PLiMAP: Proximity‐Based Labeling of Membrane‐Associated Proteins

Abstract

Peripheral membrane proteins participate in numerous biological pathways. Thus, methods to analyze their membrane-binding characteristics have become important. In this report, we detail protocols for the synthesis and utilization of a photoactivable fluorescent lipid as a reporter to monitor membrane binding of proteins. The assay, referred to as proximity-based labeling of membrane-associated proteins (PLiMAP), is based on UV activation of a fluorescent lipid reporter, which in turn crosslinks with proteins bound to membranes and renders them fluorescent. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of BODIPY-diazirine phosphatidylethanolamine (BDPE) Basic Protocol 2: Preparation of BDPE-containing liposomes Basic Protocol 3: Performing PLiMAP with a candidate protein Basic Protocol 4: Quantitation of liposome-binding properties of the candidate protein from analyzing in-gel fluorescence Support Protocol: Purification of GST-2×P4M domain of SidM protein.