Santoni V, Rabilloud T, Doumas P, Rouquié D, Mansion M, Kieffer S, Garin J, Rossignol M
Biochimie et Physiologie Moléculaire des Plantes, INRA/ENSA-M/CNRS URA 2133, Montpellier, France.
Electrophoresis. 1999 Apr-May;20(4-5):705-11. doi: 10.1002/(SICI)1522-2683(19990101)20:4/5<705::AID-ELPS705>3.0.CO;2-Q.
An extensive proteomic approach relies on the possibility to visualize and analyze various types of proteins, including hydrophobic proteins which are rarely detectable on two-dimensional electrophoresis (2-DE) gels. In this study, two methods were employed for the purification of hydrophobic proteins from Arabidopsis thaliana leaf plasma membrane (PM) model plants, prior to analysis on 2-DE immobilized pH gradient (IPG) gels. Solubilization efficiency of two detergents, (3-[(3-cholomidopropyl)-1-propanesulfonic acid (CHAPS) and C8phi, were tested for the recovery of hydrophobic proteins. An immunological approach was used to determine the efficiency of the above methods. Fractionation of proteins by Triton X-114 combined with solubilization with CHAPS resulted in the inability to detect hydrophobic proteins on 2-DE gels. The use of C8phi for protein solubilization did not improve this result. On the contrary, after treatment of membranes with alkaline buffer, the solubilization of PM proteins with detergent C8phi permitted the recovery of such proteins on 2-DE gels. The combination of membrane washing and the use of zwitterionic detergent resulted in the resolution of several integral proteins and the disappearance of peripheral proteins. In the resolution of expressed genome proteins, both large pH gradients in the first dimension and various acrylamide concentrations in the second dimension must be used. Notwithstanding, it is important to combine various sample treatments and different detergents in order to resolve soluble and hydrophobic proteins.
一种广泛的蛋白质组学方法依赖于可视化和分析各种类型蛋白质的可能性，包括在二维电泳（2-DE）凝胶上很少能检测到的疏水蛋白。在本研究中，在对2-DE固定pH梯度（IPG）凝胶进行分析之前，采用了两种方法从拟南芥叶质膜（PM）模型植物中纯化疏水蛋白。测试了两种去污剂（3-[(3-胆酰胺丙基)-1-丙烷磺酸（CHAPS）和C8phi]的增溶效率，以回收疏水蛋白。采用免疫学方法来确定上述方法的效率。用Triton X-114分离蛋白质并结合CHAPS增溶，导致在2-DE凝胶上无法检测到疏水蛋白。使用C8phi增溶蛋白质并没有改善这一结果。相反，用碱性缓冲液处理膜后，用去污剂C8phi增溶PM蛋白可使此类蛋白在2-DE凝胶上得以回收。膜洗涤和两性离子去污剂的使用相结合，使得几种整合蛋白得以分离，外周蛋白消失。在解析表达的基因组蛋白时，必须在第一维使用大的pH梯度，在第二维使用不同的丙烯酰胺浓度。尽管如此，为了解析可溶性蛋白和疏水蛋白，将各种样品处理方法和不同的去污剂结合起来很重要。
