Use of short monolithic columns for isolation of low abundance membrane proteins

• 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
• ISSN: 0021-9673
• DOI: 10.1016/j.chroma.2006.02.053

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.