(255f) Separation and Characterization of Raft Associated Membrane Species Using a Patterned Supported Lipid Bilayer

The role of lipid rafts is important in understanding
cell processes and disease states; however, identifying the key components of
these lipid domains is difficult and impedes research progress. Here we present
a new platform to sort, separate, and characterize membrane-bound molecules based
on their affinity for raft phase domains using a heterogeneous supported lipid
bilayer (SLB) consisting of patterned lipid raft and non-raft domains.  Adjacent SLBs consisting of liquid-ordered and
liquid-disordered phases are formed in predefined locations. Membrane-bound
biomolecules loaded into the device are convected laterally in the
two-dimensional plane along the heterogeneous supported bilayer by a shear
force induced from hydrodynamic flow of the bulk aqueous phase. During axial convection,
the membrane-bound biomolecules diffuse within the 2-D heterogeneous bilayer
plane, partitioning into their preferred lipid phase. Patterns for the lipid
phases are designed to facilitate the sorting and collection of separated
species. The main advantages of this new method over existing methods used to identify
and assay raft species include separating membrane species within a membrane
environment, near physiological conditions, and without artifacts associated
with detergent, high salt, or alkaline pH. These criteria are particularly crucial
to the separation of lipid-linked proteins and transmembrane proteins to avoid denaturation.
This new capability to separate membrane-bound species near native conditions
based on affinity for certain lipid phases can be used to identify intrinsic membrane
raft residents, characterize how post-translational modifications shift the
affinity of analogs to a particular lipid phase and dynamically observe the
effect of membrane composition on membrane species behavior.