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Ion exchange chromatography – basic principles and application

Cummins, Phil and Dowling, Oonagh and O'Connor, Brendan (2011) Ion exchange chromatography – basic principles and application. In: Walls, Dermot and Loughran, Sinéad T., (eds.) Protein Chromatography. Methods in Molecular Biology, 681 . Humana Publishers Ltd., pp. 215-229. ISBN 978-1-60761-913-0

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Abstract

Ion-Exchange Chromatography (IEC) allows for the separation of ionizable molecules on the basis of differences in charge properties. Its large sample-handling capacity, broad applicability (particularly to proteins and enzymes), moderate cost, powerful resolving ability, and ease of scale-up and automation have led to it becoming one of the most versatile and widely used of all liquid chromatography (LC) techniques. In this chapter, we review the basic principles of IEC, as well as the broader criteria for selecting IEC conditions. By way of further illustration, we outline protocols necessary to partially purify a serine peptidase from bovine whole brain cytosolic fraction, covering crude tissue extract preparation through to partial purification of the target enzyme using anion-exchange chromatography. Protocols for assaying total protein and enzyme activity in both pre- and post-IEC fractions are also described. The target serine peptidase, prolyl oligopeptidase (POP, EC3.4.21.26), is an 80 kDa enzyme with endopeptidase activity towards peptide substrates of ≤30 amino acids. POP is a ubiquitous post-proline cleaving enzyme with particularly high expression levels in the mammalian brain, where it participates in the metabolism of neuroactive peptides and peptide-like hormones (e.g. thyroliberin, gonadotropin-releasing hormone).

Item Type:Book Section
Refereed:Yes
Uncontrolled Keywords:Chromatographic methods; Microfluidics; Native protein isolation; Protein biochemistry; Protein quantitation; Purification schemes; Recombinant antibody production
Subjects:Biological Sciences > Biotechnology
Biological Sciences > Biochemistry
DCU Faculties and Centres:DCU Faculties and Schools > Faculty of Science and Health > School of Biotechnology
Research Initiatives and Centres > Irish Separation Science Cluster (ISSC)
Publisher:Humana Publishers Ltd.
Official URL:http://www.springer.com/new+%26+forthcoming+titles+%28default%29/book/978-1-60761-912-3
Copyright Information:© 2011 Springer (Humana)
Use License:This item is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 3.0 License. View License
Funders:Science Foundation Ireland, Enterprise Ireland
ID Code:17809
Deposited On:19 Feb 2013 10:24 by Brendan O'Connor. Last Modified 02 Nov 2016 14:03

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