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J Proteome Res. 2008 Apr;7(4):1761-6. doi: 10.1021/pr700677u. Epub 2008 Feb 20.

Rapid and effective focusing in a carrier ampholyte solution isoelectric focusing system: a proteome prefractionation tool.

Author information

1
Department of Chemistry, Dalhousie University, Halifax, Nova Scotia, Canada.

Abstract

Preparative (solution) isoelectric focusing (sIEF) is a proven technique for proteome prefractionation, but carries limitations which include the risk of protein loss from isoelectric precipitation, poor focusing, and excessively long separation times. This report describes a simple yet effective method to achieve rapid focusing (as fast as 1 h) and maximize protein recovery using a carrier ampholyte sIEF system. Cathodic drift was not present over the time course of the experiment using our eight-chamber device, and we demonstrate the effectiveness of this device for focusing proteome mixtures. We also discuss an MS-compatible acidic wash protocol, which is shown to enhance the recovery of proteins following sIEF, thus, improving detection by LC-MS/MS. These approaches overcome significant shortcomings of the technique, enabling effective prefractionation prior to MS analysis.

PMID:
18284188
DOI:
10.1021/pr700677u
[Indexed for MEDLINE]

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