TY  - JOUR
TI  - Displacement-based ELISA: Quantifying Competition between Two Binding Partners for Interaction with a His-tagged Ligand Immobilized on a Ni2+-NTA Plate
AU  - Dow, Allexa 
AU  - Prisic, Sladjana 
VL  - 6
IS  - 5
PY  - 2016
DA  - 2016/03/05
SP  - e1745
C1  - Bio-protocol 2016;6:e1745
DO  - 10.21769/BioProtoc.1745
UR  - https://doi.org/10.21769/BioProtoc.1745
AB  - 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.
KW  - ELISA
KW  - His tag
KW  - Protein-protein interaction
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False