Direct Labeling of Components in Protein Complexes by Immuno-Electron Microscopy

Immunogold labeling has been used extensively to study the localization and distribution of antigens within cells and tissue. Gold-antibody complexes are generated by charge interaction of the colloidal gold with the antibody, although the exact nature of this interaction is not fully understood. Colloidal gold can be prepared in sizes ranging from 1 nm to over 30 nm allowing a simultaneous detection of several antigens within the same electron microscope preparation. Colloidal gold conjugated antibodies can be employed to label whole cells for light microscopy and to stain ultrathin sections for electron microscopy. In addition colloidal gold probes can also be used to identify the position of subunits within isolated protein complexes. We will present here our methods of preparing colloidal gold-conjugated primary antibodies. In combination with a modified technique of negative staining these primary antibodies can then be employed to identify subunits within protein complexes.