The IgE (immunoglobulin E) receptor of cultured rat basophilic leukemia (RBL-1) cells, shown previously to be an M(r) = 45,000 to 50,000 glycoprotein, was purified by repetitive affinity chromatography on IgE-Sepharose. RBL-1 cells were either labeled exogenously with 125I by the lactoperoxidase method or endogenously by growing cells in the presence of D-[14C]glucosamine and L-[3H]leucine. Receptor was solubilized with nonionic detergent and purified on Sepharose columns containing one of several conjugated proteins. Columns with non-IgE proteins were used to remove cell constituents adhering nonselectively to protein-conjugated Sepharose 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 polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) also showed that the receptor was still heavily contaminated. These contaminants were not labeled with 125I or 14C, indicating the desirability of utilizing an endogenous protein label when the purification of plasma membrane proteins is studied. Subsequent modifications of the procedure showed that when the receptor was eluted at 4°C with 0.5 N acetic acid, 1% NP-40 (Nonidet P-40), followed by immediate neutralization, most of the binding activity was retained, which allowed further purification on a second IgE column. Receptor preparations then purified by repetitive affinity chromatography appeared to be at least 90% pure based on the distribution of 3H, 125I, or 14C radioactivity by SDS-PAGE in gels of differing, porosities, and represented at least a 5000-fold purification from the original cell extract with an overall yield of 10 to 15%.
|Number of pages||7|
|Journal||Journal of Biological Chemistry|
|State||Published - 1979|