A COVALENT FLUORESCENT-GOLD IMMUNOPROBE - SIMULTANEOUS DETECTION OF APRE-MESSENGER-RNA SPLICING FACTOR BY LIGHT AND ELECTRON-MICROSCOPY

Immunoprobes that combine a fluorescent label with a 1.4-nm gold clust er compound have been prepared by covalent conjugation with polyclonal antibody Fab' fragments. These new immunoconjugates allow the collect ion of two complementary sets of data, from fluorescence and electron microscopy, from a single labeling experiment. We find that incorporat ion of one or more fluorescein moieties into the coordinated ligands o f the 1.4-nm Nanogold gold cluster label yields a stable, dual-functio n immunolabel in which fluorescence quenching is negligible. In a seco nd synthetic strategy, Nanogold and fluorescein were separately covale ntly conjugated to Fab' fragments to yield a probe with very similar p roperties. With the use of Fab' fragments, the entire probe is smaller than a whole IgG molecule, and it exhibited excellent penetration pro perties. It was used to localize the pre-mRNA splicing factor SC35 in the HeLa cell nucleus by both fluorescence and electron microscopy.