CYTOSOL ESTRADIOL RECEPTOR IN HUMAN MAMMARY-CARCINOMA - ASSAY BASED ON ISOELECTRIC-FOCUSING IN POLYACRYLAMIDE-GEL
CYTOSOL ESTRADIOL RECEPTOR IN HUMAN MAMMARY-CARCINOMA - ASSAY BASED ON ISOELECTRIC-FOCUSING IN POLYACRYLAMIDE-GEL
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DOI：
10.1016/0003-2697(78)90243-9
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发表时间：
1978-01-01
影响因子：
2.9
通讯作者：
GUSTAFSSON, JA
中科院分区：
文献类型：
--
作者：
WRANGE, O;NORDENSKJOLD, B;GUSTAFSSON, JA
人乳腺癌细胞质中雌二醇受体复合物的有限胰蛋白酶消化将复合物的沉降系数从8改变为3-4 S。用参考蛋白校准的Sephadex柱上的高离子强度凝胶过滤显示，胰蛋白酶消化后，雌二醇受体复合物的Stokes半径从2.8 nm降低到1.9 nm。等电聚焦分析表明，8 S形式的类固醇受体复合物沉淀在玻璃柱电聚焦过程中，胰蛋白酶化的受体集中在pH 6.6作为一个尖锐的峰。建立了人乳腺癌细胞浆雌二醇受体复合物的聚丙烯酰胺凝胶等电聚焦方法。利用该技术（聚焦时间为2小时），可以在1个凝胶上分析至少8个不同的肿瘤，并且容易应用高达300 μ l的样品体积。雌二醇受体复合物的电聚焦相比，在低离子强度的蔗糖梯度离心分析，更敏感，更具体。所描述的技术等电聚焦应被视为替代蔗糖梯度离心雌二醇受体测定。
Limited trypsin digestion of the estradiol-receptor complex in human mammary cancer cytosol changed the sedimentation coefficient of the complex from 8 to 3-4 S. Gel filtration in high ionic strength on Sephadex columns calibrated with reference proteins showed that, after trypsinization, the Stokes radius of the estradiol-receptor complex decreased from 2.8 to 1.9 nm. Isoelectric focusing analysis indicated that the 8 S form of the steroid-receptor complex precipitated during electrofocusing in glass columns; the trypsinized receptor focused as a sharp peak at pH 6.6. A method was developed for isoelectric focusing of the partially trypsin-digested estradiol-receptor complex in human mammary cancer cytosol on slabs of polyacrylamide gel. With this technique (the focusing time is 2 h) at least 8 different tumors may be analyzed on 1 gel, and sample volumes up to 300 .mu.l are easily applied. Electrofocusing of the estradiol-receptor complex was compared to sucrose gradient centrifugation analysis in low ionic strength and was more sensitive and more specific. The described technique of isoelectric focusing should be considered as an alternative to sucrose gradient centrifugation for estradiol receptor assays.