A Computational Protocol to Analyze PDZ/PBM Affinity Data Obtained by High-Throughput Holdup Assay.
Fiche publication
Date publication
janvier 2021
Journal
Methods in molecular biology (Clifton, N.J.)
Auteurs
Membres identifiés du Cancéropôle Est :
Dr TRAVE Gilles, Dr NOMINE Yves
Tous les auteurs :
Jané P, Chiron L, Bich G, Travé G, Nominé Y
Lien Pubmed
Résumé
The holdup assay is an automated high-throughput comparative chromatographic retention approach that allows to measure quantitative binding intensities (BI) for a large number of domain-motif pairs and deduce equilibrium binding affinity constants. We routinely apply this approach to obtain quantitative binding specificity profiles of particular PDZ-binding motifs (PBMs) toward the full library of known human PDZ domains (the PDZome). The quality of the electropherograms extracted from the capillary electrophoresis instrument at the final step of the holdup assay may vary, influencing the accuracy and reproducibility of the measurement. By using bioinformatic tools, we can solve these issues to extract more reliable BIs by means of a better superimposition of the electropherograms. The protocol presented in this chapter describes the main principles and strategies of our curated method to process holdup data and new ways to plot and compare the BIs for the PBM-PDZ interactions. For this particular protocol, all the necessary computing commands are freely available in open Python packages.
Mots clés
Computational approach, Electropherogram superimposition, Holdup assay, PDZ–PBM interaction, Processing accuracy
Référence
Methods Mol Biol. 2021 ;2256:61-74