THE SIZE OF SITES CONTAINING SR PROTEINS IN HUMAN NUCLEI - PROBLEMS ASSOCIATED WITH CHARACTERIZING SMALL STRUCTURES BY IMMUNOGOLD LABELING

Some SR proteins are associated with eukaryotic transcripts as they mo ve from synthetic sites (transcription ''factories''), through downstr eam sites, to nuclear pores. Downstream sites can also be isolated as large nuclear ribonucleoprotein particles of similar to 200 5 (diamete r similar to 50 nm). In ultrathin sections of HeLa nuclei, indirect im munogold labeling with a specific antibody gives many small clusters o f similar to 10 gold particles (diameter 50-80 nm). We gauged errors i n estimating the diameter of underlying structures marked by immunogol d probes (lengths similar to 20 nm). We examined systematically how pr obe dimensions affected cluster diameter. Probes contained one to thre e immunoglobulin molecules, sometimes a protein A molecule, and a gold particle of 5-15 nm. We found that (a) immunolabeling particles were tightly packed, (b) reducing particle size by 5 nm reduced cluster dia meter by 10 nm, (c) reducing the number of immunoglobulins in the immu nolabeling sandwich from three to two reduced cluster diameter by simi lar to 4 nm, (d) replacing the last immunoglobulin in a sandwich with protein A increased diameter by similar to 7 nm and led to a periphera l concentration of particles, and (e) increasing the number of layers in the sandwich increased sensitivity. Assuming that underlying struct ures had diameters of 50 nm, we find that errors ranged from -20% to 50%.