Materials and Reagents

1. HCV NS5B inhibitors
   
2. Purified recombinant HCV NS5B ΔC21 soluble protein [cloned and purified according to Boyce et al. (2014) and Hung et al. (2011)]
   
3. ProteOn GLH Sensor Chip (Bio-Rad Laboratories, catalog number: 176-5013 )
   
4. HEPES solution (1 M) (Sigma-Aldrich, catalog number: H3537-1L )
   
5. MgCl₂ (1 M) (Sigma-Aldrich, catalog number: M1028-100ML )
   
6. EDTA (0.5 M) (Sigma-Aldrich, catalog number: E7889-100ML )
   
7. NaCl (5 M) (Sigma-Aldrich, catalog number: S6546-1L )
   
8. KCl (2 M) (Life Technologies, Ambion^®, catalog number: AM9640G )
   
9. Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) (Sigma-Aldrich, catalog number: C4706-10G )
   
10. Surfactant P20 (10% v/v) (General Electric Company, catalog number: BR-1000-54 )
    
11. DMSO (Sigma-Aldrich, catalog number: 472301-100ML )
    
12. ProteOn Amine Coupling Kit (Bio-Rad Laboratories, catalog number: 176-2410 ) containing 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC or EDC), sulfo-N-hydroxysuccinimide (Sulfo-NHS) and ethanolamine-HCl
    
13. Preconditioning Reagents (see Recipes)
    
14. Immobilization Buffer (see Recipes)
    
15. Running Buffer (see Recipes)
    
16. Running Buffer with 5% DMSO (see Recipes)
    

Equipment

1. ProteOn Standard & Deep-Well Microplates (Bio-Rad Laboratories, catalog numbers: 176-6020 and 176-6023 )
   
2. ProteOn Microplate Sealing Film (Bio-Rad Laboratories, catalog number: 176-6040 )
   
3. Nalgene Rapid-Flow Filter Units and Bottle Top Filters, PES Membrane, Sterile (VWR International, catalog number: 16211-056 ) (for the 1 L size)
   
4. ProteOn XPR36 instrument (Bio-Rad Laboratories)
   
5. BenchTop Centrifuge for microfuge tubes
   
6. BenchTop Centrifuge to spin ProteOn 96-well assay plate
   

Software

1. ProteOn Manager Software Version 3.1.0.6
   
2. Scrubber software designed for ProteOn data analysis (BioLogic Software)
   

Procedure