The cell surface receptor for immunoglobulin E. I. The use of repetitive affinity chromatography for the purification of a mammalian receptor.

The IgE (immunoglobulin E) receptor of cultured rat basophilic leukemia (RBL-1) cells, shown previously to be an M, = 45,000 to 50,000 glycoprotein, was purified by repetitive affinity chromatography on IgE-Sepha- rose. RBL-1 cells were either labeled exogenously with “‘1 by the lactoperoxidase method or endogenously by growing cells in the presence of o-[14C]glucosamine and L-[3H]leucine. Receptor was solubilized with nonionic detergent and purified on Sepharose columns contain- ing one of several conjugated proteins. Columns with non-IgE proteins were used to remove cell constituents adhering nonselectively to protein-conjugated Sepha- rose and columns with IgE were used to specifically bind the receptor. Initially, attempts to elute receptor from IgE columns resulted in major losses of binding activity. Analysis of 3H radioactivity using polyacryl- amide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) also showed that the receptor was still heavily contaminated. These contaminants were not labeled with “‘1 or 14C, indicating the desirability of utilizing an endogenous protein label when the purifi- cation of plasma membrane proteins is studied. Subse- quent modifications of the procedure showed that when the receptor was eluted at 4°C with 0.5