TY - JOUR
T1 - One-step isolation of plasma membrane proteins using magnetic beads with immobilized concanavalin A
AU - Lee, Yu-Chen
AU - Block, Gregory
AU - Chen, Huiwen
AU - Folch-Puy, Emma
AU - Foronjy, Robert
AU - Jalili, Roxana
AU - Jendresen, Christian Bille
AU - Kimura, Masashi
AU - Kraft, Edward
AU - Lindemose, Søren
AU - Lu, Jin
AU - McLain, Teri
AU - Nutt, Leta
AU - Ramon-Garcia, Santiago
AU - Smith, Joseph
AU - Spivak, Aaron
AU - Wang, Michael L
AU - Zanic, Marija
AU - Lin, Sue-Hwa
N1 - Keywords: Animals; Biochemistry; Cell Line, Tumor; Cell Membrane; Concanavalin A; Humans; Liver Extracts; Magnetics; Membrane Proteins; Microspheres; Rats; Streptavidin
PY - 2008
Y1 - 2008
N2 - 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.
AB - 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.
U2 - 10.1016/j.pep.2008.08.003
DO - 10.1016/j.pep.2008.08.003
M3 - Journal article
C2 - 18765283
SN - 1046-5928
VL - 62
SP - 223
EP - 229
JO - Protein Expression and Purification
JF - Protein Expression and Purification
IS - 2
ER -