Mizejewski G J, Vonnegut M, Simon R
J Chromatogr. 1980 Dec 5;202(1):113-21. doi: 10.1016/s0021-9673(00)80084-9.
A one-step batch procedure is described for purification of murine alpha-fetoprotein (AFP) by estradiol affinity chromatography. Various ratios of carbodiimide (C), diaminononame (D) and estradiol hemisuccinate (E) were tested to determine optimal conditions for AFP purification. Although yields of AFP ranged from 15 to 44% depending on the reagent ratio employed, AFP isolates free of other protein contaminants were achieved at C:D:E ratios of 10:10:1 with a 29% yield. Both estrone and estradiol proved efficient as elution agents to free AFP bound to the estradiol-Sepharose beads, but higher yields were produced with estrone. After isolation the estrogen-eluted AFP preparations were analyzed by (1) estradiol-binding assays, (2) third-party radiocoprecipitation, (3) inhibition of radioimmunoassay for estrone and estradiol and (4) exchange of unlabeled for radiolabeled estradiol. These results indicated that the steroid remained attached to the eluted AFP molecule.
本文描述了一种通过雌二醇亲和色谱法纯化小鼠甲胎蛋白（AFP）的一步批量操作程序。测试了碳二亚胺（C）、二氨基壬烷（D）和雌二醇半琥珀酸酯（E）的各种比例，以确定AFP纯化的最佳条件。尽管根据所用试剂比例，AFP的产量在15%至44%之间，但在C:D:E比例为10:10:1时可获得不含其他蛋白质污染物的AFP分离物，产量为29%。雌酮和雌二醇都被证明是有效的洗脱剂，可使与雌二醇-琼脂糖珠结合的AFP游离出来，但雌酮产生的产量更高。分离后，通过以下方法对雌激素洗脱的AFP制剂进行分析：（1）雌二醇结合测定；（2）第三方放射性共沉淀；（3）雌酮和雌二醇放射免疫测定的抑制；（4）未标记雌二醇与放射性标记雌二醇的交换。这些结果表明，类固醇仍然附着在洗脱的AFP分子上。
