Assay of nuclear estradiol receptor by exchange on glass fiber filters.
A glass fiber filter exchange assay for nuclear estradiol receptor in human endometrium has been developed. It permits both exchange and measurement of bound radioactivity to be performed without any transfer of the nuclear preparations. Suspensions of nuclei containing estradiol-receptor complexes are adsorbed onto glass fiber filters. Receptor sites, both empty and occupied by endogenous hormone, are labelled by incubation with 20 nM [3H]estradiol without (total binding) or with 2 microM radioinert estradiol (nonspecific binding). Buffer containing unbound radioactive estradiol is then drained, the filters washed, transferred into vials and counted in toluene-based scintillator. Following removal from the scintillator, DNA content of the filters can be measured by the Burton procedure. This exchange technique is easy and specific, with accuracy and precision similar to those of the technique of Bayard et al. (Bayard, F., Damilano, S., Robel, P. and Baulieu, E.E. (1978) J.Clin. Endocrinol. Metab. 46, 635-648). It offers the advantage of increased sensitivity, allowing receptor determinations on nuclear samples containing more than 10 microgram DNA. Approximately 20 measurements can be made from 50 mg tissue (wet weight).
D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000032:analysis; D004717:Endometrium / Q000378:metabolism; D004958:Estradiol / Q000378:metabolism*; D005260:Female; D005374:Filtration / Q000379:methods; D005898:Glass; D006801:Humans; D007553:Isotope Labeling; D011956:Receptors, Cell Surface / Q000302:isolation & purification*
D011956:Receptors, Cell Surface; D004958:Estradiol; D004247:DNA