RNA-Binding Protein Immunoprecipitation from Whole-Cell Extracts
RNA-Binding Protein Immunoprecipitation from Whole-Cell Extracts
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DOI：
10.1007/978-1-62703-580-4_35
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发表时间：
2014-01-01
期刊：
影响因子：
--
通讯作者：
Staiger, Dorothee
中科院分区：
文献类型：
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作者：
Koester, Tino;Staiger, Dorothee
基于RNA的调控越来越被认为是塑造细胞转录组的重要因素。与前mRNA内的顺式调节基序相互作用的RNA结合蛋白决定其靶的命运。理解由RNA结合蛋白控制的转录后网络需要鉴定其直接的体内靶点。在这里，我们描述了RNA免疫沉淀在拟南芥。用甲醛处理表达与绿色荧光蛋白融合的RNA结合蛋白的转基因植物，以“捕获”与其生理蛋白伴侣复合的RNA。用针对GFP标签的抗体对全细胞提取物进行免疫沉淀。平行地，使用不相关的抗体进行模拟免疫沉淀。从免疫沉淀物洗脱共沉淀的RNA，并通过实时PCR鉴定。相对于仅表达GFP的植物的免疫沉淀和与不相关抗体的模拟免疫沉淀的富集指示特异性结合。
RNA-based regulation is increasingly recognized as an important factor shaping the cellular transcriptome. RNA-binding proteins that interact with cis-regulatory motifs within pre-mRNAs determine the fate of their targets. Understanding posttranscriptional networks controlled by an RNA-binding protein requires the identification of its immediate in vivo targets. Here we describe RNA immunoprecipitation in Arabidopsis thaliana. Transgenic plants expressing an RNA-binding protein fused to green fluorescent protein are treated with formaldehyde to "trap" RNAs in complexes with their physiological protein partners. A whole-cell extract is subjected to immunoprecipitation with an antibody against the GFP tag. In parallel, a mock immunoprecipitation is performed using an unrelated antibody. Coprecipitated RNAs are eluted from the immunoprecipitate and identified via real-time PCR. Enrichment relative to immunoprecipitation from plants expressing GFP only and mock immunoprecipitation with an unrelated antibody indicates specific binding.