[29] Preparation and use of antibodies against insoluble membrane proteins

Publisher Summary This chapter focuses on the preparation and use of antibodies against insoluble membrane proteins. The chapter describes some special problems encountered in preparing and using antibodies directed against membrane proteins. Most of these problems arise from the poor solubility of membrane proteins in aqueous media. Many membrane proteins are poor immunogens, and antigen–antibody reactions are then relatively weak. These difficulties can be largely overcome by slightly modifying the procedures normally used with soluble antigens. The first step in the purification of a membrane protein is almost always the isolation of the membrane with which the protein is associated. Because of the insolubility of most membrane proteins in aqueous media, their isolation must be performed in the presence of suitable detergents. Purification of the membrane protein should be thorough to avoid later problems; because many membrane proteins are weak immunogens, the presence of even small amounts of a more antigenic contaminant in the preparation used for immunization can lead to a strong, unwanted side reaction of the resulting serum.