Cross-linked RNA Immunoprecipitation
RNA免疫共沉淀
发布: 2013年03月05日第3卷第5期 DOI: 10.21769/BioProtoc.398 浏览次数: 24571
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参见作者原研究论文
The authors used this protocol in:
Jul 2012
Abstract
This method is for the immunoprecipitation of Flag-Tagged RNA binding proteins from mammalian cell lines and isolation of the bound RNAs for analysis by quantitative real-time PCR. The RNA binding protein of interest should be tagged with the M2 Flag-tag and expressed in the mammalian cell line of interest (Knuckles et al., 2012). However, specific antibodies for the protein of interest can be used in conjunction with Sepharose G-beads.
Materials and Reagents
- Anti-Flag M2 Affinity gel (Sigma Aldrich, catalog number: A2220 )
- RNase inhibitor (Life Technologies, InvitrogenTM, catalog number: N8080119 or Bioline, catalog number: BIO-65028 )
- Complete Protease Inhibitor Cocktail Tablets (F. Hoffmann-La Roche, catalog number: 05 892 970 001 )
- RnaseZap (Life Technologies, Ambion®, catalog number: AM9780 )
- Trizol reagent (Life Technologies, InvitrogenTM, catalog number: 15596-026 )
- General chemicals (Sigma Aldrich)
- DNase I recombinant, RNase-free inc. buffer (F. Hoffmann-La Roche, catalog number: 04716728001 )
- BioScriptTM (Bioline, catalog number: BIO-27036 )
- Formaldehyde
- Fetal calf serum
- Glycine
- SDS
- HEPES
- TritonX-100
- EDTA
- DTT
- NaCl
- IP lysis buffer (see Recipes)
- RIP buffer (see Recipes)
Equipment
- Sonicator (the model is not critical but preferably a devise with a probe ≤ 5 mm in diameter)
- Tube Rotator
- Shaker
- Centrifuge
- PCR machine
- Heating block
- 15 ml conical tubes
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Vogt, M. and Taylor, V. (2013). Cross-linked RNA Immunoprecipitation. Bio-protocol 3(5): e398. DOI: 10.21769/BioProtoc.398.
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分类
生物化学 > RNA > RNA-蛋白质相互作用
生物化学 > 蛋白质 > 免疫检测
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