基于位移的酶联免疫吸附试验：配体竞争结合Ni2+-NTA板上His标签能力的定量分析

Allexa Dow; Sladjana Prisic

doi:10.21769/BioProtoc.1745

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Peer-reviewed

Displacement-based ELISA: Quantifying Competition between Two Binding Partners for Interaction with a His-tagged Ligand Immobilized on a Ni²⁺-NTA Plate

基于位移的酶联免疫吸附试验：配体竞争结合Ni2+-NTA板上His标签能力的定量分析

AD Allexa Dow

SP Sladjana Prisic email

发布: 2016年03月05日第6卷第5期 DOI: 10.21769/BioProtoc.1745 浏览次数: 9621

评审: Valentine V TrotterAnonymous reviewer(s)

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Abstract

The displacement assay was designed to quantify the direct competition between two homologous ribosomal proteins from Mycobacterium tuberculosis, S18-1 and S18-2, for interaction with their cognate binding partner, ribosomal protein S6 (Prisic et al., 2015). The S18 proteins were dialyzed in two physiologically relevant conditions (i.e. in the presence of Zn²⁺ or with EDTA to chelate Zn²⁺) and then allowed to compete for binding to S6 which was maintained in limiting concentration. The result was obtained through an ELISA, where S6-His is first bound to a Ni²⁺-NTA plate, followed by addition of S18-2 in excess to S6, then by addition of increasing concentrations of S18-1. The percentage of S18-2 that remained bound to S6 was quantified with antibodies specific to the S18-2 protein and secondary antibodies, in chemiluminescent ELISA. In this way displacement of S18-2 protein by the S18-1 protein was reported as a percentage of the full strength signal achieved through saturation of S6 with S18-2. At its foundation, this method exploits a native protein-protein interaction and could be applied to other systems where two or more proteins compete for binding to a target ligand as above.

Keywords: ELISA (ELISA)

His tag (他的标签)

Protein-protein interaction (蛋白质-蛋白质相互作用)

Materials and Reagents

HisPur^TM Cobalt Spin Columns (Thermo Fisher Scientific, catalog number: 89969 )
Pierce™ Polyacrylamide Spin Desalting Columns, 7 K MWCO, 0.7 ml (Thermo Fisher Scientific, catalog number: 89849 )
5 PRIME^TM Ni²⁺-NTA HisPrime^TM plates (Thermo Fisher Scientific, catalog number: 2400730 )
ProTEV Plus (Promega Corporation, catalog number: V6101 )
cOmplete His-Tag Purification Resin (Sigma-Aldrich, Roche Diagnostics, catalog number: 05893682001 )
BioRad Protein Concentration Assay (Bio-Rad Laboratories, catalog number: 5000002 )
Albumin, Bovine, Fraction V (pH 7) (BSA) (Affymetrix, catalog number: 10857 50 gm )
Rabbit Polyclonal Antibody raised to peptide antigen from competing protein (i.e. S18-2) (NeoPeptide, custom made against peptide PGQDRQRRAALCP)
Goat Anti-Rabbit IgG, Peroxidase Conjugated Secondary Antibody (Thermo Fisher Scientific, Pierce^TM, catalog number: 31460 )
SuperSignal^TM West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, catalog number: 34080 )
HEPES Sodium Salt (Thermo Fisher Scientific, Fisher BioReagents, catalog number: BP410-500 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: 793590-500 g )
Tween^® 20 (enzyme grade) (Thermo Fisher Scientific, Fisher BioReagents, catalog number: BP337-500 )
2-mercaptoethanol (Thermo Fisher Scientific, Fisher BioReagents, catalog number: BP176-100 )
EDTA disodium salt (Thermo Fisher Scientific, Fisher BioReagents, catalog number: BP120-500 )
ZnSO₄^.7H₂O (Sigma-Aldrich, catalog number: Z4750-100 g )
5x HEPES/KCl (see Recipes)
Microscale Thermophoresis Buffer (MST) buffer (see Recipes)
MST with Zn²⁺ (see Recipes)
MST with EDTA (see Recipes)

Equipment

Multichannel pipettor (20-300 µl) (Gilson, model: FA10016 )
Microplate reader with absorbance and chemiluminescent capacity (BioTek Instruments, model: Synergy 2 )
Benchtop microplate shaker (IKA^® Works, model: MS 3 digital )

Procedure

English

中文翻译

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如何引用

Dow, A. and Prisic, S. (2016). Displacement-based ELISA: Quantifying Competition between Two Binding Partners for Interaction with a His-tagged Ligand Immobilized on a Ni²⁺-NTA Plate. Bio-protocol 6(5): e1745. DOI: 10.21769/BioProtoc.1745.