One-step isolation of plasma membrane proteins using magnetic beads with immobilized concanavalin A.
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.
Lee
Yu-Chen
YC
Department of Molecular Pathology, Unit 89, Box 89, The University of Texas, MD Anderson Cancer Center, Houston, TX 77030-4009, USA.
Block
Gregory
G
Chen
Huiwen
H
Folch-Puy
Emma
E
Foronjy
Robert
R
Jalili
Roxana
R
Jendresen
Christian Bille
CB
Kimura
Masashi
M
Kraft
Edward
E
Lindemose
Søren
S
Lu
Jin
J
McLain
Teri
T
Nutt
Leta
L
Ramon-Garcia
Santiago
S
Smith
Joseph
J
Spivak
Aaron
A
Wang
Michael L
ML
Zanic
Marija
M
Lin
Sue-Hwa
SH