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Displacement Chromatography of Proteins
Publication Name:
Current Protocols in Protein Science
Unit Number:
Unit 8.9
DOI:
10.1002/0471140864.ps0809s59
Online Posting Date:
February, 2010 Abstract
This unit discusses the important parameters in designing and optimizing a separation by ion-exchange displacement chromatography, including preparing the sample and choosing a matrix, column, and buffer. Protocols are provided for testing a column, determining binding and elution conditions, displacing the sample, and cleaning, regenerating, and storing of displacement columns. Curr. Protoc. Protein Sci. 59:8.9.1-8.9.14. © 2010 by John Wiley & Sons, Inc.
Keywords: displacement; HPLC; ion-exchange chromatography
Table of Contents
- Introduction
- Strategic Planning
- Basic Protocol: Displacement Chromatography of Proteins Using Ion-Exchange Resin
- Support Protocol: Analysis of Displacement Chromatography Fractions by Second Dimension Reversed-Phased HPLC
- Commentary
- Literature Cited
- Figures
- Tables
Materials
Basic Protocol: Displacement Chromatography of Proteins Using Ion-Exchange Resin
Materials
- Appropriate buffers and solvents (see
- Column and resin (chromatography matrix; see
- Regeneration buffer (e.g., 2.0 M NaCl or other suitable salt)
- Loading buffer without sample
- Protein solution for purification, dissolved in loading buffer
- Displacer in loading buffer
- Buffer for displacer removal and column cleaning (e.g., REGENERATE, available from SACHEM)
- Column cleaning solutions:
- For anion-exchange columns:
- Acid, such as 15% acetic acid or 100 mM betaine (pH = 2.0)
- Base, such as 10 mM NaOH or 100 mM Na
3 PO4 (pH = 11.5) - For cation-exchange columns:
- Base, such as 100 mM KOH
- Acid, such as 15% acetic acid or 100 mM betaine (pH = 2.0)
- Nonionic detergent (e.g., NP-40, Triton X-100)
- Buffered 2 M NaCl
- Chromatographic system (see
NOTE: Longer columns tend to provide better recoveries. The aspect ratio (length to width) should be at least 50:1. Flow rates are low, so there are rarely problems with high backpressure.
Support Protocol: Analysis of Displacement Chromatography Fractions by Second Dimension Reversed-Phased HPLC
Materials
- Protein sample resulting from displacement chromatography HPLC solvents, for example:
- Mobile phase A = 95% HPLC grade Water, 5% Acetonitrile 0.05 % TFA (trifluoroacetic acid)
- Mobile phase B = 95% HPLC grade Acetonitrile, 5% HPLC grade Water 0.05% TFA
NOTE: The sample may be run neat or diluted in deionized water. It should not be dissolved in an organic solvent or it may not bind sufficiently to the stationary phase (matrix). The sample should not be dissolved in detergent-containing solutions.
NOTE: The reverse-phase mobile phases (solvents) are by convention installed on the HPLC channels A and B. The A solvent is typically the aqueous solvent (water) and the B solvent by convention is the organic solvent (e.g., acetonitrile, methanol, propanol).
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Figures
-
Figure 8.9.1Displacement sequence in protein separation. The three stages of protein displacement chromatography are shown in the figure. Following the initial loading of the sample in the selected equilibration buffer, the protein mixture starts to separate into zones. Once the displacer molecule is added to the buffer, the zones become very discrete bands of highly purified protein. The column is then regenerated and reused. -
Figure 8.9.2Typical displacement chromatography train. Weakly bound components may elute off the column in advance of the displacement train, which is composed of the more tightly bound components. In the literature, displacement bands are often represented as vertical bars. In practice, the component band shapes are truncated triangles with the steepness of the sides being dependent on such factors as flow rate and specific binding interactions of the components with the matrix and each other. -
Figure 8.9.3Calculation of column capacity. “A” refers to the total time from appearance of sample breakthrough until appearance of displacer breakthrough. “B” is the total time between breakthrough of salt gradient as measured by conductivity and appearance of displacer breakthrough. -
Figure 8.9.4Example of an ideal displacement chromatogram.
Literature Cited
| Literature Cited | |
| Cramer, S.M., Moore, J.A., Kundu, A., Li, Y., and Jayaraman, G. 1995. US Patent #5,478,924. Displacement Chromatography of Proteins using Low Molecular Weight Displacers. | |
| Frenz, J., van der Schrieck, P., and Horváth, C. 1985. Investigation of operating parameters in high-performance displacement chromatography. J. Chromatogr. 330:1-17. | |
| Horvath, C.S., Nahum, A., and Frenz, J. 1981. High performance displacement chromatography. J. Chromatogr. 218:365-393. | |
| Jayaraman, G., Li, Y., Moore, J.A., and Cramer, S.M. 1995. Ion-exchange displacement chromatography of proteins dendritic polymers as novel displacers. J. Chromatogr. A 702:143-155. | |
| Jayaraman, G., Li, Y., Kundu, A., Moore, J., and Cramer, S.M. 1997. Displacement chromatography of proteins using low molecular weight anionic displacers. Biotechnol. Adv. 15:749. | |
| Nagele, E., Vollmer, M., Horth, P., and Vad, C. 2004. 2D-LC/MS techniques for the identification of proteins in highly complex mixtures. Expert Rev. Proteomics 1:37-46. | |
| Tiselius, A., 1943. Displacement development in adsorption analysis. Ark. Kemi. Mineral Geol. 16A:1-18. | |
| Tugcu, N. 2007. "Purification of proteins using displacement chromatography". In Methods in Molecular Biology: Vol 421 Affinity Chromatography: Methods and Protocols. 2nd ed. (M. Zachariou, ed.) pp. 71-89. Humana Press, Totowa, N.J. | |
| Internet Resources | |
| http://www.sacheminc.com/industries/biotechnology/teaching-tools.html | |
| Displacement Chromatography 101. | |
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