Detecting ordered domain formation (lipid rafts) in model membranes using tempo

Short-range fluorescence quenching has proven to be an effective method to detect the presence of coexisting ordered and disordered state lipid domains in model membranes. In this approach a fluorescent group and fluorescence-quenching molecule are incorporated into the lipid bilayer of interest. In a typical experiment, the fluorophore chosen partitions into ordered domains to a significant degree, whereas the quencher partitions more favorably into disordered domains. Thus, in the presence of lipid mixtures forming coexisting ordered and disordered domains, fluorophore and quencher segregate so that fluorescence intensity is much stronger than in homogeneous lipid bilayers lacking separate domains. The small nitroxide-labeled molecule tempo (2,2,6,6 tetramethylpiperidine- 1-oxyl) is a useful quencher for such experiments. Protocols for using tempo to detect ordered domains and ordered domain thermal stability are described. The advantages and disadvantages of use of tempo as opposed to nitroxide-labeled lipids are also described.