Parish C R, Classon B J, Tsagaratos J, Walker I D, Kirszbaum L, McKenzie I F
Anal Biochem. 1986 Aug 1;156(2):495-502. doi: 10.1016/0003-2697(86)90284-8.
A procedure is described for fractionating detergent lysates of cells based on the ability of (NH4)2SO4 to induce phase separation of detergents such as Triton X-100, sodium deoxycholate, and sodium cholate, into detergent-rich and detergent-depleted phases. An analysis of six murine lymphocyte cell surface molecules revealed that the partitioning in Triton X-100 of each molecule was highly dependent upon the (NH4)2SO4 concentration, each antigen partitioning into the detergent-rich phase at a defined salt concentration. In contrast, none of the six molecules appeared in the detergent-rich phase of a Triton X-114 phase separation, even though two of the molecules, namely Ly-2/3 and L3T4, are well-characterized integral membrane proteins. It was also observed that (NH4)2SO4 resulted in the partitioning of many nonmembrane proteins into the detergent-rich phase, indicating that the procedure can be used to fractionate all cellular proteins. By judicious choice of (NH4)2SO4 concentrations, precipitation of cellular proteins at two different (NH4)2SO4 concentrations, and combining the method with subcellular fractionation prior to detergent solubilization, substantial enrichment and concentration of particular cellular proteins could be achieved.
本文描述了一种基于硫酸铵（(NH4)2SO4）诱导去污剂（如 Triton X-100、脱氧胆酸钠和胆酸钠）相分离成富含去污剂和去污剂耗尽相的能力，对细胞去污剂裂解物进行分级分离的方法。对六种小鼠淋巴细胞细胞表面分子的分析表明，每种分子在 Triton X-100 中的分配高度依赖于硫酸铵浓度，每种抗原在特定盐浓度下分配到富含去污剂的相中。相比之下，六种分子中没有一种出现在 Triton X-114 相分离的富含去污剂相中，尽管其中两种分子，即 Ly-2/3 和 L3T4，是特征明确的整合膜蛋白。还观察到硫酸铵导致许多非膜蛋白分配到富含去污剂的相中，这表明该方法可用于分级分离所有细胞蛋白。通过明智地选择硫酸铵浓度、在两种不同的硫酸铵浓度下沉淀细胞蛋白，并在去污剂溶解之前将该方法与亚细胞分级分离相结合，可以实现特定细胞蛋白的大量富集和浓缩。
