Immunofluorescence Labeling of Lipid-Binding Proteins CERTs to Monitor Lipid Raft Dynamics.

Abstract

Fluorescence microscopy is a powerful and widely used tool in molecular biology. Over the years, the discovery and development of lipid-binding fluorescent probes has established new research possibilities to investigate lipid composition and dynamics in the cell. For instance, fluorescence microscopy has allowed the investigation of lipid localization and density in specific cell compartments such as membranes or organelles. Often, the characteristics and the composition of lipid-enriched structures are determined by analyzing the distribution of a fluorescently labeled lipid probe, which intercalates in lipid-enriched platforms, or specifically binds to parts of the lipid molecule. However, in many cases antibodies targeting proteins have higher specificity and are easier to generate. Therefore, we propose to use both antibodies targeting lipid transporters and lipid binding probes to better monitor lipid membrane changes. As an example, we visualize lipid rafts using the fluorescently labeled-B-subunit of the cholera toxin in combination with antibodies targeting ceramide-binding proteins CERTs, central molecules in the metabolism of sphingolipids.