Work

Protocol for Nanodisc single-molecule pull-down assay to detect protein-lipid interactions

Year: 2026

Type: article

Abstract: Phospholipids function as signaling molecules regulating numerous processes within the cellular environment. Identifying and characterizing the protein players involved in lipid-mediated functions remains a central focus. Here, we present a protocol for evaluating lipid-protein interactions using mammalian whole-cell lysates expressing proteins of interest in a single-molecule pull-down (SiMPull) assay. We describe steps for preparing materials and slide chambers, performing SiMPull, and analyzing data. Nanodiscs and total internal reflection fluorescence (TIRF) microcopy enable the detection of interactions at single-molecule resolution. For complete details on the use and execution of this protocol, please refer to Arauz et al. 1 and Reyes-Ordoñez et al. 2 • Procedures for assembling and purifying lipid Nanodiscs • Steps for preparing quartz slide: cleaning, passivating, and immobilizing Nanodiscs • Instructions for performing TIRF microscopy to capture protein-Nanodisc interactions • Guidance on single-molecule data processing and analysis Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Phospholipids function as signaling molecules regulating numerous processes within the cellular environment. Identifying and characterizing the protein players involved in lipid-mediated functions remains a central focus. Here, we present a protocol for evaluating lipid-protein interactions using mammalian whole-cell lysates expressing proteins of interest in a single-molecule pull-down (SiMPull) assay. We describe steps for preparing materials and slide chambers, performing SiMPull, and analyzing data. Nanodiscs and total internal reflection fluorescence (TIRF) microcopy enable the detection of interactions at single-molecule resolution.

Source: STAR Protocols

DOI: 10.1016/j.xpro.2026.104377

Authors: Adriana Reyes-Ordoñez, Shweta Shree, Stephen G. Sligar, Jie Chen

Institutions: University of Illinois Urbana-Champaign, Cancer Center at Illinois

Language: English

Topic: Lipid Membrane Structure and Behavior

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $1,550

Funders: National Institutes of Health, National Institute of General Medical Sciences