Chromatofocusing

Alan Williams1

1 Pharmacia Biotech, Piscataway, New Jersey
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 8.5
DOI:  10.1002/0471140864.ps0805s02
Online Posting Date:  May, 2001
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Abstract

This unit discusses choice of chromatofocusing medium and buffers, determination of the quantity of medium needed, and preparation of CF buffers. A protocol is provided for a typical chromatofocusing experiment, and support protocols describe regeneration of the chromatofocusing column and several methods for separation of the polybuffer from the purified protein.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Separation of Proteins by Chromatofocusing
  • Support Protocol 1: Regeneration of the CF Column
  • Support Protocol 2: Removal of Polybuffer from Protein
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Separation of Proteins by Chromatofocusing

  Materials
  • CF gel medium PBE 94 or PBE 118 or prepacked CF column Mono P (Pharmacia Biotech; see , Table 8.5.1, and Table 8.5.2)
  • CF start buffer (see and Table 8.5.1)
  • CF elution buffer (see and Table 8.5.1)
  • Protein sample to be purified
  • Sintered‐glass filter funnel, medium porosity
  • Side‐arm flask
  • Chromatographic system including:
    •  Appropriate chromatographic column
    •  Fluid delivery system with appropriate pump
    •  Sample injection valve
    •  UV monitor suitable for use at 280 nm
    •  pH monitor with flowthrough electrode (optional)
    •  Recorder
    •  Fraction collector
  • Additional reagents and equipment for checking packed chromatographic bed (unit 8.4), desalting via gel‐filtration chromatography (unit 8.3), and protein assay (Chapter 3)

Support Protocol 1: Regeneration of the CF Column

  • 1 M NaCl
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Figures

Videos

Literature Cited

Literature Cited
   Gianazza, E. and Righetti, P.G. 1980. Size and charge distribution of macromolecules in living systems. J. Chromatogr. 193:1‐8.
   Hutchens, W.T. 1989. Chromatofocusing. In Protein Purification: Principles, High Resolution Methods, and Applications (J.C. Janson and L. Rydén, eds.) pp. 149‐174. VCH Publishers, New York.
  Pharmacia Biotech. 1985. FPLC Ion Exchange and Chromatofocusing: Principles and Methods. Pharmacia Biotech AB, Uppsala, Sweden.
   Scopes, R.K. 1994. Protein Purification, Principles and Practice, 3rd ed., pp. 81‐85. Springer‐Verlag, New York.
   Sluyterman, L.A. Æ. and Elgersma, O. 1978. Chromatofocusing: Isoelectric focusing on ion exchange columns. I. General principles. J. Chromatogr. 150:17‐30.
   Sluyterman, L.A. Æ. and Wijdenes, J. 1978. Chromatofocusing: Isoelectric focusing on ion exchange columns. II. Experimental verification. J. Chromatogr. 150:31‐44.
Key Reference
   Pharmacia Biotech. 1984. Chromatofocusing with Polybuffer and PBE. Pharmacia Biotech AB, Uppsala, Sweden.
  An excellent discussion of CF theory including practical techniques and model applications for the separation of proteins by CF.
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