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Native holdup (nHU) to measure binding affinities from cell extracts.
Zambo, Boglarka; Morlet, Bastien; Negroni, Luc; Trave, Gilles; Gogl, Gergo.
Afiliação
  • Zambo B; Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U1258/CNRS UMR 7104/Université de Strasbourg, 1 rue Laurent Fries, BP 10142, Illkirch F-67404, France.
  • Morlet B; Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U1258/CNRS UMR 7104/Université de Strasbourg, 1 rue Laurent Fries, BP 10142, Illkirch F-67404, France.
  • Negroni L; Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U1258/CNRS UMR 7104/Université de Strasbourg, 1 rue Laurent Fries, BP 10142, Illkirch F-67404, France.
  • Trave G; Équipe Labellisée Ligue 2015, Département de Biologie Structurale Intégrative, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U1258/CNRS UMR 7104/Université de Strasbourg, 1 rue Laurent Fries, BP 10142, Illkirch F-67404, France.
  • Gogl G; Équipe Labellisée Ligue 2015, Département de Biologie Structurale Intégrative, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U1258/CNRS UMR 7104/Université de Strasbourg, 1 rue Laurent Fries, BP 10142, Illkirch F-67404, France.
Sci Adv ; 8(51): eade3828, 2022 Dec 21.
Article em En | MEDLINE | ID: mdl-36542723
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.

Texto completo: 1 Base de dados: MEDLINE Tipo de estudo: Qualitative_research Idioma: En Ano de publicação: 2022 Tipo de documento: Article

Texto completo: 1 Base de dados: MEDLINE Tipo de estudo: Qualitative_research Idioma: En Ano de publicação: 2022 Tipo de documento: Article