Proteomics

Dataset Information

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


ABSTRACT: Characterizing macromolecular interactions is essential for understanding cellular processes, yet nearly all methods used to detect protein interactions from cells are qualitative. Here, we introduce the native holdup (nHU) approach to quantify equilibrium binding constants and explore binding mechanisms of protein interactions from cell extracts. Compared to other pulldown-based assays, nHU requires less sample preparation and can be coupled to any analytical methods, such as western blotting (nHU-WB) or mass spectrometry (nHU-MS) as readouts. 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. This challenges the unwritten paradigm stating that biophysical parameters like binding constants cannot be accurately determined from cells or cellular extracts. We discuss the strengths and limitations of nHU and provide simple protocols that can be implemented in most laboratories.

INSTRUMENT(S): Orbitrap Exploris 480, LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell

DISEASE(S): Brain Cancer

SUBMITTER: Luc Negroni  

LAB HEAD: Gergo Gogl

PROVIDER: PXD036024 | Pride | 2023-03-11

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
220627_GG_01_rep1_dil10.msf Msf
220627_GG_01_rep1_dil10.raw Raw
220627_GG_01_rep2_dil10.msf Msf
220627_GG_01_rep2_dil10.raw Raw
220627_GG_01_rep3_dil10.msf Msf
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Publications

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

Zambo Boglarka B   Morlet Bastien B   Negroni Luc L   Trave Gilles G   Gogl Gergo G  

Science advances 20221221 51


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 mas  ...[more]

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