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Sample preparation for two‐dimensional gel electrophoresis
Author(s) -
Shaw Margaret M.,
Riederer Beat M.
Publication year - 2003
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200300471
Subject(s) - isoelectric focusing , sample preparation , electrophoresis , sample (material) , chromatography , two dimensional gel electrophoresis , protocol (science) , membrane protein , chemistry , gel electrophoresis , proteomics , membrane , biochemistry , enzyme , pathology , gene , medicine , alternative medicine
The choice of sample preparation protocol is a critical influential factor for isoelectric focusing which in turn affects the two‐dimensional gel result in terms of quality and protein species distribution. The optimal protocol varies depending on the nature of the sample for analysis and the properties of the constituent protein species (hydrophobicity, tendency to form aggregates, copy number) intended for resolution. This review explains the standard sample buffer constituents and illustrates a series of protocols for processing diverse samples for two‐dimensional gel electrophoresis, including hydrophobic membrane proteins. Current methods for concentrating lower abundance proteins, by removal of high abundance proteins, are also outlined. Finally, since protein staining is becoming increasingly incorporated into the sample preparation procedure, we describe the principles and applications of current (and future) pre‐electrophoretic labelling methods.