title: Protein separation by electrophoretic-electroosmotic focusing on supported lipid bilayers.
abstract: An electrophoretic-electroosmotic focusing (EEF) method was developed and used to separate membrane-bound proteins and charged lipids based on their charge-to-size ratio from an initially homogeneous mixture. EEF uses opposing electrophoretic and electroosmotic forces to focus and separate proteins and lipids into narrow bands on supported lipid bilayers (SLBs). Membrane-associated species were focused into specific positions within the SLB in a highly repeatable fashion. The steady-state focusing positions of the proteins could be predicted and controlled by tuning experimental conditions, such as buffer pH, ionic strength, electric field, and temperature. Careful tuning of the variables should enable one to separate mixtures of membrane proteins with only subtle differences. The EEF technique was found to be an effective way to separate protein mixtures with low initial concentrations, and it overcame diffusive peak broadening to allow four bands to be separated simultaneously within a 380 μm wide isolated supported membrane patch.
authors.affiliation: Department of Chemistry, Texas A&M University, 3255 TAMU, College Station, Texas 77843, USA.
authors.forename: Chunming
authors.initials: C
authors.lastname: Liu
authors.forename: Christopher F
authors.initials: CF
authors.lastname: Monson
authors.forename: Tinglu
authors.initials: T
authors.lastname: Yang
authors.forename: Hudson
authors.initials: H
authors.lastname: Pace
authors.forename: Paul S
authors.initials: PS
authors.lastname: Cremer
mesh_terms: D004560:Electricity; D005456:Fluorescent Dyes / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008051:Lipid Bilayers / Q000737:chemistry*; D008565:Membrane Proteins / Q000302:isolation & purification*; D009994:Osmolar Concentration; D013696:Temperature
chemical_list: D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D008565:Membrane Proteins