High-Resolution Two-Dimensional Polyacrylamide Gel Electrophoresis: A Tool for Identification of Polymorphic and Modified Linker Histone Components

Abstract

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is a powerful technique used for separation, detection and quantification of complex protein mixtures expressed within cellular systems. Whereas a single-dimension electrophoretic separation allows to detect individual proteins in cell extracts mainly based on a single physicochemical parameter only and may be therefore inadequate for effective resolution of protein components, two-dimensional analysis is fully capable of simultaneous separation of majority protein constituents employing at least two independent parameters for their separation. Depending on the nature of protein complexes to be resolved, 2D-PAGE may comprise isoelectric focusing (IEF) or acetic acid-urea polyacrylamide gel electrophoresis (AU-PAGE) in the first dimension followed by sodium dodecyl suflate polyacrylamide gel electrophoresis (SDS-PAGE) in the second one. Therefore, this technique combines protein fractionation according to isoelectric point in the IFE or size and net charge in the AU-PAGE with the separation in conformity with their molecular weights in the SDS-PAGE gels. A combination of the IEF in the first dimension with the SDS-PAGE in the second dimension, originally introduced by O’Farrell (1975) for total cellular protein separation in Escherichia coli, is currently widely used in the majority of proteome analyses (Gorg et al., 2004; Weiss & Gorg, 2009). The fusion of AU-PAGE with the SDS-PAGE is especially handy for the identification of charged proteins of low to medium size in heterogeneous protein preparations containing a limited number of components such as cellular acid-soluble proteins including histones (Goldknopf & Busch, 1975). The one-dimension electrophoretic techniques separate the bulk of protein into distinct units independently of their conformation and keep them from forming the aggregates. At highly acidic pH of AUPAGE system, around pH 3.5, the positively charged proteins of similar size can be resolved from each other according to their charges. Addition of urea which is an efficient agent for breaking non-covalent protein interactions improve the protein solubility and facilitates electrophoretic migration. At an alkaline pH of the SDS-PAGE system, routinely above pH