Ion‐Exchange Chromatography

Alan Williams1, Verna Frasca1

1 Amersham Pharmacia Biotech, Piscataway
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 8.2
DOI:  10.1002/0471140864.ps0802s15
Online Posting Date:  May, 2001
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Abstract

This unit outlines the basic steps in planning and carrying out ion‐exchange chromatography to separate proteins. Protocols describe both batch adsorption and column chromatography in conjunction with either step‐ or linear elution gradients. Support protocols describe (1) pilot experiments to determine initial conditions for batch or column chromatography (i.e., pH required for binding, change in pH or salt concentration required for elution, and available capacity of a medium), (2) calculation of the dynamic capacity of an ion‐exchange column, (3) methods for producing continuous gradients of pH and salt concentration to elute proteins from ion‐exchange columns, (4) regeneration of used ion‐exchange media, and (5) storage of ion‐exchange media.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Batch Adsorption and Step‐Gradient Elution with Increasing Salt Concentration
  • Alternate Protocol 1: pH‐Based Step‐Gradient Elution
  • Basic Protocol 2: Column Chromatography with Linear Gradient Elution
  • Support Protocol 1: Test Tube Pilot Experiment to Determine Starting Conditions for Ion‐Exchange Chromatography
  • Support Protocol 2: Measurement of Dynamic (Column) Capacity and Breakthrough Capacity of Ion‐Exchange Columns
  • Support Protocol 3: Gradient‐Formation Techniques
  • Support Protocol 4: Cleaning and Regeneration of Ion‐Exchange Media
  • Support Protocol 5: Storage of Ion‐Exchange Media
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Batch Adsorption and Step‐Gradient Elution with Increasing Salt Concentration

  Materials
  • QAE Sephadex A‐25 (Amersham Pharmacia Biotech) or equivalent anion‐exchange gel
  • Binding buffer: 20 mM Tris⋅Cl, pH 7.5 (or other buffer as determined empirically; see protocol 4)
  • Protein sample to be purified
  • Wash buffer: 20 mM Tris⋅Cl (pH 7.5)/100 mM NaCl (or other buffer/salt solution as determined empirically; see protocol 4)
  • Elution buffer: 20 mM Tris⋅Cl (pH 7.5)/350 mM NaCl (or other buffer/salt solution as determined empirically; see protocol 4)
  • Regeneration buffer: 20 mM Tris⋅Cl (pH 7.5)/2 M NaCl (also see protocol 8)
  • Boiling water bath (optional)
  • 500‐ml sintered‐glass filter funnel, medium porosity
  • Three 2000‐ml side‐arm flasks
  • Conductivity meter

Alternate Protocol 1: pH‐Based Step‐Gradient Elution

  Materials
  • Liquid chromatography system (FPLC or HPLC)
  • Elution buffer: binding buffer (see protocol 1 and protocol 4) containing 1 M NaCl
  • Binding buffer (see protocol 1 and protocol 4)
  • RESOURCE Q chromatography column (1‐ml packed bed volume; Amersham Pharmacia Biotech)
  • Protein sample to be purified
  • Conductivity meter
  • 0.22‐µm filter

Basic Protocol 2: Column Chromatography with Linear Gradient Elution

  Materials
  • 20 mM piperazine, pH 5.0, 5.5, and 6.0 (prepare from 100 mM stock)
  • 20 mM 1,3‐bis[tris{hydroxymethyl}methylamino]propane (bis‐Tris propane), pH 6.5 and 7.0 (prepare from 100 mM stock)
  • 20 mM Tris⋅Cl, pH 7.5, 8.0, and 8.5 ( appendix 2E; prepare from 100 mM stock)
  • Q Sepharose Fast Flow (50% slurry in 20% ethanol; Amersham Pharmacia Biotech) or equivalent anion exchange resin in appropriate buffer
  • Protein sample to be purified, containing known quantity of target and total protein
  • 4 M NaCl
  • 15‐ml test tubes
  • Centrifuge with rotor accommodating 15‐ml test tubes (optional)

Support Protocol 1: Test Tube Pilot Experiment to Determine Starting Conditions for Ion‐Exchange Chromatography

  • Ion‐exchange gel of unknown capacity, and column
  • Elution buffer capable of eluting target protein in single step of salt concentration or pH (e.g., 2 M NaCl; see protocol 4)
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Figures

Videos

Literature Cited

Literature Cited
   Karlsson, E., Rydén, L., and Brewer, J. 1998. Ion exchange chromatography. In Protein Purification: Principles, High Resolution Methods and Applications, 2nd ed. (J.C. Janson and L. Rydén, eds.) pp. 145‐205. John Wiley & Sons, New York.
   Gianazza, E. and Righetti, P.G. 1980. Size and charge distribution of macromolecules in living systems J. Chromatog. 193:1‐8.
   Peterson, E.A. and Sober, H.A. 1956. Chromatography of proteins. I. cellulose ion exchange adsorbents. J. Amer. Chem. Soc. 78:751‐755.
   Pharmacia Biotech 1995. Ion Exchange Chromatography: Principles and Methods, ed. AA. Pharmacia Biotech AB, Uppsala, Sweden.
   Pharmacia Biotech 1997. Application Note 18‐1124‐57: Use of Sodium Hydroxide for Cleaning and Sanitizing Chromatography Media and Systems. Pharmacia Biotech, Uppsala, Sweden.
Key References
   Cooper, E.H., Turner, R., Webb, J.R., Lindblom, H., and Fägerstam, L. 1985. Fast liquid protein chromatography scale‐up procedures for the preparation of low molecular weight proteins from urine. J. Chromatogr. 327:269‐277.
  Good example of methods development with respect to optimization of resolution.
   Pharmacia Biotech 1985. FPLC Ion Exchange and Chromatofocusing: Principles and Methods. Pharmacia Biotech AB, Uppsala, Sweden.
  Contains detailed discussions of experimental approach, methodology, and applications for protein purification.
   Pharmacia Biotech, 1995. See above.
  Concise descriptions of theory and practice in planning and implementing ion‐exchange purification.
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