title: Lectin-Magnetic Beads for Plasma Membrane Isolation.
abstract: Plasma membrane proteins mainly function to transmit external signals into the cell. Many plasma membrane receptor tyrosine kinases (e.g., HER2 and EGFR) are known to mediate oncogenic progression, making them prime targets for cancer therapy. Recently, it has become important to identify plasma membrane proteins that are differentially expressed in normal versus cancer cells, in drug-sensitive versus drug-resistant cells, or among tumor cells that metastasize to different organ sites because these differentially expressed membrane proteins may lead to the identification of therapeutic targets or diagnostic markers. In addition, there is an increased interest in identifying cell-surface proteins that could serve as markers for stem cells, progenitor cells, or cells of different lineages. Traditionally, membrane isolation requires multiple centrifugation steps to isolate different organelles based on their density. With the advent of affinity matrix technology, it is possible to separate organelles based on their molecular differences. A defining characteristic of the plasma membrane is that plasma membrane proteins are more extensively glycosylated than are intracellular membrane proteins. As a result, affinity chromatography employing lectin, a carbohydrate-binding protein, is commonly used to isolate plasma membrane proteins. We have extended this concept for plasma membrane isolation by using concanavalin A (ConA), a lectin with mannose specificity. Here we describe a protocol that uses immobilized ConA bound to magnetic beads to isolate plasma membranes from homogenized cell lysates. The captured plasma membrane proteins are then solubilized from the ConA-magnetic beads by detergents in the presence of a competing sugar, methyl α-mannopyranoside.
authors.affiliation: Department of Translational Molecular Pathology, University of Texas, M.D. Anderson Cancer Center, Houston, Texas 77030.
authors.forename: Yu-Chen
authors.initials: YC
authors.lastname: Lee
authors.affiliation: Department of Translational Molecular Pathology, University of Texas, M.D. Anderson Cancer Center, Houston, Texas 77030.
authors.forename: Hsuan-Chen
authors.initials: HC
authors.lastname: Liu
authors.affiliation: Department of Translational Molecular Pathology, University of Texas, M.D. Anderson Cancer Center, Houston, Texas 77030.
authors.forename: Carol
authors.initials: C
authors.lastname: Chuang
authors.affiliation: Department of Translational Molecular Pathology, University of Texas, M.D. Anderson Cancer Center, Houston, Texas 77030.
authors.forename: Sue-Hwa
authors.initials: SH
authors.lastname: Lin
mesh_terms: D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000737:chemistry; D003208:Concanavalin A / Q000378:metabolism*; D055767:Immobilized Proteins / Q000378:metabolism*; D008280:Magnetics; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008863:Microspheres; D011485:Protein Binding
chemical_list: D055767:Immobilized Proteins; D008562:Membrane Glycoproteins; D003208:Concanavalin A