通过间接免疫吸附富集合成组蛋白H5的多核糖体。H5 mRNA的纯化。
Enrichment of histone H5-synthesizing polysomes by indirect immunoadsorption. Purification of H5 mRNA.
作者信息
Perucho M, Molgaard H V, Ruiz-Carrillo A
出版信息
J Biol Chem. 1982 Jan 25;257(2):958-63.
A method of purifying H5 mRNA from immature hen erythrocytes is described. Polysomes from red blood cells of anemic animals were allowed to react with affinity-chromatography-purified rabbit anti-H5 antibodies and immunoadsorbed to cellulose-insolubilized sheep and anti-rabbit antibodies. This method produced a 10- to 15-fold enrichment of the H5 mRNA, which was further purified by sucrose gradient centrifugation, poly(U)-Sepharose chromatography, and size fractionation in denaturing polyacrylamide gels. The H5 mRNA activity was essentially pure, as shown by translation in a reticulocyte cell-free system and immunoprecipitation with H5-antibodies. The proportion of H5 mRNA has been estimated to lie in the range 0.4-0.6% of all cellular mRNAs. These values are in agreement with the estimated relative synthesis of H5 in the immature erythrocyte.
描述了一种从未成熟母鸡红细胞中纯化H5 mRNA的方法。使贫血动物红细胞中的多核糖体与亲和层析纯化的兔抗H5抗体反应，并免疫吸附到纤维素固定化的羊抗兔抗体上。该方法使H5 mRNA富集了10至15倍，通过蔗糖梯度离心、聚（U）-琼脂糖凝胶层析和变性聚丙烯酰胺凝胶中的大小分级进一步纯化。如在网织红细胞无细胞系统中翻译并用H5抗体进行免疫沉淀所示，H5 mRNA活性基本纯净。据估计，H5 mRNA在所有细胞mRNA中的比例在0.4%至0.6%之间。这些值与未成熟红细胞中H5的估计相对合成量一致。
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