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Updated: Apr 26, 2026

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy
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Protocol for Nanodisc single-molecule pull-down assay to detect protein-lipid interactions.

Adriana Reyes-Ordoñez1, Shweta Shree2, Stephen G Sligar3

  • 1Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA.

STAR Protocols
|February 19, 2026
PubMed
Summary

This study introduces a single-molecule pull-down (SiMPull) assay protocol to identify protein interactions with phospholipids in cellular environments. The method uses Nanodiscs and TIRF microscopy for high-resolution detection of these crucial lipid-protein interactions.

Keywords:
BiophysicsCell BiologyCell cultureSingle-molecule Assays

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Area of Science:

  • Cellular Biology
  • Biochemistry
  • Molecular Interactions

Background:

  • Phospholipids are key signaling molecules regulating cellular processes.
  • Identifying proteins involved in lipid-mediated functions is critical for understanding cell signaling.
  • Current methods may lack the resolution to characterize these interactions effectively.

Purpose of the Study:

  • To present a detailed protocol for evaluating lipid-protein interactions.
  • To enable the characterization of protein players involved in phospholipid signaling.
  • To utilize advanced microscopy and nanotechnology for high-resolution analysis.

Main Methods:

  • Development of a single-molecule pull-down (SiMPull) assay protocol.
  • Utilizing mammalian whole-cell lysates expressing proteins of interest.
  • Employing Nanodiscs and total internal reflection fluorescence (TIRF) microscopy for detection.

Main Results:

  • The protocol allows for the preparation of materials and slide chambers for SiMPull assays.
  • Successful execution of the SiMPull assay enables data acquisition.
  • Single-molecule resolution detection of lipid-protein interactions is achieved.

Conclusions:

  • The presented SiMPull assay protocol provides a robust method for studying lipid-protein interactions.
  • This technique facilitates the identification and characterization of proteins involved in phospholipid signaling.
  • The use of Nanodiscs and TIRF microscopy enhances the resolution and sensitivity of interaction detection.