Roy E J, McEwen B S
Steroids. 1977 Nov;30(5):657-69. doi: 10.1016/0039-128x(77)90055-1.
A nuclear exchange assay was developed for brain estrogen receptors. The assay employs an extraction procedure which solubilizes essentially all of the estradiol from brain cell nuclei: purified cell nuclei are evenly dispersed in a hypotonic buffer prior to the addition of an equal volume of 0.8M KC1. Experiments with an in vivo injection of 3H-estradiol established that this procedure extracts even normally salt-resistant binding. For exchange, aliquots of the extract are incubated with 3H-estradiol or 3h-estradiol plus 100-fold excess unlabeled estradiol. Bound 3H-estradiol is separated from free 3H-estradiol on Sephdex LH-20 columns. Loss of estradiol binding activity can occur with brain nuclear extracts under conditions required for exchange. This loss of binding is inhibited by the addition of bacitracin to the incubation buffer. The exchange is complete within 5 hrs at 25 degrees C and specific binding activity is stable for at least 16 hrs. The assay was validated by comparing levels of macromolecular-bound radioactivity after an in vivo injection of 3H-estradiol and levels determined by exchange after an injection of unlabeled estradiol. Scatchard analysis confirmed the high affinity nature of the binding measured by exchange.
已开发出一种用于检测脑雌激素受体的核交换分析方法。该分析方法采用一种提取程序，该程序可将脑细胞核中的基本上所有雌二醇溶解：在加入等体积的0.8M KCl之前，将纯化的细胞核均匀分散在低渗缓冲液中。体内注射³H-雌二醇的实验表明，该程序甚至能提取出通常对盐具有抗性的结合物。为了进行交换，将提取物的等分试样与³H-雌二醇或³H-雌二醇加100倍过量的未标记雌二醇一起孵育。结合的³H-雌二醇与游离的³H-雌二醇在Sephdex LH - 20柱上分离。在交换所需的条件下，脑细胞核提取物可能会出现雌二醇结合活性的丧失。向孵育缓冲液中添加杆菌肽可抑制这种结合活性的丧失。在25℃下，交换在5小时内完成，特异性结合活性至少稳定16小时。通过比较体内注射³H-雌二醇后大分子结合放射性水平与注射未标记雌二醇后通过交换测定的水平，验证了该分析方法。Scatchard分析证实了通过交换测量的结合具有高亲和力特性。
