Antibodies to the Golgi complex and the rough endoplasmic reticulum

Abstract

Rabbits were immunized with membrane fractions from either the Golgi complex or the rough endoplasmic reticulum (RER) by injection into the popliteal lymph nodes. The antisera were then tested by indirect immunofluorescence on tissue culture cells or frozen, thin sections of tissue. There were may unwanted antibodies to cell components other than the RER or the Golgi complex, and these were removed by suitable absorption steps. These steps were carried out until the pattern of fluorescent labeling was that expected for the Golgi complex or RER. Electron microscopic studies, using immunoperoxidase labeling of normal rat kidney (NRK) cells, showed that the anti-Golgi antibodies labeled the stacks of flattened cisternae that comprise the central feature of the Golgi complex, many of the smooth vesicles around the stacks, and a few coated vesicles. These antibodies were directed, almost entirely, against a single polypeptide with an apparent molecular weight of 135,000. The endoplasmic reticulum (ER) in NRK cells is an extensive, reticular network that pervades the entire cell cytoplasm and includes the nuclear membrane. The anit-RER antibodies labeled this structure alone at the light and electron microscopic levels. They were largely directed against four polypeptides with apparent molecular weights of 29,000, 58,000, 66,000, and 91,000. Some examples are presented, using immunofluorescence microscopy, where these antibodies have been used to study the Golgi complex and RER under a variety of physiological and experimental condition . For biochemical studies, these antibodies should prove useful in identifying the origin of isolated membranes, particularly those from organelles such as the Golgi complex, which tend to lose their characteristic morphology during isolation.