narrow pH range two-dimensional electrophoresis. Proteomics 2, 58–68 (2002).

    Article  CAS  Google Scholar 

  13. Weber, G. & Bocek, P. Recent developments in preparative free flow isoelectric focusing. Electrophoresis 19, 1649–1653 (1998).

    Article  CAS  Google Scholar 

  14. Obermaier, C. et al. Free-flow isoelectric focusing of proteins remaining in cell fragments following sonication of thyroid carcinoma cells. Electrophoresis 26, 2109–2116 (2005).

    Article  CAS  Google Scholar 

  15. Grego, B., Nice, E.C. & Simpson, R.J. Use of scanning diode-array detector with reversed-phase microbore columns for the real-time spectral-analysis of aromatic-amino-acids in peptides and proteins at the submicrogram level—applications to peptide and protein microsequencing. J. Chromatography 352, 359–368 (1986).

    Article  CAS  Google Scholar 

  16. Knowles, M.R. et al. Multiplex proteomic analysis by two-dimensional differential in-gel electrophoresis. Proteomics 3, 1162–1171 (2003).

    Article  CAS  Google Scholar 

  17. Shaw, J. et al. Evaluation of saturation labelling two-dimensional difference gel electrophoresis fluorescent dyes. Proteomics 3, 1181–1195 (2003).

    Article  CAS  Google Scholar 

  18. Greengauz-Roberts, O. et al. Saturation labeling with cysteine-reactive cyanine fluorescent dyes provides increased sensitivity for protein expression profiling of laser-microdissected clinical specimens. Proteomics 5, 1746–1757 (2005).

    Article  CAS  Google Scholar 

  19. Burggraf, D., Weber, G. & Lottspeich, F. Free flow-isoelectric focusing of human cellular lysates as sample preparation for protein analysis. Electrophoresis 16, 1010–1015 (1995).

    Article  CAS  Google Scholar 

  20. Hoffmann, P. et al. Continuous free-flow electrophoresis separation of cytosolic proteins from the human colon carcinoma cell line LIM 1215: a non two- dimensional gel electrophoresis-based proteome analysis strategy. Proteomics 1, 807–818 (2001).

    Article  CAS  Google Scholar 

  21. Amigorena, S., Drake, J.R., Webster, P. & Mellman, I. Transient accumulation of new class II MHC molecules in a novel endocytic compartment in B lymphocytes. Nature 369, 113–120 (1994).

    Article  CAS  Google Scholar 

  22. Weber, G. & Bocek, P. Optimized continuous flow electrophoresis. Electrophoresis 17, 1906–1910 (1996).

    Article  CAS  Google Scholar 

  23. Zischka, H. et al. Improved proteome analysis of Saccharomyces cerevisiae mitochondria by free-flow electrophoresis. Proteomics 3, 906–916 (2003).

    Article  CAS  Google Scholar 

  24. Bondy, B., Bauer, J., Seuffert, I. & Weber, G. Sodium chloride in separation medium enhances cell compatibility of free flow electrophoresis. Electrophoresis 16, 92–97 (1995).

    Article  CAS  Google Scholar 

  25. Simpson, R.J. et al. Proteomic analysis of the human colon carcinoma cell line (LIM 1215): development of a membrane protein database. Electrophoresis 21, 1707–1732 (2000).

    Article  CAS  Google Scholar 

  26. Liotta, L.A., Ferrari, M. & Petricoin, E. Clinical proteomics: written in blood. Nature 425, 905 (2003).

    Article  CAS  Google Scholar 

  27. Wong, C., Sridhara, S., Bardwell, J.C. & Jakob, U. Heating greatly speeds Coomassie blue staining and destaining. Biotechniques 28, 426–432 (2000).

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Joint Proteomics Laboratory, Ludwig Institute for Cancer Research (Melbourne Branch) and The Walter and Eliza Hall Institute of Medical Research, Parkville, 3050, Victoria, Australia

    Robert L Moritz & Richard J Simpson

Authors
  1. Robert L Moritz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Richard J Simpson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Richard J Simpson.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Moritz, R., Simpson, R. Liquid-based free-flow electrophoresis–reversed-phase HPLC: a proteomic tool. Nat Methods 2, 863–873 (2005). https://doi.org/10.1038/nmeth1105-863

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth1105-863

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing