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lncRNA与蛋白互作技术：RNA-Protein Pull-Down/RIP

2013-07-05 11:29阅读：

http://blog.sina.cn/dpool/blog/u/2156458864

lncRNA与蛋白互作技术：RNA-Protein Pull-Down/RIP
lncRNA与蛋白互作技术：RNA-Protein <wbr>Pull-Down/RIP
1、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。
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。
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
lncRNA与蛋白互作技术：RNA-Protein <wbr>Pull-Down/RIP

Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit


Description:
Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit
Trade Name:
Upstate (Millipore)
Qty/Pk:
12 assays
Product 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
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Key Applications:
RNA Binding Protein Immunoprecipitation (RIP)
Usage Statement:
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
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Components:
  • 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 water

View All »
Brand Family:
Upstate
Presentation:
Two boxes containing all necessary reagents to perform 12 individual RNA-binding protein immunoprecipitation (RIP) reactions.
Storage Conditions:
Upon receipt, store components at the temperatures indicated on the labels. Kit components are stable for 6 months from date of shipment when stored as directed.
Packaging:
RIP Kit capacity: 12 RNA-binding protein immunoprecipitation assays
Product Name:
Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit
Materials Required but Not Delivered:
Magna Grip™ Rack 8 well (Cat.# 20-400) (Now Available!) or similar magnetic rack.
3、Abcam

RNA Immunoprecipitation (RIP)

RIP protocol PDF
Interest 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.

Reagents

Nuclear isolation buffer
1.28 M sucrose
40 mM Tris-HCl pH 7.5
20 mM MgCl2
4% Triton X-100
RIP buffer
150 mM KCl
25 mM Tris pH 7.4
5 mM EDTA
0.5 mM DTT
0.5% NP40
100 U/ml RNAase inhibitor SUPERASin (add fresh each time)
Protease inhibitors (add fresh each time)

RIP protocol:

  1. Cell Harvesting (optional treatment of cells with formaldehyde to cross-link in vivo Protein-RNA complexes)
  2. Nuclei isolation and nuclear pellets lysis
  3. Shearing of chromatin
  4. Immunoprecipitation to purify the RNA binding protein (RBP) of interest together with the bound RNA
  5. Washing off unbound material
  6. Purification of RNA that is bound to immunoprecipitated RBP
  7. Reverse transcription (RT) of RNA to cDNA and analysis (qPCR of cDNA if target is known; if target is not known create cDNA libraries, microarrays and sequencing can be used for analysis)

1. Cell Harvesting

  1. Grow cells of the tissue culture cell line of interest to confluency and treat cells as required for the experiment.
  2. If a cross-linking step is required this will require optimization of the fixation time, check out the Cross-linking section of ourChIP protocol for details.
  3. Harvest cells by trypsinization and resuspended in PBS (e.g. 10x7 cells in 2 ml PBS), freshly prepared nuclear isolation buffer (2 ml) and water (6 ml), keep on ice for 20 min (with frequent mixing).
  4. *One or more negative controls should be maintained throughout the experiment, e.g. no-antibody sample or immunoprecipitation from knockout cells or tissue, knockdown cells are not recommended for negative control experiments

2. Nuclei isolation and nuclear pellets lysis

  1. Pellet nuclei by centrifugation at 2,500 g for 15 min.
  2. Resuspend nuclear pellet in freshly prepared RIP buffer (1 ml).
  3. *Avoid contamination using RNase-free reagents such as RNase-free tips, tubes and reagent bottles; also use ultraPURE distilled, DNase-free, RNase-free water to prepare buffers and solutions.

3. Shearing of chromatin

  1. Split resuspended nuclei into two fractions of 500 ml each (for Mock and IP).
  2. Mechanically shearing using a dounce homogenizer with 15–20 strokes.
  3. *Different cell lines might require optimization of shearing conditions.
  4. Pellet nuclear membrane and debris by centrifugation at 13,000 rpm for 10 min.
  5. *Freeze an aliquot of lysate in liquid nitrogen for reference RNA isolation. *Stringent washing of protein A/G bead pellets is important and might need to be optimised.

4. RNA Immunoprecipitation

  1. Add antibody to protein of interest (2 to 10 ug) to supernatant (6 mg-10 mg) and incubate for 2 hr (to overnight) at 4oC with gentle rotation.
  2. Add protein A/G beads (40 µl) and incubate for 1 hr at 4oC with gentle rotation.
  3. *The amount of antibody that is added and the incubation time might need to be optimised depending on the protein of interest and antibody. If an antibody is working in IP, this is a good indication that it will work in RIP.

5. Washing off unbound material

  1. Pellet beads at 2,500 rpm for 30 s, remove supernatant, and resuspend beads in 500 ml RIP buffer.

  3. Repeat for a total of three RIP washes, followed by one wash in PBS.
  4. *Freeze five percent of the beads for SDS PAGE analysis after the second wash (e.g. use 5 μl of bead slurry
    if you have 100 μl total bead slurry volume)

6. Purification of RNA that was bound to immunoprecipitated RBP

  1. Isolate coprecipitated RNAs by resuspending beads in TRIzol RNA extraction reagent (1 ml) according to manufacturer’s instructions (further information can be found in our RNA isolation protocol).
  2. Elute RNA with nuclease-free water (e.g. 20 μl).
  3. *Add approximately 15-25 μl (depending on yield) of either DEPC treated TE buffer or water to the RNA pellet.
    Eluted RNA can be stored at -80°C.
  4. Protein isolated by the beads can be detected by western blot analysis (further information can be found in our Western blot protocol).
If a cross-linking step has been used (1.2), the cross-link should now be reversed. Check out the reverse cross-links section of ourChIP protocol for details.

7. Reverse transcription (RT) of RNA to cDNA and analysis

  1. Reverse transcription of DNAse treated RNA according to manufacturer’s instructions (further information on DNAse treatment and Reverse transcription can be found in our RNA isolation protocol).
  2. If target is known use qPCR of cDNA; if target is not known create cDNA libraries, microarrays and sequencing can be used for analysis.
  3. *The control experiments should give no detectable products after PCR amplification, and high-throughput sequencing of these control libraries should return very few unique sequences.

Useful references:

Further information on the RIP protocol can be found at:
A. M. Khalila, M. Guttmana, M. Huarte, M. Garbera, A. Rajd, D. R. Morales, K. Thomas, A. Pressera, B. E. Bernstein, A. v. Oudenaardend, A. Regeva, E. S. Lander, and J. L. Rinn, “Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression.” PNAS July 14 2009.
D. G. Hendrickson, D. J. Hogan, H. L. McCullough, J. W. Myers, D. Herschlag, J. E. Ferrell, and P. O. Brown, “Concordant Regulation of Translation and mRNA Abundance for Hundreds of Targets of a Human microRNA.” PLoS Biology 2009.
D. G. Hendrickson, D. J. Hogan, D. Herschlag, J. E. Ferrell, and P. O. Brown, “Systematic Identification of mRNAs Recruited to Argonaute 2 by Specific microRNAs and Corresponding Changes in Transcript Abundance.” PLoS One 2008.
J. L. Rinn, M. Kertesz, J. K. Wang, S. L. Squazzo, X. Xu, S. A. Brugmann, L. H. Goodnough, J. A. Helms, P. J. Farnham, E. Segal, and H. Y. Chang “Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs.” Cell 129:1311–1323, 2007.
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