Two‐dimensional electrophoresis with immobilized pH gradients of leaf proteins from barley (Hordeum vulgare): Method, reproducibility and genetic aspects

Abstract

Leaf proteins from 14 barley cultivars (Hordeum vulgare) were analyzed by two-dimensional electrophoresis with immobilized pH gradients (IPG 4-7 and IPG 6-10) in the first dimension. Highly reproducible two-dimensional patterns were obtained, owing to constant spot positions along the isoelectric focusing axis. A number of variety-specific protein spots were detected, allowing us to discriminate barley cultivars not only into main groups but into individual cultivars.

Two-Dimensional Polyacrylamide Gel Electrophoresis - A Practical Perspective

“…However, carrier ampholytes-based pH gradient made in soft unsupported tube gels (typically 4 % acrylamide) are not really stable, depending on lot of ampholytes, and prone to cathodic drift (a progressive loss of basic proteins during long running of electrofocusing under electric field), leading to low reproducibility and requiring of careful control of application of electric field. The key development in improving reproducibility of 2-D electrophoresis was the introduction of immobilized pH gradients (IPG) that almost replaced carrier ampholyte-based pH gradient in tube gel (Gorg et al, 1988a;Gorg et al, 1988b). Although procedure for casting of IPG gels was described in details (Bossi et al, 1988;Gianazza, 1999), precast IPG gels with wide or various narrow range of pH are now commercially available (but more expensive).…”

Comparison of protein solubilization methods suitable for proteomic analysis of soybean seed proteins

“…Protein was extracted by vortexing 100 mg of seed powder with 300 l of lysis solution (8 M urea, 4% CHAPS, 2% ampholyte [pH [3][4][5][6][7][8][9][10]) and was sonicated for 40 min at room temperature. The extract was centrifuged at 20,800g for 10 min.…”

“…The precipitate was collected by centrifugation (20,800g, 20 min, 4°C), and the pellet was washed twice with 0.1 M ammonium acetate in methanol, with ice-cold 80% acetone, and Wnally once with cold 70% ethanol. The pellet was resuspended in 0.5 to 1.0 ml of extraction solution (8 M urea, 2 M thiourea, 2% CHAPS, 2% Triton X-100, 50 mM DTT, 0.5% [w/v] ampholytes [pH [3][4][5][6][7][8][9][10]) by pipetting and vortexing at 25 to 30°C. The samples were incubated for 1 h at room temperature with agitation, and the extract was used for protein determination and 2D analysis.…”

“…Although several methods for 2D analysis of plant and seed proteins have been reported in a variety of crops [6][7][8][9][10][11][12][13], only a limited number of methods have been reported for soybean seed protein analysis [14][15][16]. In this study, we compared four diVerent methods of soybean seed protein extraction for their compatibility with 2D-PAGE analysis and with respect to their eYciency in solubilizing proteins and subsequent identiWcation by MALDI-TOF-MS and LC-MS.…”

Expression in Multigene Families. Analysis of Chloroplast and Mitochondrial Proteases

“…Washed membranes were precipitated again and resuspended in storage buffer (0.3 M sorbitol, 10 mM HEPES-KOH, pH 8.0, 10 mM MgCl 2 , 10 mM NaCl) to 1 to 2 mg chlorophyll/mL, and either used immediately or stored at 270°C. Proteins were extracted from membranes and separated from pigments by resuspension in 20% TCA and 1% b-mercaptoethanol in acetone, as described previously (Gorg et al, 1988), and solubilized in two-dimensional sample buffer (8 M urea, 2 M thiourea, 2% w/v CHAPS, 1% w/v NP-40, 100 mM dithiothreitol). Isoelectric focusing was performed using 18-cm IPG strips (Amersham Biosciences) covering the pI range of 5 to 6.…”