Supported Bilayer Electrophoresis under Controlled Buffer Conditions

摘要

A pH controlled flow cell device was constructed to allow electrophoreticnmovement of charged lipids and membrane associated proteins in supported phospholipidnbilayers. The device isolated electrolysis products near the electrodes from thenelectrophoresis process within the bilayer. This allowed the pH over the bilayer region tonremain within (0.2 pH units or better over many hours at salt concentrations up to 10nmM. Using this setup, it was found that the electrophoretic mobility of a dye conjugatednlipid (Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (TR-DHPE))nwas essentially constant between pH 3.3 and 9.3. In contrast, streptavidin, which wasnbound to biotinylated lipids, shifted from migrating cathodically at acidic pH values tonmigrating anodically under basic conditions. This shift was due to the modulation of thennet charge on the protein, which changed the electrophoretic forces experienced by the macromolecule. The addition of anpolyethylene glycol (PEG) cushion beneath the bilayer or the increase in the ionic strength of the buffer solutionnresulted in a decrease of the electroosmotic force experienced by the streptavidin with little effect on the Texas Red-DHPE.nAs such, it was possible in part to control the electrophoretic and electroosmotic contributions to streptavidin independently ofnone another.