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.
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
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
Figure 8.2.2 Liquid column chromatography system with gradient maker.
Figure 8.2.4 Typical chromatogram for determining the capacity of an ion‐exchange column. The volume of eluant ( x) at 50% full‐scale deflection is used to calculate the breakthrough capacity (QB50).
Figure 8.2.6 Peristaltic pump accommodating three pumping channels for continuous gradient formation.
|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.|
|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.|