Use of short monolithic columns for isolation of low abundance membrane proteins.
Convective interaction media (CIM) monoliths provide a stationary phase with a high binding capacity for large molecules and are capable of high flow rates at a very low pressure drop. Used as anion- and cation-exchangers or with affinity ligands such as antibodies, these columns have the potential for processing large volumes of complex biological mixtures within a short time. In the present report, monoclonal antibodies against several rat liver plasma membrane proteins were bound and cross-linked to protein A or protein G CIM affinity columns with a bed volume of only 60 microL. Antigens recognized by bound antibodies and co-eluting (interacting) proteins were rapidly isolated in a single step from either total plasma membrane extracts or subfractions isolated using anion-exchange CIM disk-shaped columns. The isolated antigens and co-eluting proteins were subsequently identified by immunoblot or by LC-MS/MS.
Division of Medical Oncology, Department of Medicine, Rhode Island, Hospital, Providence, RI 02903, USA.
Marijana
M
Rucevic
James G
JG
Clifton
Feilei
F
Huang
Xuesong
X
Li
Helen
H
Callanan
Douglas C
DC
Hixson
Djuro
D
Josic
D000818:Animals; D015703:Antigens, CD / Q000302:isolation & purification; D015815:Cell Adhesion Molecules / Q000302:isolation & purification; D002462:Cell Membrane / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000295:instrumentation* / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D002853:Chromatography, Liquid; D018819:Dipeptidyl Peptidase 4 / Q000302:isolation & purification; D008099:Liver / Q000648:ultrastructure; D008114:Liver Neoplasms, Experimental / Q000737:chemistry; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000302:isolation & purification*; D009419:Nerve Tissue Proteins / Q000737:chemistry; D051381:Rats; D011916:Rats, Inbred F344; D013205:Staphylococcal Protein A / Q000737:chemistry
D015703:Antigens, CD; C069847:CD66 antigens; D015815:Cell Adhesion Molecules; C029152:G-substrate; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D013205:Staphylococcal Protein A; D018819:Dipeptidyl Peptidase 4