Two–dimensional polyacrylamide gel electrophoresis, with immobilized pH gradients in the first dimension, of barley seed proteins: Discrimination of cultivars with different malting grades

Abstract

The suitability of high-resolution two-dimensional gel electrophoresis for barley cultivar discrimination and for classification with respect to their malting properties was studied. Seed proteins of 14 barley cultivars with different malting qualities were extracted with urea/dithiothreitol/Nonidet P-40 buffer and subjected to two-dimensional gel electrophoresis with immobilized pH gradients in the first dimension (IPG-DALT). The results of IPG-DALT were compared to the protein patterns obtained by a standard technique, sodium dodecyl sulfate polyacrylamide gel electrophoresis of hordeins. Sodium dodecyl sulfate-gel electrophoresis yielded seven different "B" and four different "C" hordein patterns; "A" and "D" hordein patterns were uniform in all cultivars tested. Four cultivars could be distinguished unequivocally, the others were classified into three groups containing between two and five cultivars. In contrast to these findings. IPG-DALT yielded three different "A", eight different "B", four different "C" and two different "D" hordein patterns. When the "A", "B", "C" and "D" hordein patterns were combined, ten cultivars exhibited unique hordein patterns whereas the remaining ones were classified into two groups containing two cultivars each. Moreover, when albumin and globulin proteins were used for evaluation in addition to the hordeins, all cultivars could be discriminated by IPG-DALT. IPG-DALT, performed on small-scale and/or ready-made gels, proved to be an ideal complementary system to one-dimensional electrophoretic methods for routine seed testing purposes because of its speed, reliability, and simplicity. IPG-DALT was also applied to study the relationship between the different polypeptide patterns and the malting quality. Although cultivars with identical one-dimensional protein patterns but different malting quality could be successfully differentiated by IPG-DALT, a direct correlation between specific protein spots or protein patterns to the malting quality was not found within the cultivars tested.

Changes in Cytoskeletal Protein Composition Indicative of an Epithelial-Mesenchymal Transition in Human Micrometastatic and Primary Breast Carcinoma Cells

“…Whole cell homogenates were separated by isoelectric focusing using in-gel rehydration buffer consisting of 7 mol/L urea, 2 mol/L thiorurea, 2% CHAPS, 0.5% IPG Buffer, and 10 mmol/L DTT (pH 4-7) immobilized IPG-Strip (Amersham Biosciences, Freiburg, Germany) essentially as described by Gö rg et al (10,11), followed by 12% SDS-PAGE using a Bio-Rad Protean II XL chamber. To visualize protein spots and to identify differentially expressed polypeptides by visual inspection, gels obtained from triplicate experiments were silver-stained as described before (12) and scanned using an Epson expression 1680 pro scanner.…”

Two-Dimensional Polyacrylamide Gel Electrophoresis - A Practical Perspective

“…This gradient drift usually causes reduction in reproducibility. Together with low mechanical stability, batch-to-batch variability, and the probability of the soft gel to be stretched or broken, Immobiline pH gradient was introduced by Gorg and colleagues (Gorg, 1993;Gorg et al, 1995;Gorg et al, 1998;Gorg et al, 2000;Gorg et al, 1992;Gorg et al, 1988b). More description can be found in section 3.1.3.…”

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.…”