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Rapid and Effective Focusing in a Carrier Ampholyte Solution Isoelectric Focusing System: A Proteome Prefractionation Tool

2008; American Chemical Society; Volume: 7; Issue: 4 Linguagem: Inglês

10.1021/pr700677u

1535-3907

John C. Tran, Alan A. Doucette,

Biosensors and Analytical Detection

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.