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...

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Bibliographic Details
Published in:Journal of Chromatography A 2006-08, Vol.1123 (2), p.199-204
Main Authors: Rucevic, Marijana, Clifton, James G., Huang, Feilei, Li, Xuesong, Callanan, Helen, Hixson, Douglas C., Josic, Djuro
Format: Article
Language:English
Subjects:
Analytical, structural and metabolic biochemistry
Animals
Antigens, CD - isolation & purification
Biological and medical sciences
Cell Adhesion Molecules - isolation & purification
Cell Membrane - chemistry
Chromatography, Affinity - instrumentation
Chromatography, Affinity - methods
Chromatography, Ion Exchange - methods
Chromatography, Liquid
Dipeptidyl Peptidase 4 - isolation & purification
Fundamental and applied biological sciences. Psychology
General aspects, investigation methods
Immunoaffinity chromatography
Liver - ultrastructure
Liver Neoplasms, Experimental - chemistry
Mass Spectrometry
Membrane Proteins - isolation & purification
Monoliths
Nerve Tissue Proteins - chemistry
Plasma membrane proteins
Proteins
Rats
Rats, Inbred F344
Staphylococcal Protein A - chemistry
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Summary: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 μL. 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.
ISSN:0021-9673
DOI:10.1016/j.chroma.2006.02.053