Fluorescence polarization is a method to determine membrane fluidity using a hydrophobic fluorescent dye that intercalates into the fatty acid bilayer. A spectrofluorometer is used to polarize UV light as a vertical excitation beam which passes through the dye-labeled membrane where the dye fluoresces. The beams perpendicular and horizontal to the excitation light are then collected and analyzed. Membrane structural properties are largely due to the packing of the fatty acids in the lipid bilayer that determines the membrane biophysical parameters. Staphylococcus aureus contains straight-chain (SCFAs) and branched-chain (BCFAs) fatty acids in the membrane and alters the proportion of membrane fluidizing BCFAs and stabilizing SCFAs as a response to a variety of stresses. Herein, we describe a method for determination of membrane fluidity in S. aureus using diphenylhexatriene, one of the most used fluorescent dyes for this purpose.
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