RT Journal Article
SR Electronic
T1 Protein-protein interactions on membrane surfaces analysed using pull-downs with supported bilayers on silica beads
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.12.06.471516
DO 10.1101/2021.12.06.471516
A1 Andhare, Devika S.
A1 Khurana, Himani
A1 Pucadyil, Thomas J.
YR 2022
UL http://biorxiv.org/content/early/2022/01/30/2021.12.06.471516.abstract
AB Discovery-based proteomics workflows that identify novel interactors rely on immunoprecipitations or pull-downs with genetically-tagged bait proteins immobilized on appropriate matrices. But strategies to analyse protein interactions on a diffusible membrane surface combined with the practical ease of pull-downs remain unavailable. Such strategies are important to analyse protein complexes that mature in composition and stability because of diffusion-based encounter between participant proteins. Here, we describe a generic pull-down strategy to analyse such complexes using chelating lipid-containing supported bilayers formed on silica beads. These templates can display desired His-tagged bait proteins on a diffusible membrane surface. Using clathrin-mediated endocytosis as a paradigm, we find that the clathrin-binding adaptor protein epsin1 displayed as bait on these templates pulls down significantly higher amounts of clathrin from brain lysates than when immobilized on conventional matrices. Together, our results establish the potential of such templates as superior matrices for analysing protein-protein interactions and resultant complexes formed on membrane surfaces.Competing Interest StatementThe authors have declared no competing interest.