No peptide left behind: the "out of range" recovery in IPG-IEF fractionation
Elisabetta Iori1, Marcello Rattazzi, Renato Millioni
1Department of Medicine, University of Padova, Via Giustiniani 2, 35121, Padua, Italy.
Abstract:
IEF is often used in multidimensional shotgun proteomics and the narrow range of 3.5-4.5 is the recommended pH interval for the fractionation of tryptic peptides. Usually, even if IEF is performed in IPG strip with a narrow range pH, the entire sample must be loaded onto the strip, including the "out of IPG range" peptides. We describe a simple protocol to recover at least a part of these missing peptides and show that this recovery significantly influences the overall fractionation result, increasing the number of the identified proteins and the protein coverage.
More Related Videos
Related Concept Videos
Two-dimensional Gel Electrophoresis
The first dimension separation uses the isoelectric focusing or IEF technique performed on immobilized pH gradient (IPG) strips that separate proteins according to their isoelectric points.
Biological samples, such...
Insensitive Nuclei Enhanced by Polarization Transfer (INEPT)
Ion-Exchange Chromatography


