Tools for Studying Membranes
• Enrichment of membrane factions
• Isolation of membrane components
• Characterization of membrane components
• Reconstitution of membrane components
Enrichment of Membrane Fractions
• Disruption of source material
• Tool = Homogenization
• Separation/enrichment
• Tool = Centrifugation
• Purification
• Tool = Detergents
• Reconstitution
• Tool = lipid bilayer model system
Detergents
• Water soluble surfactants (reduce surface tension)
• Amphiphilic in nature
• Effect in solubilizing membrane components
• More soluble in water compared to lipids
• Grouped by function (ionic, zwitterionic, nonionic)
• Impurities are common in detergents (!!!!)
Action of Detergents
• Act through micelle formation (spherical assembly)
• Partial exposure of hydrophobic moieties
• Occurs at a defined concentration (and Temperature)
• CMC critical micellar concentration
• Below CMC - all monomers
Practical Aspects
• Detergents enable to bring membrane proteins and lipids in the aqueous phase
• Steps of actions are concentration dependent
• Intercalation to mixed micelles (above CMC necessary)
• Detergent concentration and detergent-to-protein ratio influence extraction
• Membrane solubilization is roughly at CMC
Membrane Reconstitution
• Monolayer model systems provided insight how lipid components and physical parameters (pH, ion strength, T) influence one leaflet of a membrane
• Planar bilayer model systems provided insight into electrochemical aspects, transport events, and membrane protein behavior
• Curved, closed-in bilayer model systems (liposomes) provided insight mostly into membrane protein behavior
Lipid Monolayers
• Amphipathic molecules (lipids) line up at an air-water interface
• Access to inside of lipid bilayer• Control of composition and amount of lipid• Model system to study constraints of membrane
behavior by lipid components
Planar Lipid Bilayer
• Separation of two compartments
• Studies of electrical properties, transport, osmosis, and smaller proteins
• Electrophysiological studies per-patch clamp