Unité 5-Luminy, Centre de Biochimie et de Biologie Moléculaire, Case 901, F-13288, Marseille Cedex 9, France.
Planta. 1984 Nov;162(5):427-33. doi: 10.1007/BF00393455.
A polyclonal antiserum was raised against highly purified RNA polymerase II from soybean embryos. Pure RNA polymerase II was fractionated on sodium dodecyl sulfate-polyacrylamide gels and transferred onto nitrocellulose sheets, incubated with the immune antiserum and then with iodinated protein A. Autoradiograms showed that the immune antiserum recognized all subunits of RNA polymerase II. Subunits 42, 27 and 16 kdalton were particularly reactive. Application of this transfer technique to protein extracts from soybean embryos or from cultured soybean cells allowed the identification of subunits of RNA polymerase II in the extracts. Analysis of the staining of the bands on the autoradiograms for increasing amounts of pure RNA polymerase II demonstrated that the transfer was quantitative, so that standard curves could be drawn to estimate the unknown amounts of enzyme in the extracts.
针对大豆胚中高度纯化的 RNA 聚合酶 II，制备了多克隆抗血清。将纯 RNA 聚合酶 II 在十二烷基硫酸钠-聚丙烯酰胺凝胶上进行分级分离，然后转移到硝酸纤维素片上，与免疫抗血清孵育，然后与碘化蛋白 A 孵育。放射自显影显示，免疫抗血清识别 RNA 聚合酶 II 的所有亚基。亚基 42、27 和 16 千道尔顿特别具有反应性。将这种转移技术应用于大豆胚或培养的大豆细胞的蛋白质提取物中，可以鉴定提取物中的 RNA 聚合酶 II 亚基。用纯 RNA 聚合酶 II 的逐渐增加量分析放射自显影图谱上条带的染色，表明转移是定量的，因此可以绘制标准曲线来估计提取物中未知酶的量。
