title: The synthesis and characterization of a nuclear membrane affinity chromatography column for the study of human breast cancer resistant protein (BCRP) using nuclear membranes obtained from the LN-229 cells.
abstract: BCRP expression has been reported in glioblastoma cell lines and clinical specimens and has been shown to be expressed both in purified nuclei and in the soluble cytoplasmic fraction. To date, the nuclear BCRP has not been characterized. Our laboratory has previously developed an online chromatographic approach for the study of binding interactions between ligands and protein, cellular membrane affinity chromatography. To this end, we have immobilized the nuclear membrane fragments onto an immobilized artificial membrane stationary phase (IAM), resulting in the nuclear membrane affinity chromatography (NMAC) column. Initial characterization was carried out on the radio flow detector, as well as the LC-MSD, using frontal displacement chromatography techniques. Etoposide, a substrate for BCRP, was initially tested, to determine the functional immobilization of BCRP. Frontal displacement experiments with multiple concentrations of etoposide were run and the binding affinity was determined to be 4.54 μM, which is in close agreement with literature. The BCRP was fully characterized on the NMAC column and this demonstrates that for the first time the nuclear membranes have been successfully immobilized.
authors.affiliation: Biomedical Research Center, National Institute on Aging, National Institutes of Health, 251 Bayview Boulevard, Suite 100, Baltimore, MD 21224, USA.
authors.forename: K-L
authors.initials: KL
authors.lastname: Habicht
authors.forename: C
authors.initials: C
authors.lastname: Frazier
authors.forename: N
authors.initials: N
authors.lastname: Singh
authors.forename: R
authors.initials: R
authors.lastname: Shimmo
authors.forename: I W
authors.initials: IW
authors.lastname: Wainer
authors.forename: R
authors.initials: R
authors.lastname: Moaddel
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000032:analysis / Q000737:chemistry*; D001943:Breast Neoplasms / Q000737:chemistry; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D005047:Etoposide / Q000737:chemistry; D005260:Female; D006801:Humans; D008024:Ligands; D008567:Membranes, Artificial; D009363:Neoplasm Proteins / Q000032:analysis / Q000737:chemistry*; D009685:Nuclear Envelope / Q000737:chemistry*; D011485:Protein Binding
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D008024:Ligands; D008567:Membranes, Artificial; D009363:Neoplasm Proteins; D005047:Etoposide