RNA结合蛋白免疫共沉淀法（RIP）检验结合到衰老相关分泌表型（SASP）因子 的mRNAAUF1

Elise Alspach; Sheila A. Stewart

doi:10.21769/BioProtoc.1481

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RNA-binding Protein Immunoprecipitation (RIP) to Examine AUF1 Binding to Senescence-Associated Secretory Phenotype (SASP) Factor mRNA

RNA结合蛋白免疫共沉淀法（RIP）检验结合到衰老相关分泌表型（SASP）因子的mRNAAUF1

EA Elise Alspach

SS Sheila A. Stewart email

发布: 2015年05月20日第5卷第10期 DOI: 10.21769/BioProtoc.1481 浏览次数: 14550

评审: HongLok LungJyotiska ChaudhuriAnonymous reviewer(s)

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Abstract

Immunoprecipitation and subsequent isolation of nucleic acids allows for the investigation of protein:nucleic acid interactions. RNA-binding protein immunoprecipitation (RIP) is used for the analysis of protein interactions with mRNA. Combining RIP with quantitative real-time PCR (qRT-PCR) further enhances the RIP technique by allowing for the quantitative assessment of RNA-binding protein interactions with their target mRNAs, and how these interactions change in different cellular settings. Here, we describe the immunoprecipitation of the RNA-binding protein AUF1 with several different factors associated with the senescence-associated secretory phenotype (SASP) (Alspach and Stewart, 2013), specifically IL6 and IL8. This protocol was originally published in Alspach et al. (2014).

Materials and Reagents

Tissue culture
1. DMEM (Sigma-Aldrich, catalog number: D6429 ) 
2. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F2442 )
3. Medium M199 (Sigma-Aldrich, catalog number: M7528 )
4. Penicillin/streptomycin (Sigma-Aldrich, catalog number: P4333 ) 
5. BJ human foreskin fibroblasts 
6. Bleomycin sulfate (Sigma-Aldrich, catalog number: B8416 )
7. BJ culture media (see Recipes)
General materials
1. Bradford Protein Assay reagent (Bio-Rad Laboratories, catalog number: 500-0006 )
2. Polyclonal anti-AUF1 antibody (EMD Millipore, catalog number: 07260MI ) 
3. Normal Rabbit IgG antibody (Cell Signaling Technology, catalog number: 2729 )
4. Protein A Dynabeads (Life Technologies, catalog number: 10002D ) 
5. MCLB (mammalian cell lysis buffer) (see Recipes)
6. NT2 buffer (see Recipes)
7. Buffer A (see Recipes)
8. Buffer B (see Recipes)
9. Buffer C (see Recipes)
Immunoprecipitation reagents
1. Tris 
2. EDTA 
3. NP40 
4. NaCl 
5. Phosphate-buffered saline (PBS) 
6. SDS 
7. Deoxycholate 
8. MgCl₂ 
9. NaH₂PO₄
10. RNAse OUT (Life Technologies, catalog number: 10777-019 ) 
11. Proteinase K (Sigma-Aldrich, catalog number: P6556 )
12. RiboPure RNA isolation kit (Life Technologies, catalog number: AM1924 )
qRT-PCR reagents
1. dNTPs 
2. Hexanucleotide mix (Roche Diagnostics, catalog number: 11277081001 )
3. SuperScript II Reverse Transcriptase (Life Technologies, catalog number: 18064-014 )
4. Power SYBR GREEN PCR master mix (Life Technologies, catalog number: 4367659 )
Primers
1. IL6 forward: 5′-ACA TCCTCGACGGCA TCTCA-3′
2. IL6 reverse: 5′-TCACCAGGCAAGTCTCCTCA-3′
3. IL8 forward: 5′-GCTCTGTGTGAAGGTGCAGT-3′
4. IL8 reverse: 5′-TGCACCCAGTTTTCCTTGGG-3′

Equipment

Tissue Culture
1. 37 °C forced-air incubator maintained at 5% CO₂ and 5% O₂ (Thermo Fisher Scientific, Forma Series II water jacketed CO₂ incubator)
2. Laminar-flow biosafety cabinet (Labconco Purifier Class II Biosafety Cabinet)
Immunoprecipitation
1. Spectrophotometer capable of reading at 450 and 595 nm
2. Dyna-mag magnetic bead separator (Life Technologies, catalog number: 12321D )
3. Micro centrifuge
4. End-over-end tube rotator (Thermo Fisher Scientific, catalog number: 400110Q )
qRT-PCR
1. PCR machine (Bio-Rad Laboratories, model: C1000 thermo cycler ) 
2. Real-time PCR detection system (Bio-Rad Laboratories, model: CFX96 Real Time System )
General equipment
1. Tissue culture cell scrapers 
2. Refrigerated micro centrifuge 
3. Heat block capable of reaching 70 °C

Procedure

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如何引用

Alspach, E. and Stewart, S. A. (2015). RNA-binding Protein Immunoprecipitation (RIP) to Examine AUF1 Binding to Senescence-Associated Secretory Phenotype (SASP) Factor mRNA. Bio-protocol 5(10): e1481. DOI: 10.21769/BioProtoc.1481.