Practice question

Experimental investigation of lipid dynamics in plasma membranes of living cells requires fluorescence techniques capable of micrometer spatial resolution and millisecond temporal. FRAP fluorescence recovery after photobleaching involves incorporating fluorescent lipid analogs NBD-phosphatidylethanolamine BODIPY-phosphatidylcholine DiI dialkylcarbocyanine into bilayer at low mole fraction, or fluorescent protein fusions GFP-tagged for proteins. Focused laser beam bleaches circular spot one to four micrometer diameter irreversible photodestruction, subsequent recovery of fluorescence intensity monitored at low laser power as unbleached fluorophores diffuse from adjacent regions into bleached zone following Fick laws. Analysis yields diffusion coefficient D and mobile fraction Mf reflecting barriers cytoskeleton fences eighty nanometer corrals and lipid raft domains ordered phases slowing recovery. Complementary methods single particle tracking gold conjugated lipids high speed video, fluorescence correlation spectroscopy FCS confocal fluctuation. Western blotting separates denatured subunits by SDS-PAGE immunodetection does not report movement, X-ray crystallography determines static atomic coordinates crystalline lattice, PCR amplifies DNA. FRAP remains gold standard quantifying lateral movement of lipids and proteins supporting fluidity concept and membrane organization measurement.