采用表面等离子体共振研究HCV NS5B抑制因子

Melanie  Wong; Giuseppe A.  Papalia

doi:10.21769/BioProtoc.1044

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Peer-reviewed

A Surface Plasmon Resonance Method to Study HCV NS5B Inhibitors

采用表面等离子体共振研究HCV NS5B抑制因子

Melanie Wong

Giuseppe A. Papalia email

发布: 2014年02月20日第4卷第4期 DOI: 10.21769/BioProtoc.1044 浏览次数: 8855

评审: Anonymous reviewer(s)

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Abstract

Surface Plasmon Resonance (SPR) technology is a well-established platform used to evaluate the kinetic parameters of protein-small molecule interactions. Below, we describe the use of the ProteOn XPR36 biosensor from Bio-Rad (Hercules, CA) to evaluate the binding of small molecule inhibitors to recombinant NS5B protein. The high pI (> 9) of this construct allows for chemical immobilization using HEPES-buffered saline at pH 7.5. This is in contrast to traditional biosensor protocols that use both low pH and ionic strength. The use of a more physiological buffer to immobilize this enzyme leads to improved surface activity.

Keywords: Surface Plasmon Resonance (SPR) (表面等离子体共振（SPR）)

ProteOn XPR36 (质子xpr36)

BIosensor (生物传感器)

NS5B (NS5B)

Small molecule inhibitor (小分子抑制剂)

Materials and Reagents

HCV NS5B inhibitors
Purified recombinant HCV NS5B ΔC21 soluble protein [cloned and purified according to Boyce et al. (2014) and Hung et al. (2011)]
ProteOn GLH Sensor Chip (Bio-Rad Laboratories, catalog number: 176-5013 )
HEPES solution (1 M) (Sigma-Aldrich, catalog number: H3537-1L )
MgCl₂ (1 M) (Sigma-Aldrich, catalog number: M1028-100ML )
EDTA (0.5 M) (Sigma-Aldrich, catalog number: E7889-100ML )
NaCl (5 M) (Sigma-Aldrich, catalog number: S6546-1L )
KCl (2 M) (Life Technologies, Ambion^®, catalog number: AM9640G )
Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) (Sigma-Aldrich, catalog number: C4706-10G )
Surfactant P20 (10% v/v) (General Electric Company, catalog number: BR-1000-54 )
DMSO (Sigma-Aldrich, catalog number: 472301-100ML )
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
Preconditioning Reagents (see Recipes)
Immobilization Buffer (see Recipes)
Running Buffer (see Recipes)
Running Buffer with 5% DMSO (see Recipes)

Equipment

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

Software

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

Procedure

English

中文翻译

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如何引用

Wong, M. and Papalia, G. A. (2014). A Surface Plasmon Resonance Method to Study HCV NS5B Inhibitors . Bio-protocol 4(4): e1044. DOI: 10.21769/BioProtoc.1044.