In vitro RNA-protein Binding Assay by UV Crosslinking

Materials and Reagents

1. Linearized DNA template for transcription of the RNA of interest
   
2. MAXIscript in vitro Transcription Kit (Applied Biosciences, catalog number: AM1308M ) (This kit contain ATP, CTP, GTP, α-P32UTP and DNase I)
   
3. RNasin (Promega Corporation, catalog number: N2611 )
   
4. RNase T1 (Applied Biosciences, catalog number: 10109193001 )
   
5. 0.5 M EDTA (Life Technologies, Gibco^®, catalog number: 15575-038 )
   
6. ddH₂O (GeneMate Loyalty, catalog number: UPW-1000 )
   
7. HEPES (Sigma-Aldrich, catalog number: H3375 )
   
8. MgCl₂ (Sigma-Aldrich, catalog number: M-2393 )
   
9. Glycerol (Sigma-Aldrich, catalog number: G7893-1L )
   
10. DTT (Sigma-Aldrich, catalog number: D9779-5G )
    
11. Tris-Base (Thermo Fisher Scientific, catalog number: BP152-1 )
    
12. SDS (Sigma-Aldrich, catalog number: L-4390 )
    
13. Bromphenol blue (Sigma-Aldrich, catalog number: B5525 )
    
14. TEMED
15. Ammoniampersulfate
    
16. 5x binding buffer (see Recipes)
    
17. 2x SDS loading buffer (see Recipes)
    
18. 10x TGE buffer (see Recipes)
    
19. 6% TGE gel (see Recipes)
    

Equipment

1. SPIN-PureTM column (G-50) (PireBiotech SCW50-50 DEPC-water)
   
2. SDS-PAGE system (Bio-Rad, catalog number: 165-1802 )
   
3. UV Stratalinker 1800 (Stratagene, catalog number: 474645 )
   
4. Film processor (Konica Minolta, model: SRX-101A )
   
5. Centrifuges (Eppendorf, catalog number: 5810R )
   
6. Scintillation Counter (Beckman, catalog number: LS6500 )
   
7. Bench top Radiation Shield with 6-1/4" base (Cole-Parmer, catalog number: WU-36218-00 )
   
8. 37 °C water bath
   

Procedure