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

Abstract

AbstractCharacterizing 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.