The preparation and development of cellular membrane affinity chromatography columns.
Cellular membrane affinity chromatography is a technique that is based on the immobilization of a target trans-membrane protein onto a stationary phase. The target protein is isolated by homogenization and solubilization of a source (e.g., cell line) followed by immobilization on either the immobilized artificial membrane-phosphatidyl choline (IAM-PC) stationary phase or the surface of an open tubular capillary during a dialysis step. The procedure typically takes 3-4 d for the IAM-PC stationary phase, whereas the open-tubular method takes an extra week for the preparation of the capillary. The resulting columns can then be used to characterize binding sites on the target protein through frontal chromatographic and/or nonlinear chromatographic studies using a wide variety of ligands including small molecules and polypeptides. The columns have been used in drug discovery as well as in the screening of tobacco smoke condensates.
Moaddel
Ruin
R
Laboratory of Clinical Investigation, Gerontology Research Center, National Institute on Aging/NIH, 5600 Nathan Shock Drive, Baltimore, Maryland 21224, USA. moaddelru@mail.nih.gov
Wainer
Irving W
IW
D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity / Q000295:instrumentation* / Q000379:methods*; D006224:Cricetinae; D004285:Dogs; D055808:Drug Discovery; D006801:Humans; D008567:Membranes, Artificial
D008567:Membranes, Artificial