Open AccessThe Interaction of Proline-Rich Ligands with Profilin Probed with an Enzyme-Linked Immunosorbent Assay
Author(s) -
Sylvie Veniere,
Christophe Ampè,
Joël Vandekerckhove,
Anja Lambrechts
Publication year - 2009
Publication title -
slas discovery
Language(s) - English
Resource type - Journals
eISSN - 2472-5560
pISSN - 2472-5552
DOI - 10.1177/1087057109332594
Subject(s) - profilin , ternary complex , chemistry , biochemistry , recombinant dna , enzyme , microbiology and biotechnology , biology , gene , cytoskeleton , actin cytoskeleton , cell
To detect interactions of different proline-rich ligands with profilins, the authors developed a simple analytical antibody-based screening method. Profilin I or profilin IIa was coated in microplates, and ligand binding was monitored via antibody detection. Using purified components, the authors show that the assay is very sensitive as nanomolar concentrations of recombinant profilin ligands can be used. They further apply this technique to detect interaction of profilin with various proline-rich partners, either endogenously present or ectopically expressed as tagged fusions, using lysates. With this assay, the authors identify Shootin1 as a novel profilin IIa partner. In addition, they demonstrate that this assay can be used for studying competition or ternary complex formation. In conclusion, they developed a sensitive, easy-to-use, and versatile method for the study of the interaction between profilin and different ligands.