Lechuga Ana, Berjón-Otero Mónica, Salas Margarita, Redrejo-Rodríguez Modesto
Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones científicas y Universidad Autónoma de Madrid, Madrid, Spain.
Bio Protoc. 2018 Jan 5;8(1):e2678. doi: 10.21769/BioProtoc.2678.
This protocol analyzes the direct interaction between two DNA-binding proteins by pull-down co-immunoprecipitation. One of the proteins is overexpressed in as HA-tagged recombinant protein and cell-free extracts are immunoprecipitated in HA-affinity resin. Cell extracts are treated with nuclease to degrade DNA and RNA, which rules out nucleic acid-mediated indirect interaction. Then, a second immunoprecipitation step is performed using the purified putative partner protein. Co-immunoprecipitated proteins can be detected either by Coomassie Blue staining and/or Western blotting (WB) if a specific antibody is available. Moreover, many DNA/RNA binding proteins are highly electropositive, which can hinder WB under standard conditions, as has been shown in histones and histone-like proteins. In this case, we show that the high isoelectric point of the putative partner results in a poor transfer. Tips to troubleshot WB transfer of highly electropositive DNA-binding proteins are provided.
本方案通过下拉式免疫共沉淀分析两种DNA结合蛋白之间的直接相互作用。其中一种蛋白以HA标签重组蛋白的形式在细胞中过表达，然后将无细胞提取物在HA亲和树脂中进行免疫沉淀。细胞提取物用核酸酶处理以降解DNA和RNA，这排除了核酸介导的间接相互作用。然后，使用纯化的假定伴侣蛋白进行第二步免疫沉淀。如果有特异性抗体，共免疫沉淀的蛋白可以通过考马斯亮蓝染色和/或蛋白质免疫印迹（WB）检测。此外，许多DNA/RNA结合蛋白具有高度正电性，这在标准条件下会阻碍蛋白质免疫印迹，如组蛋白和组蛋白样蛋白所示。在这种情况下，我们表明假定伴侣的高电荷导致转移效果不佳。本文提供了针对高正电性DNA结合蛋白蛋白质免疫印迹转移故障排除的提示。
