Improve Research Reproducibility A Bio-protocol resource
Peer-reviewed
tooltip

Co-immunoprecipitation of Flag-TLR3 or Myc-MSR1 with HCV RNA

Flag-TLR3 或 Myc-MSR1 与 HCV RNA的免疫共沉淀

DY Daisuke Yamane
HD Hiromichi Dansako
SL Stanley M. Lemon

发布: 2014年03月05日第4卷第5期 DOI: 10.21769/BioProtoc.1061 浏览次数: 10911

评审: Fanglian He

download pdf PDF PDF
ask Q&A
引用
收藏
Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of PLOS Pathogens, featuring study using the protocol.

May 2013

Advertisement

Special Issue on Protocols for Organoid Research
“科研可重复性与创新”系列网络研讨会
Technologies for RNA Detection
Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

HIV-1病毒样颗粒芽殖试验

HIV-1病毒样颗粒芽殖试验

Nathan H Vande Burgt [...] Paul Bates

2013年07月20日 11267 阅读

病毒覆盖检测（Far-Western蛋白杂交）

病毒覆盖检测（Far-Western蛋白杂交）

Kun-Tong Jia [...] Jian-Guo He

2013年10月05日 16250 阅读

基于磁珠分选及固相吸附分析针对HIV-1 gp120核心蛋白的抗体

基于磁珠分选及固相吸附分析针对HIV-1 gp120核心蛋白的抗体

Jidnyasa Ingale and Richard T Wyatt

2015年10月05日 7501 阅读

Abstract

Co-immunoprecipitation assay of TLR3-Flag or Myc-MSR1 with HCV RNA is used to identify direct interaction of viral RNA with host proteins that recognize viral RNA to initiate interferon (IFN) signaling, a crucial antiviral response of the host cells. Both Toll-like receptor 3 (TLR3) and class-A scavenger receptor type 1 (MSR1) proteins recognize viral double-stranded RNA (dsRNA) which may be released into the extracellular milieu or spread from HCV-infected cells to uninfected neighbor cells via cell-to-cell contact, resulting in IFN-β activation that restricts viral propagation. We have found that MSR1 binds extracellular dsRNA, mediating its endocytosis and transport toward the endosome where it is engaged by TLR3, thereby triggering IFN responses in both infected and uninfected cells. We used this assay to demonstrate the pivotal role of MSR1 in mediating TLR3-recognition of the HCV RNA. The assay described in this protocol is based on the conventional protein immunoprecipitation protocol with conditioned buffers that prevent nonspecific RNA degradation by RNase present in the lysate. RNA molecules associated with the Flag-tagged protein were trapped by a specific antibody followed by Protein G capture, extracted and detected quantitatively by RT-PCR assay, followed by agarose-gel electrophoresis for visualization. This method can also be applied to detection of other protein-RNA interactions.

Keywords: PAMP receptor (PAMP受体) search Toll-like receptor (Toll样受体) search Viral RNA (病毒RNA) search Interferon signaling adaptor molecule (干扰素信号适配器分子) search Scavenger receptor (清道夫受体) search

Materials and Reagents

  1. Huh-7.5 cells (obtained from Apath, LLC) expressing TLR3-Flag (or other cells stably/transiently expressing Flag/Myc-tagged protein)
  2. DMEM (Life Technologies, catalog number: 11995065 )
  3. 10% heat-inactivated FBS (Life Technologies, catalog number: 26140079 )
  4. Penicillin-streptomycin (Life Technologies, catalog number: 15140-148 )
  5. L-Glutamine (Life Technologies, catalog number: 25030-081 )
  6. Non-essential amino acids (Life Technologies, catalog number: 11140050 )
  7. HCV strain HJ3-5 stock prepared in cell culture medium (see Reference 2)
  8. DPBS (Life Technologies, catalog number: 14190-144 )
  9. RNase inhibitor (RNaseOUT) (Life Technologies, catalog number: 10777-019 )
  10. Protease inhibitor (Complete Protease Inhibitor Cocktail Tablets) (Roche Diagnostics, catalog number: 1697498 )
  11. Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
  12. Anti-Flag (M2) and anti-Myc (9E10) monoclonal antibodies (Sigma-Aldrich, catalog numbers: F1804 and M4439 ) and mouse IgG control (Life Technologies, catalog number: 02-6502 )
  13. Protein G sepharose (General Electric Company , catalog number: 17-0618-01 )
  14. TRIzol (Life Technologies, catalog number: 15596-026 )
  15. Molecular biology grade water (Corning, catalog number: 46-000-CM )
  16. Agarose (GenePure LE Agarose) (BioExpress, GeneMate, catalog number: E-3120-500 )
  17. Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, catalog number: 500-0006 )
  18. Platinum® Taq DNA Polymerase (Life Technologies)
  19. Culture medium (see Recipes)
  20. Lysis buffer (see Recipes)

Equipment

  1. 100 mm plates (FalconTM, catalog number: 353003 )
  2. Cell scraper (FalconTM, catalog number: 353085 )
  3. Electrophoresis Gel Box
  4. Tube Rotator (Fisher Scientific, catalog number: 05-450-200 )
  5. Centrifuge (Eppendorf, catalog number: 5415R )
  6. Superscript III One-step RT-PCR system (Life Technologies, catalog number: 12574-018 )

Procedure

English
中文翻译

文章信息

版权信息

© 2014 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

  1. Yamane, D., Dansako, H. and Lemon, S. M. (2014). Co-immunoprecipitation of Flag-TLR3 or Myc-MSR1 with HCV RNA. Bio-protocol 4(5): e1061. DOI: 10.21769/BioProtoc.1061.
  2. Dansako, H., Yamane, D., Welsch, C., McGivern, D. R., Hu, F., Kato, N. and Lemon, S. M. (2013). Class A scavenger receptor 1 (MSR1) restricts hepatitis C virus replication by mediating toll-like receptor 3 recognition of viral RNAs produced in neighboring cells. PLoS Pathog 9(5): e1003345.

    3. ris ris Download Citation in RIS Format

分类

微生物学 > 微生物生物化学 > 蛋白质 > 免疫检测

生物化学 > RNA > RNA-蛋白质相互作用

生物化学 > 蛋白质 > 免疫检测 > 免疫沉淀（IP）

您对这篇实验方法有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

post 发布问题
0 Q&A
pen 提交稿件