Native holdup (nHU) to measure binding affinities from cell extracts.

Date publication

décembre 2022

Journal

Science advances

Auteurs

Membres identifiés du Cancéropôle Est :
Dr TRAVE Gilles , Dr NEGRONI Luc , Mr MORLET Bastien , Dr GOGL Gergo

Tous les auteurs :
Zambo B, Morlet B, Negroni L, Trave G, Gogl G

Lien Pubmed

https://www.ncbi.nlm.nih.gov/pubmed/36542723

Résumé

Characterizing macromolecular interactions is essential for understanding cellular processes, yet most methods currently used to detect protein interactions from cells are qualitative. Here, we introduce the native holdup (nHU) approach to estimate equilibrium binding constants of protein interactions directly from cell extracts. Compared to other pull-down-based assays, nHU requires less sample preparation and can be coupled to any analytical methods as readouts, such as Western blotting or mass spectrometry. We use nHU to explore interactions of SNX27, a cargo adaptor of the retromer complex and find good agreement between in vitro affinities and those measured directly from cell extracts using nHU. We discuss the strengths and limitations of nHU and provide simple protocols that can be implemented in most laboratories.

Référence

Sci Adv. 2022 12 21;8(51):eade3828