new tech--RNA pull down protein(RNA IP方法).doc

lncRNA 与蛋白互作技术：RNA-Protein Pull-Down/RIP(2013-07-05 11:29:49)lncRNA 与蛋白互作技术：RNA-Protein Pull-Down/RIP1、Thermo fisher 试剂盒近日，赛默飞世尔科技全新推出了一款 Pierce Magnetic RNA-Protein Pull-Down Kit，让研究人员能够以末端标记的 RNA作为诱饵，轻松富集蛋白质-RNA 的相互作用与抗体捕获相比，这种方法的优势在于脱硫生物素化的目标 RNA能够直接富集 RBP（或复合物）蛋白质与 RNA 的相互作用是许多细胞功能的核心，如蛋白质合成、mRNA 组装、病毒复制、细胞发育调控等了解它们之间相互作用的分子机制对理解这些生物学过程非常重要然而，之前的分析方法往往受限于使用放射性标记，或实验步骤过多，不仅耗时费力，也增加了实验结果的不稳定此试剂盒利用脱硫生物素末端标记的 RNA 和链霉亲和素磁珠标记的来高效富集 RNA 结合蛋白（RBP）与抗体捕获相比，这种方法的优势在于脱硫生物素化的目标 RNA 能够直接富集 RBP（或复合物）此外，试剂盒还提供了经过验证的对照，适用于标记和 pull-down 分析，也与多个下游应用兼容，如 Western blotting 和质谱（MS）。

试剂盒中包含了 Pierce RNA 3’-End Desthiobiotinylation Kit它利用 T4 RNA 连接酶将单个脱硫生物素化的胞苷二磷酸连接到单链 RNA 的 3’端3’端的末端标记不干扰 RNA 结构，因此，比标记核苷酸的随机掺入更加理想每个标记反应适合 50 pmol RNA；不过，如有必要的话，标记反应也可扩展（从 1 pmol 到 1 nmol）标记反应需要 20 倍过量的脱硫生物素化核苷酸对于不太复杂的 RNA，孵育时间可为 37°C 30 分钟，若是更长或更复杂的 RNA，时间也延长到 4-16°C 过夜通过改变 RNA 与核苷酸的比例，延长孵育时间，或在标记反应中添加 DMSO，可优化复杂 RNA 的标记效率RBP 的富集过程经过优化，相当简单首先将 RNA 与链霉亲和素磁珠结合之后在蛋白质-RNA 结合缓冲液中平衡 RNA 结合的磁珠，再加入蛋白裂解液随后添加适当的缓冲液、涡旋振荡，并在磁力架上分离，洗涤磁珠最后样品可通过非变性的生物素洗脱缓冲液或 SDS-PAGE 上样缓冲液洗脱，用于下游分析此试剂盒的特点在于：直接：直接利用末端标记的 RNA 捕获核糖核蛋白复合物；不需要使用抗体进行 pull-down;简单：RBP 富集过程无需离心；步骤简化，在 RNA 标记反应后手工操作不到 3 小时 灵活：使用不同长度的体外转录 RNA或合成 RNA进行标记；可成功富集内源、过表达和体外翻译的裂解液中蛋白; 特异：磁珠背景低；未处理的 RNA或突变 RNA不会明显富集特定的 RBP; 经济：比单独购买合成的末端标记 RNA、磁珠和试剂更为经济; 完整：包含标记和富集组分及分析缓冲液；阳性对照 RNA、阴性对照 RNA和 RBP抗体也包含在内此试剂盒包含的试剂足够 20次 RNA标记反应和 20次蛋白质-RNA pull-down 分析使用。

2、milipore 试剂盒Magna RIP™ RNA-Binding Protein Immunoprecipitation KitDescription:Magna RIP™ RNA-Binding Protein Immunoprecipitation KitTrade Name:Upstate (Millipore)Qty/Pk:12 assaysProduct Overview:RNA-binding protein immunoprecipitation (RIP) is the RNA analog of the more well-known ChIP application (chromatin immunoprecipitation), which identifies DNA targets of DNA-binding proteins in an in-vivo cellular context. RIP can be used to identify specific RNA molecules (of many types) associated with specific nuclear or cytoplasmic binding proteins. These experiments involve immunoprecipitation of endogenously formed complexes of RNA-binding proteins and co-isolation of any RNA species associated with that RNA-binding protein. Purification of these RNA species allows interrogation and identification of mRNAs (and potentially non-coding RNAs associated with them) and can be directly measured using down stream applications including quantitative reverse transcription polymerase chain reaction (RT-PCR), microarray analysis (RIP-chip) and “deep-sequencing” or 2nd-generation sequencing based platforms (RIP-Seq).Features & Benefits:-Protein A/G magnetic beads, optimized to bind nucleic acid-protein immune complexes-RNAse inhibitors and RNAse-free reagents-Negative controls« CollapseKey Applications:RNA Binding Protein Immunoprecipitation (RIP)« CollapseComponents: Magnetic Beads Protein A/G RIP Wash Buffer RIP Lysis Buffer 0.5 M EDTA 10% SDS Salt Solution I Salt Solution II Precipitate Enhancer Normal Mouse IgG Rabbit IgG Purified Protease Inhibitor Cocktail 200X RNase Inhibitor Proteinase K (10 mg/mL) Nuclease free waterView All »3、AbcamRNA Immunoprecipitation (RIP)RIP protocol PDFInterest in RNA-protein interactions is booming as we begin to appreciate the role of RNA, not just in well-established processes such as transcription, splicing, and translation, but also in newer fields such as RNA interference and gene regulation by non-coding RNAs. RIP is an antibody-based technique used to map RNA–protein interactions in vivo by immunoprecipitating the RNA binding protein of interest together with its associated RNA and allows identification of bound transcripts. RIP precipitates a specific RNA binding protein (RBP) and associated RNA (mRNAs, non coding RNAs, viral RNAs) that can be detected by real- time PCR, microarrays or e.g. sequencing. Here is a RIP protocol adapted from Khalila et al. PNAS 2009, Hendrickson et al. 2009, Hendrickson et al. 2008 and from Rinn et al. Cell 2007.ReagentsNuclear isolation buffer1.28 M sucrose40 mM Tris-HCl pH 7.520 mM MgCl24% Triton X-100RIP buffer150 mM KCl25 mM Tris pH 7.45 mM EDTA0.5 mM DTT0.5% NP40100 U/ml RNAase inhibitor SUPERASin (add fresh each time) Protease inhibitors (add fresh each time)。