کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1176267 961839 2008 4 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Plant protein isolation and stabilization for enhanced resolution of two-dimensional polyacrylamide gel electrophoresis
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
Plant protein isolation and stabilization for enhanced resolution of two-dimensional polyacrylamide gel electrophoresis
چکیده انگلیسی

Two-dimensional polyacrylamide gel electrophoresis (2D–PAGE) is the common method of choice for proteomic analysis. By introducing several small changes, a method was developed that not only improved the resolution and reproducibility of 2D–PAGE but also shortened the time of analysis. Precipitation by alkaline phenol and methanol/ammonium acetate was the choice for protein extraction. However, instead of precipitating the proteins overnight at –20 °C, it was carried out for 2 to 3 h at –80 °C. Ethanol was used for the final wash of the protein precipitate instead of routinely used acetone. Dithiothreitol (DTT) was used in all solutions from the beginning, considerably improving the solubilization of precipitated proteins. Solubilization was further improved by using a mixture of detergents and denaturants at high concentrations along with large amounts of DTT. Both in-gel rehydration and cup-loading methods were used for isoelectric focusing (IEF). For in-gel rehydration, samples reduced with DTT were diluted with sample buffer containing 2-hydroxyethyl disulfide (2-HED) (1:3) or were cup-loaded on a strip rehydrated with sample buffer containing 2-HED. Glycerol (5%) was used in the sample buffer, and the focusing was performed at 15 °C. The applicability of the method was demonstrated using several soybean tissues.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 379, Issue 2, 15 August 2008, Pages 192–195
نویسندگان
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