By James H. Clark Ph. D., Ernest J. Peck Jr. Ph. D. (auth.)
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Sample text
Anyone faced with nonlinear Scatchard plots after considering the points discussed above (Sects. f. 1-5) should use these sources for a thorough analysis of interacting systems. 3. Receptor States and 3H-Steroid Exchange Most biologic systems contain both occupied and unoccupied receptors. The assessment of both forms is important if an· accurate picture of the relationship between steroid binding and physiology is to emerge. , 1972a). , 1973a). Subsequently the 3H-steroid exchange technique has been widely employed (for references, see Sects.
1971). , 1975a). Although these procedures are rapid and generally effective for the separation of free from bound S, they are not without fault. One should employ them cautiously, always bearing in mind the dangers inherent in determining equilibrium values under nonequilibrium conditions. As discussed earlier, both occupied and unoccupied sites can be measured by 3Hestradiol exchange. , 1972a) was modified for the measurement of cytosol-estrogen receptors by Katzenellenbogen et al. (1973).
Saturation analysis is performed as described above for the 3H-estradiol exchange assay. The nuclear fractions are incubated with shaking at 4°C for 18-24 h. 5 ml TO buffer is added, and the samples are centrifuged at 800 g for 10 min. 0 ml ethanol. The radioactivity in the ethanol extract is determined as explained for the 3fI-estradiol exchange procedure. 3) Nuclear Binding and Retention of Receptor-Hormone Complexes The interactions between RnS complexes and nuclear binding sites are poorly understood.