Very alkaline immobilized pH gradients for two‐dimensional electrophoresis of ribosomal and nuclear proteins

Abstract

Basic proteins normally lost by the cathodic drift of carrier ampholyte focusing, or separated by NEPHGE with limited reproducibility, could be well separated by two-dimensional (2-D) electrophoresis under equilibrium conditions using immobilized pH gradients (IPGs) 4-10 and 6-10 using a previously published protocol (Görg et al., Electrophoresis 1988, 9, 531-546). In the present study we have extended the pH gradient to pH 12 with IPGs 8-12, 9-12 and 10-12 for the analysis of very basic proteins. Different optimization steps with respect to pH engineering, gel composition and running conditions, such as substitution of acrylamide by dimethylacrylamide and addition of isopropanol with and without methylcellulose to the IPG rehydration solution (in order to suppress the reverse electroosmotic flow) were necessary to obtain highly reproducible 2-D patterns of ribosomal proteins from HeLa cells and mouse liver. Histones from chicken erythrocyte nuclei as well as total cell extracts of erythrocytes were also successfully separated under steady-state conditions. Due to the selectivity of isoelectric focusing in IPG 9-12, where the more acidic proteins abandon the gel, the tedious procedure of nuclei preparation prior to histone extraction can be omitted.

Strategies for the enrichment and identification of basic proteins in proteome projects

“…IPG monomers were prepared as 0.2 M solutions in n-propanol as described previously [19,34] or purchased commercially (IPG monomer pK . 12; Aldrich, St. Louis, MO, USA).…”

“…12; Aldrich, St. Louis, MO, USA). 17 cm pH 9-12 IPG dry strips were produced using a modification of the recipe described by Molloy et al [18] for pH 8-11 IPGs (Table 1) and by Görg et al, [19]. Gel sheets were washed as described [18][19], dried, and cut into 3 mm thick strips.…”

“…These include active water transport towards the anode (reverse electroendosmosis) caused by the strong positive charge of basic acrylamido buffers, as well as the hydrolysis of acrylamide to acrylic acid at alkaline pH and the migration of reducing agents such as DTT, leading to streaky 2-DE patterns [1]. These effects have been overcome to some extent by changing the composition of the IPG strips to utilize N,N-dimethylacrylamide (DMA; [19]) or N-acryloylaminoethoxyethanol (AAEE; [20]), and by adding isopropanol or glycerol to 2-DE sample buffers [19,21], as well as utilizing nonionic reducing agents, such as tributyl phosphine [22]. However, it is by no means standard methodology to reproducibly separate proteins with pIs greater than pH 9.0.…”

Proteomics in human cancer research

“…There are already some promising results in this respect. Acrylamide N-substituted derivatives such as dimethylacrylamide, found to be more resistant to hydrolysis, are now available [17]. The absence of a suitable acrylamide buffer above pH~10.5 is, however, still a major problem.…”

Global proteome analysis of hepatitis B virus expressing human hepatoblastoma cell line HepG2

“…The development of proteomic techniques has provided powerful tools for studying complex cell biological processes and alterations in global protein expression [Görg et al, 1997]. Methods have evolved for parallel high throughput analyses of gene and protein expression, leading to the identification of markers of disease progression unrecognized previously, and diseaseassociated protein targets that contribute to pathogenesis.…”