Key Laboratory of Protein Chemistry and Developmental Biology of National Education Committee, College of Life Sciences, Hunan Normal University, Changsha, PR China.
Electrophoresis. 2012 Jan;33(2):316-24. doi: 10.1002/elps.201100364.
SDS is mostly used to enhance the solubilization and extraction of membrane proteins due to its strong detergency and low cost. Nevertheless, SDS interferes with the subsequent procedures and needs to be removed from the samples. In this work, a special gradient gel electrophoresis (GGE) system was developed to remove SDS from the SDS-solubilized protein samples. As a proof-of-principle experiment, the GGE system was designed to be composed of an agarose loading layer, six polyacrylamide fractionation layers with different concentrations and a high-concentration polyacrylamide sealing layer. The advantages of the GGE system are that it not only can electrophoretically remove SDS efficiently so that the protein loss resulted from the repeated gel washing after electrophoresis was avoided, but also can reduce the complexity of the sample, prevent the precipitation of proteins after loading and avoid the loss of proteins with low molecular weight during the electrophoresis. Using GGE system, about 85% of SDS in the sample and gel was electrophoretically removed and the proteins were fractionated. Compared with the two representative gel-based sample cleanup methods reported in literature, GGE-based strategy significantly improved the identification efficiency of proteins in terms of the number and coverage of the identified proteins.
SDS 主要由于其强大的去污能力和低成本而被用于增强膜蛋白的溶解和提取。然而，SDS 会干扰后续的步骤，因此需要从样品中去除。在这项工作中，开发了一种特殊的梯度凝胶电泳（GGE）系统来去除 SDS-溶解蛋白样品中的 SDS。作为原理验证实验，设计了 GGE 系统，由琼脂糖加载层、六个具有不同浓度的聚丙烯酰胺分层层和一个高浓度聚丙烯酰胺密封层组成。GGE 系统的优点不仅在于能够有效地电泳去除 SDS，从而避免了电泳后反复凝胶洗涤导致的蛋白质损失，而且还可以降低样品的复杂性，防止加载后蛋白质沉淀，并避免在电泳过程中丢失低分子量蛋白质。使用 GGE 系统，大约 85%的 SDS 在样品和凝胶中被电泳去除，并对蛋白质进行了分层。与文献中报道的两种有代表性的基于凝胶的样品净化方法相比，基于 GGE 的策略在鉴定蛋白质的数量和覆盖率方面显著提高了蛋白质的鉴定效率。
