Fractionation of detergent lysates of cells by ammonium sulphate-induced phase separation

Abstract

A procedure is described for fractionating detergent lysates of cells based on the ability of (NH 4) 2SO 4 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 (NH 4) 2SO 4 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 (NH 4) 2SO 4 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 (NH 4) 2SO 4 concentrations, precipitation of cellular proteins at two different (NH 4) 2SO 4 concentrations, and combining the method with subcellular fractionation prior to detergent solubilization, substantial enrichment and concentration of particular cellular proteins could be achieved.