Alexander D C, Miller W L
J Biol Chem. 1981 Dec 25;256(24):12628-31.
Polyadenylated mRNA from pituitary glands of castrate male sheep was used to demonstrate the presence of an mRNA coding for the beta-subunit of follicle-stimulating hormone (FSH). The mRNA fraction was translated in a wheat germ membrane-free system in the presence of [35S]cysteine. Affinity-purified antibodies, raised against the reduced and carbamylmethylated beta-subunit of ovine FSH (RCM-oFSH beta), were used to extract an in vitro translation product of Mr approximately equal to 15,000, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibody binding of the product was readily blocked by unlabeled RCM-oFSH beta, but not by the RCM derivatives of the alpha- or beta-subunits of ovine luteinizing hormone. Tryptic peptide analysis using reversed phase high performance liquid chromatography yielded very similar patterns for the immunospecific translation product and authentic oFSH beta. FSH beta translation product was found to account for approximately 0.1-0.4% of the total isotope incorporation when mRNA preparations from castrate male sheep were used. Backgrounds for the immunoextraction procedure were kept at approximately 0.02% by using a unique two-cycle specific extraction method.
来自去势公羊垂体的聚腺苷酸化mRNA被用于证明编码促卵泡激素（FSH）β亚基的mRNA的存在。mRNA组分在存在[35S]半胱氨酸的小麦胚芽无膜系统中进行翻译。针对还原和氨甲酰甲基化的绵羊FSH（RCM-oFSHβ）β亚基产生的亲和纯化抗体，用于提取通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳判断的Mr约为15,000的体外翻译产物。未标记的RCM-oFSHβ可轻易阻断产物的抗体结合，但绵羊促黄体生成素α或β亚基的RCM衍生物则不能。使用反相高效液相色谱进行的胰蛋白酶肽分析显示，免疫特异性翻译产物和 authentic oFSHβ的模式非常相似。当使用来自去势公羊的mRNA制剂时，发现FSHβ翻译产物约占总同位素掺入量的0.1-0.4%。通过使用独特的双循环特异性提取方法，免疫提取程序的背景保持在约0.02%。
