Department of Sericulture, Karnatak University, Dharwad, 580 003, Karnataka, India.
Appl Biochem Biotechnol. 2013 Feb;169(4):1459-66. doi: 10.1007/s12010-012-0067-0. Epub 2013 Jan 13.
The outstanding capability of two-dimensional gel electrophoresis in separating all types of proteins basically depends on the efficiency of sample preparation. Sample preparation is one of the most critical steps in two-dimensional gel electrophoresis. Unfortunately, due to severe solubility, resolution of protein on gel is usually hampered, and thus, analysis remains a difficult task. However, technically several problems are generally encountered during protein extraction and isoelectric focusing. In the present investigation, we emphasized on evaluation and comparison of six different protein solubilization methods intended for resolving and analyzing silkworm hemolymph proteins by two-dimensional gel electrophoresis. Our findings revealed that the buffer composition of 8 M urea, 4 % 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 40 mM Tris base, 65 mM dithiothreitol, and 0.2 % ampholyte can effectively solubilize and yields maximum protein spots.
二维凝胶电泳在分离各种类型蛋白质方面的出色能力基本上取决于样品制备的效率。样品制备是二维凝胶电泳中最关键的步骤之一。不幸的是，由于严重的可溶性问题，蛋白质在凝胶上的分辨率通常受到阻碍，因此分析仍然是一项艰巨的任务。然而，在技术上，在蛋白质提取和等电聚焦过程中通常会遇到几个问题。在本研究中，我们重点评估和比较了六种不同的蛋白质溶解方法，旨在通过二维凝胶电泳解析和分析家蚕血液蛋白。我们的研究结果表明，由 8M 尿素、4% 3-[(3-胆酰胺丙基)二甲氨基]-1-丙磺酸、40mM Tris 碱、65mM 二硫苏糖醇和 0.2%两性电解质组成的缓冲液可以有效地溶解并获得最大的蛋白质斑点。
