A mild method for the purification of guinea pig peritoneal macrophage Fc gamma receptors. Affinity chromatography and elution of the receptor with reducing agents.
A method for the purification of Fc gamma receptors from guinea pig peritoneal macrophages using mild conditions is described. The method is based on the observation that reduction and alkylation of IgG disulfide bonds partially or completely abrogate their binding to Fc gamma receptors. Cell lysates were directly applied to Sepharose-IgG or Sepharose-TNP-Ab(IgG) and the specifically bound Fc gamma receptor was eluted from adsorbents by incubation with reducing agents (2-mercaptoethanol or dithiothreitol). Alternatively, cell lysates were first treated with IgG and then applied to Protein A-Sepharose and the receptor was eluted with reducing agents. Yields of the purified Fc gamma receptor preparations and their activities were considerably higher than when the receptor was eluted from affinity chromatography gels with acetic acid or other acidic buffers or chaotropic agents. The best results were obtained when Fc gamma receptor-IgG complexes were applied to Protein A-Sepharose. No significant difference in the subunit structure was observed using SDS-PAGE when receptor preparations obtained by elution with reducing agents were compared with preparations obtained by elution with acidic buffers.
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D004229:Dithiothreitol; D006168:Guinea Pigs; D007143:Immunoglobulin Heavy Chains / Q000276:immunology*; D007142:Immunoglobulin gamma-Chains / Q000276:immunology*; D046911:Macromolecular Substances; D008264:Macrophages / Q000276:immunology*; D011961:Receptors, Fc / Q000302:isolation & purification*; D017452:Receptors, IgG; D013438:Sulfhydryl Compounds
D007143:Immunoglobulin Heavy Chains; D007142:Immunoglobulin gamma-Chains; D046911:Macromolecular Substances; D011961:Receptors, Fc; D017452:Receptors, IgG; D013438:Sulfhydryl Compounds; D004229:Dithiothreitol