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The detection of protein complexes by coimmunoprecipitation or two-hybrid analysis is often limited to cytosolic and soluble proteins, while interaction between membrane proteins or proteins and lipids is hampered by solubilization artefacts or absence of appropriate antibodies to detect a complex. More recently, the proximity ligation assay (PLA) using antibodies for in situ detection of protein complexes in cells and cross-linkable lipid analogs that can be endowed with molecular tags for pull-down assyas were techniques utilized to identify and monitor interaction between proteins and lipids. We have developed a novel technique termed "cross-link/PLA" combining a cross-linkable ceramide analog with PLA and anti-ceramide antibody to visualize lipid-protein complexes in ceramide-rich platforms (CRPs), a particular type of lipid raft. This chapter will discuss experimental protocols and data analysis to use cross-link/PLA for detection and visualization of lipid-protein complexes in CRPs and other types of lipid rafts.

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.

Extracellular vesicles (EVs) are secreted by eukaryotic cells and serve as carriers for a variety of cell signaling factors, including RNAs, proteins, and lipids. We described a unique population of EVs, the membrane of which is highly enriched with the sphingolipid ceramide. We suggested that ceramide in the EV membrane is organized in ceramide-rich platforms (CRPs), a type of lipid raft that mediates interaction of ceramide with ceramide-associated proteins (CAPs). Here, we describe methods using anti-ceramide antibody to isolate ceramide-enriched EVs and detect exosomes after uptake into recipient cells. In addition, we discuss methods for EV analysis using nanoparticle tracking and mass spectrometry. The methods can be extended to the isolation of other types of EVs and "mobile rafts" transported by EVs from donor to recipient cells using antibodies against lipids specific for these EVs.