NDLI: One-step isolation of plasma membrane proteins using magnetic beads with immobilized concanavalin A1

Content Provider	PubMed Central
Author	Lee, Yu-chen Block, Gregory Chen, Huiwen Emma, Folch-puy Robert, Foronjy Jalili, Roxana Bille, Jendresen Christian Kimura, Masashi Kraft, Edward Lindemose, Søren Lu, Jin Mclain, Teri Nutt, Leta Santiago, Ramon-garcia Smith, Joseph Spivak, Aaron Wang, Michael L. Zanic, Marija Lin, Sue-hwa
Abstract	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.
Related Links	http://dx.doi.org/10.1016/j.pep.2008.08.003
Ending Page	229
Page Count	7
Starting Page	223
File Format	PDF
ISSN	10465928
e-ISSN	10960279
Journal	Protein expression and purification
Issue Number	2
Volume Number	62
Language	English
Publisher Date	2008-12-01
Access Restriction	Open
Subject Keyword	Biotechnology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Biotechnology

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4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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