Plasma membrane isolation using immobilized concanavalin A magnetic beads.
Isolation of highly purified plasma membranes is the key step in constructing the plasma membrane proteome. Traditional plasma membrane isolation method takes advantage of the differential density of organelles. While differential centrifugation methods are sufficient to enrich for plasma membranes, the procedure is lengthy and results in low recovery of the membrane fraction. Importantly, there is significant contamination of the plasma membranes with other organelles. The traditional agarose affinity matrix is suitable for isolating proteins but has limitation in separating organelles due to the density of agarose. Immobilization of affinity ligands to magnetic beads allows separation of affinity matrix from organelles through magnets and could be developed for the isolation of organelles. We have developed a simple method for isolating plasma membranes using lectin concanavalin A (ConA) magnetic beads. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. The ConA magnetic beads are used to bind glycosylated proteins present in the membranes. The bound membranes are solubilized from the magnetic beads with a detergent containing the competing sugar alpha methyl mannoside. In this study, we describe the procedure of isolating rat liver plasma membranes using sucrose density gradient centrifugation as described by Neville. We then further purify the membrane fraction by using ConA magnetic beads. After this purification step, main liver plasma membrane proteins, especially the highly glycosylated ones and proteins containing transmembrane domains could be identified by LC-ESI-MS/MS. While not described here, the magnetic bead method can also be used to isolate plasma membranes from cell lysates. This membrane purification method should expedite the cataloging of plasma membrane proteome.
Department of Molecular Pathology, University of Texas, M.D. Anderson Cancer Center, Houston, TX 77030, USA.
Yu-Chen
YC
Lee
Martina
M
Srajer Gajdosik
Djuro
D
Josic
Sue-Hwa
SH
Lin
D000818:Animals; D002457:Cell Extracts / Q000737:chemistry / Q000302:isolation & purification*; D002462:Cell Membrane / Q000737:chemistry*; D003208:Concanavalin A / Q000737:chemistry*; D055767:Immobilized Proteins / Q000737:chemistry*; D008099:Liver / Q000166:cytology; D060526:Magnetic Fields; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D008863:Microspheres; D010449:Peptide Mapping; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry; D059748:Proteolysis; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D014357:Trypsin
D002457:Cell Extracts; D055767:Immobilized Proteins; D008565:Membrane Proteins; D003208:Concanavalin A; D014357:Trypsin; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase