Protocolo de ensayo de captura de molécula única Nanodisc para detectar interacciones proteína-lípido

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.