Immunogold localisation of the intermediate chain within the protein complex of cytoplasmic dynein

It was our goal to determine the location of the intermediate chain within the complex of cytoplasmic dynein by immunoelectron microscopy. To do so we generated two monoclonal antibodies (m74-1 and m74-2) specific for the intermediate chain, Both antibodies recognised the intermediate chain by sodium dodecyl sulphate-polyacrylamide gel electrophoresis immunoblot and ELISA assays of native and denatured proteins. When sucrose density gradient-purified cytoplasmic dynein from bovine brain was incubated with the gold-conjugated monoclonal antibodies, m74-1 and m74-2, and examined by negative staining, the gold label was found opposite the globular heads at the base of the V-shaped stalk of the motor complex, The labelling of the intermediate chain is the first mapping of a component within cytoplasmic dynein, The identification of the intermediate chain at the base of the complex supports a possible docking function of the intermediate chain. (C) 1996 Academic Press