The interaction of proline-rich ligands with profilin probed with an enzyme-linked immunosorbent assay.
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.
Veniere
Sylvie
S
Department of Medical Protein Research, VIB, Ghent, Belgium.
Ampe
Christophe
C
Vandekerckhove
Joël
J
Lambrechts
Anja
A
D000818:Animals; D001667:Binding, Competitive; D019921:Biotinylation; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002457:Cell Extracts; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008024:Ligands; D008840:Microfilament Proteins / Q000378:metabolism; D015335:Molecular Probes / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D051304:Profilins / Q000378:metabolism*; D011392:Proline / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D018909:src Homology Domains; D000098662:Vasodilator-Stimulated Phosphoprotein
D015815:Cell Adhesion Molecules; D002457:Cell Extracts; D008024:Ligands; D008840:Microfilament Proteins; D015335:Molecular Probes; D009419:Nerve Tissue Proteins; D010455:Peptides; D010750:Phosphoproteins; D051304:Profilins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000098662:Vasodilator-Stimulated Phosphoprotein; D011392:Proline