Please use this identifier to cite or link to this item: http://hdl.handle.net/10125/40213

Displacement-based ELISA: Quantifying Competition between Two Binding Partners for Interaction with a His-tagged Ligand Immobilized on a Ni 2+ -NTA Plate

Item Summary

Title: Displacement-based ELISA: Quantifying Competition between Two Binding Partners for Interaction with a His-tagged Ligand Immobilized on a Ni 2+ -NTA Plate
Authors: Dow, Allexa
Prisic, Sladjana
Issue Date: Mar 2016
Publisher: bio-protocol
Related To: http://www.bio-protocol.org/e1745
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 Zn^2+ or with EDTA to chelate Zn^2+) 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 Ni^2+-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.
Pages/Duration: 7
URI/DOI: http://hdl.handle.net/10125/40213
Appears in Collections:Department of Microbiology Faculty & Researcher Works



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