Identification of RNA-binding Proteins
RNA结合蛋白质的识别
发布: 2016年09月05日第6卷第17期 DOI: 10.21769/BioProtoc.1920 浏览次数: 14879
评审: Antoine de MorreeZhen ShiKate Hannan
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参见作者原研究论文
The authors used this protocol in:
Jun 2013
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Abstract
This protocol describes the extraction of RNA-binding proteins (RBPs) from cell lysates. In order to pull down target RBPs, 5-bromo-UTP (BrUTP)-incorporated RNA probes are used, which are generated by in vitro transcription. The schematic diagram (Flowchart) with procedure is indicated (Figure1 and Figure 2).
Figure 1. Schematic diagram of procedure (A-H). Flow chart of experimental procedure is indicated at A-H.
Figure 2. Linearization of plasmids by restricted enzyme. The plasmid is cut at restriction sites adjacent to its cloning element.
5-bromo-UTP-incorporated RNA (5-bromo-utp-incorporated RNA)
Mass spectrometry (质谱法)
Pull-down assay (拉法)
Materials and Reagents
- Microcentrifuge tubes (1.5 to 2.0 ml)
- Desalting MobiSpin columns (MoBiTec, catalog number: M105035F )
- DNA template (e.g., pBluescript vector encoding the target sequences such as non-coding elements IL-6 3’UTR and TNF-α 3’UTR)
- Reagents for in vitro transcription kits (TAKARA BIO, catalog number: 6140 )
- 5-bromouridine 5’-triphosphate (sodium salt) (Cayman Chemical, catalog number: 18140 )
- 50 mM ATP solution
- 50 mM GTP solution
- 50 mM CTP solution
- 50 mM UTP solution
- RNaseOUTTM recombinant ribonuclease inhibitor (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10777-019 )
- T7 RNA polymerase
- DNase I (RNase free) (New England Biolabs, catalog number: M0303S )
- Protease inhibitor cocktail (Sigma-Aldrich, catalog number: MSSAFE )
- Protease inhibitor cocktail (NACALAI TESQUE, catalog number: 0 4080 )
- TRIzol® Reagent (Thermo Fisher Scientific, AmbionTM, catalog number: 15596-026 )
- Anti-BrdU antibody (IIB5) (Abcam, catalog number: ab8152 )
- Protein G Sepharose 4 fast flow (GE Healthcare, catalog number: 17061801 )
- Nuclease-free PBS (NACALAI TESQUE, catalog number: 14249 )
- Nuclease-free water (Thermo Fisher Scientific, AmbionTM, catalog number: 4387936 )
- Dithiothreitol (DTT) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0861 )
- Chloroform (NACALAI TESQUE, catalog number: 08401-65 )
- DEPC water (Thermo Fisher Scientific, AmbionTM, catalog number: AM9916 )
- 96% ethanol or 70% ethanol (NACALAI TESQUE, catalog number: 09666-85 )
- NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific, catalog number: 78833 )
- 0.25% trypsin-EDTA solution (Thermo Fisher Scientific, catalog number: 25200056 )
- 1 M Tris-HCl, pH 7.4 (NACALAI TESQUE, Gibco®, catalog number: 35436-01 )
- 5 M NaCl (NACALAI TESQUE, catalog number: 31320-05 )
- 0.1 M spermidine (Sigma-Aldrich, catalog number: S2626-1G )
- 10% Nonidet(R) P-40 (NACALAI TESQUE, catalog number: 25223-04 )
- 0.5 M EDTA (NACALAI TESQUE, catalog number: 06894-14 )
- 1 M MgCl2 (NACALAI TESQUE, catalog number: 20942-34 )
- 1% Tween 20 (NACALAI TESQUE, catalog number: 35624-15 )
- PBS, pH 7.0 (NACALAI TESQUE, catalog number: 14249-24 )
- 1% SDS (NACALAI TESQUE, catalog number: 30562-04 )
- Coomassie brilliant blue (CBB) (Thermo Fisher Scientific, catalog number: 20278 )
- 10x transcription buffer (see Recipes)
- Bead washing buffer (see Recipes)
- RNA-binding buffer (see Recipes)
- RNA-protein wash buffer (see Recipes)
- Lysis buffer (see Recipes)
- Elution buffer (see Recipes)
Equipment
- MALDI-QIT-TOF (Shimadzu Europa GmbH, model: AXIMA Resonance )
- Microcentrifuge capable of reaching up to 16,000 x g
- Vortex mixer
- Centrifuge capable of reaching up to 2,000 x g
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Masuda, K. and Kishimoto, T. (2016). Identification of RNA-binding Proteins. Bio-protocol 6(17): e1920. DOI: 10.21769/BioProtoc.1920.
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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 > 分离和纯化
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