Protocol

Protein Purification Protocols

Volume 244 of the series Methods in Molecular Biology pp 233-237

Chromatofocusing

  • Timothy J. MantleAffiliated withDepartment of Biochemistry, Trinity College
  • , Patricia NooneAffiliated withDepartment of Biochemistry, Trinity College

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Abstract

Chromatofocusing (1) is essentially ion-exchange chromatography (conventionally using an anion exchanger) where the elution conditions are obtained by dropping the pH so that the proteins elute in order of their isoelectric points (pI). Although in principle this can be achieved by equilibrating an ion exchanger at a relatively alkaline pH and then titrating the resin with a buffer at a lower, more acidic pH, in practice it is more usual to utilize amphoteric buffers titrated to the lower pH to generate a more linear pH gradient. Because the pH gradient is generated gradually as the eluting buffer moves down the column, it is possible to add large volumes to the column or to add a second batch during the run and, in either case, single identical components with a particular pI will focus and elute as a single peak.