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bioRxiv · 10.1101/2021.07.07.451367

Rapid extraction and kinetic analysis of protein complexes from single cells

Sarıkaya, S.·Dickinson, D. J.
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Abstract

Proteins contribute to cell biology by forming dynamic, regulated interactions, and measuring these interactions is a foundational approach in biochemistry. We present a rapid, quantitative in vivo assay for protein-protein interactions, based on optical cell lysis followed by time-resolved single-molecule analysis of protein complex binding to an antibody-coated substrate. We show that our approach has better reproducibility, higher dynamic range, and lower background than previous single-molecule pull-down assays. Furthermore, we demonstrate that by monitoring cellular protein complexes over time after cell lysis, we can measure the dissociation rate constant of a cellular protein complex, providing information about binding affinity and kinetics. Our dynamic single-cell, single-molecule pull-down method thus approaches the biochemical precision that is often sought from in vitro assays, while being applicable to native protein complexes isolated from single cells in vivo.

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Sarıkaya, S., Dickinson, D. J.. 2021-07-07. Rapid extraction and kinetic analysis of protein complexes from single cells. https://doi.org/10.1101/2021.07.07.451367

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