Immunopurification and analysis of protein and RNA components of mRNP in mammalian cells.
Immunopurification and analysis of protein and RNA components of mRNP in mammalian cells.
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DOI：
10.1385/1-59259-750-5:115
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发表时间：
2004-01-01
期刊：
影响因子：
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通讯作者：
Maquat, Lynne E
中科院分区：
文献类型：
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作者：
Lejeune, Fabrice;Maquat, Lynne E
我们描述了一种基本的，快速的，可靠的技术来分离和表征核糖核蛋白（RNP）使用抗体的组成蛋白。该抗体用于在促进RNP完整性的条件下从总细胞或核和细胞质细胞组分中免疫纯化RNP。其他RNP蛋白的存在以及转录本可以分别通过Western blotting和逆转录聚合酶链反应进行分析。免疫纯化前的RNase处理可用于评估蛋白-蛋白相互作用对RNA的依赖性。我们还描述了一种使用β -巯基乙醇的修饰，有助于分析与抗体轻链或重链结合的蛋白质。
We describe a basic, fast, and reliable technique to isolate and characterize ribonucleoprotein (RNP) using antibody to a constituent protein. The antibody serves to immunopurify RNP from total cells or nuclear and cytoplasmic cell fractions under conditions that promote RNP integrity. The presence of other RNP proteins as well as transcripts can then be analyzed by Western blotting and reverse transcription polymerase chain reaction, respectively. RNase treatment before immunopurification can be used to assess the dependence of protein-protein interactions on RNA. We also describe a modification using beta-mercaptoethanol that facilitates analyzing proteins that comigrate with antibody light or heavy chains.