Filter assay method for the estrogen receptor protein: Detection of both filled and unfilled estrogen binding sites
✍ Scribed by Lawrence Cousens; Barnet Eskin
- Publisher
- Elsevier Science
- Year
- 1982
- Tongue
- English
- Weight
- 829 KB
- Volume
- 121
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
New filter assay methods are presented for quantitating both cytoplasmic and nuclear forms of the estrogen receptor protein. These methods exploit the strong adsorption of this protein to glass-fiber filters, which appears to occur without loss of steroid binding affinity. A "direct assay protocol" is described that detects only unfilled (nonliganded) estrogen binding sites. In addition, a convenient "exchange assay protocol" has been developed that detects, in addition, those receptors present whose binding sites have already bound nonradioactive estradiol. For the exchange assay, an extract containing receptor is adsorbed to a filter, which is washed free of unbound steroid and then equilibrated for a prolonged period with an excess volume of buffer containing radioactive estradiol. After brief washing in steroid-free buffer, the radioactivity adsorbed to the filter is measured to determine the amount of receptor present. These assays can be used at either 4 or 23°C over a broad range of salt concentrations. The background of nonspecific binding is extremely low, due in part to the almost negligible affinity of free estradiol for the glass-fiber support.