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 a native holdup (nHU) approach that requires minimal sample preparation, investigates binding mechanisms, and quantifies equilibrium binding constants of protein interactions from cell extracts. We use nHU coupled with western blotting (nHU-WB) and mass spectrometry (nHU-MS) to explore interactions of SNX27, a cargo adaptor of the retromer complex. We find good agreement between affinities measured using minimal binding fragments 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):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell

DISEASE(S): Brain Cancer

SUBMITTER: Luc Negroni  

LAB HEAD: Gilles Travé

PROVIDER: PXD034790 | Pride | 2023-03-11

REPOSITORIES: Pride