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

## TL;DR

This paper provides a detailed protocol for detecting protein-lipid interactions using a single-molecule pull-down assay with Nanodiscs and TIRF microscopy.

## Contribution

The paper introduces a new protocol for studying lipid-protein interactions at single-molecule resolution using mammalian cell lysates and Nanodiscs.

## Key findings

- Nanodiscs and TIRF microscopy enable detection of protein-lipid interactions at single-molecule resolution.
- The protocol includes steps for preparing, immobilizing, and analyzing lipid Nanodiscs for interaction studies.

## 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

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.