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Methods Mol Biol. 2011;748:227-41. doi: 10.1007/978-1-61779-139-0_16.

Use of colloidal silica-beads for the isolation of cell-surface proteins for mass spectrometry-based proteomics.

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  • 1Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada.

Abstract

Chaney and Jacobson first introduced the colloidal silica-bead protocol for the coating of cellular plasma membranes in the early 1980s. Since then, this method has been successfully incorporated into a wide range of in vitro and in vivo applications for the isolation of cell-surface proteins. The principle is simple - cationic colloidal silica microbeads are introduced to a suspension or monolayer of cells in culture. Electrostatic interactions between the beads and the negatively charged plasma membrane, followed by cross-linking to the membrane with an anionic polymer, ensure attachment and maintain the native protein conformation. Cells are subsequently ruptured, and segregation of the resulting plasma membrane sheets from the remaining- cell constituents is achieved by ultracentrifugation through density gradients. The resulting membrane-bead pellet is treated with various detergents or chaotropic agents (i.e., urea) to elute bound proteins. If proteomic profiling by mass spectrometry is desired, proteins are denatured, carbamidomethylated, and digested into peptides prior to chromatography.

PMID:
21701978
[PubMed - indexed for MEDLINE]
PMCID:
PMC3706334
Free PMC Article

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