AVS 49th International Symposium
    Biomaterials Tuesday Sessions
       Session BI+SS-TuM

Paper BI+SS-TuM10
Purification of Mobile, Membrane-tethered Proteins in Micropatterned Supported Lipid Bilayers

Tuesday, November 5, 2002, 11:20 am, Room C-201

Session: Platforms for Non-fouling and Patterned Surfaces
Presenter: L. Kam, Stanford University
Authors: L. Kam, Stanford University
T.D. Perez, Stanford University
W.J. Nelson, Stanford University
S.G. Boxer, Stanford University
Correspondent: Click to Email
Supported lipid bilayers are a unique system for studying fluidic membranes in a controllable in vitro format. A variety of methods for tethering proteins to supported bilayers provide a powerful reductionist model of cell-cell recognition and activation. However, contemporary methods for preparing membrane-tethered protein systems typically incur an immobile fraction; this population complicates and, at worst, subverts interpretation of experimental results. Here, we present a method for separating a population of mobile, GPI-tethered protein from an immobile fraction in a supported lipid bilayer. A GPI-modified protein based on the cell-cell adhesion protein E-cadherin was introduced into Egg PC vesicles by detergent dialysis. On a glass substrate, two adjacent and connected regions of supported lipid bilayer were created using a converging flow configuration. One region contained both mobile and immobile populations of GPI/cadherin while the other contained Egg PC alone. An electric field applied tangentially to the surface induced migration of the mobile, but not immobile, protein into the region of Egg PC, generating a purified population of these proteins which may then be isolated for analysis or further experimentation free from the immobile fraction. Importantly, this method is independent of the specific factors influencing protein mobility and thus generally applicable.