One-step isolation of plasma membrane proteins using magnetic beads with immobilized concanavalin A
| Autor: | Emma Folch-Puy, Teri McLain, Yu Chen Lee, Jin Lu, Gregory J. Block, Joseph Smith, Roxana Jalili, Marija Zanic, Edward Kraft, Robert F. Foronjy, Huiwen Chen, Masashi Kimura, Sue Hwa Lin, Aaron Spivak, Søren Lindemose, Michael L. Wang, Santiago Ramón-García, Christian Bille Jendresen, Leta K. Nutt |
|---|---|
| Rok vydání: | 2008 |
| Předmět: |
Lysis
Nucleotidase activity Biochemistry Article Cell membrane Magnetic Bead Separation Magnetics Cell Line Tumor medicine Concanavalin A Animals Humans Magnetic beads Chromatography biology Cell Membrane Membrane Proteins Microspheres Rats Plasma membrane isolation medicine.anatomical_structure Membrane Membrane protein Biotinylation biology.protein Streptavidin Liver Extracts Lectin Biotechnology |
| Zdroj: | Digital.CSIC. Repositorio Institucional del CSIC instname |
| ISSN: | 1096-0279 |
| Popis: | Yuchen Lee et al. We have developed a simple method for isolating and purifying plasma membrane proteins from various cell types. This one-step affinity-chromatography method uses the property of the lectin concanavalin A (ConA) and the technique of magnetic bead separation to obtain highly purified plasma membrane proteins from crude membrane preparations or cell lines. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. When these ConA magnetic beads were used to enrich plasma membranes from a crude membrane preparation, this procedure resulted in 3.7-fold enrichment of plasma membrane marker 5′-nucleotidase activity with 70% recovery of the activity in the crude membrane fraction of rat liver. In agreement with the results of 5′-nucleotidase activity, immunoblotting with antibodies specific for a rat liver plasma membrane protein, CEACAM1, indicated that CEACAM1 was enriched about threefold relative to that of the original membranes. In similar experiments, this method produced 13-fold enrichment of 5′-nucleotidase activity with 45% recovery of the activity from a total cell lysate of PC-3 cells and 7.1-fold enrichment of 5′-nucleotidase activity with 33% recovery of the activity from a total cell lysate of HeLa cells. These results suggest that this one-step purification method can be used to isolate total plasma membrane proteins from tissue or cells for the identification of membrane biomarkers. © 2008 Elsevier Inc. The work was supported by National Institutes of Health Grant 2 R25 CA09481 |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect