“…Dithiothreitol Preparation and labelling of cytosol Overlying fat, connective tissue and the urethra were removed from the prostate glands and the glands were chopped finely. The tissue was then washed in medium A (10 mM-Tris-HCl, 0-1 mM-EDTA, 0-25 mM-dithiothreitol, pH 7-4) and homogenized as described by Evans & Pierrepoint (1975) at a tissue : medium ratio of 1 : 10 (w/v). Assessment of binding characteristics Sucrose density-gradient ultracentrifugation Cytosol (1 ml) was incubated with tritiated oestradiol-17ß, oestradiol-17 , oestrone or oestriol (0-74 nmol/1), in the presence or absence of a 100-fold excess of unlabelled steroid, at 0°C for 4-6 h. The labelled cytosol (400 µ ) was layered on to 5-20% sucrose density gradients, prepared in medium A and centrifuged at 114 000 # for 18 h at 3°C using a Beckmann SW50.1 swinging-bucket rotor in an L265B ultracentrifuge.…”