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

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

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Article in STAR protocols, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.

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4 · The record

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5 · Who and what money

Authors and funding

4 authors.

Adriana Reyes-OrdoñezDepartment of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA.

Shweta ShreeDepartment of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA.

Stephen G SligarDepartment of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA; Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA; Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA; Cancer Center at Illinois, University of Illinois at Urbana-Champaign, Urbana, IL, USA.

Jie ChenDepartment of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA; Cancer Center at Illinois, University of Illinois at Urbana-Champaign, Urbana, IL, USA; Department of Biomedical and Translational Sciences, Carle Illinois College of Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, USA. Electronic address: jiechen@illinois.edu.

Funding

No grant is acknowledged in the PubMed record.

6 · The paper itself

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.

Indexed as

LipidsNanostructuresProteinsSingle Molecule ImagingAnimalsHumansMicroscopy, FluorescenceProtein BindingLipidsProteinsBiophysicsCell BiologyCell cultureSingle-molecule Assays

Identifiers

PMID41712387

DOI10.1016/j.xpro.2026.104377

PMCPMC12926638

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