title: A novel analytical tool for quantification of estrogenicity in river water based on fluorescence labelled estrogen receptor alpha.
abstract: A novel combined procedure for estrogen-affinity purification and labelling of estrogen receptor alpha ligand-binding domain with Cy 5.5 cystein reactive dye was established. By using this procedure, mainly functional proteins are recovered. It can be easily adapted to a large variety of other proteins for which ligand-coated affinity materials are available. The labelled receptor was used in a total internal reflection fluorescence-based binding inhibition assay for determination of the impact of pollutants in river water on the receptor. The great advantage compared to conventional methods is that the total effect on the receptor is measured instead of concentrations of single compounds and that even currently unknown ligands are found as well. Therefore, the obtained signal is related to the response of the organism, which is exposed to the water. The limit of detection was found to be 0.139 nM of estradiol equivalents. The assay also provides a highly sensitive tool for pharmaceutical research and can be adapted to diagnostic applications.
authors.affiliation: Institute of Physical and Theoretical Chemistry, Eberhard Karls University of Tübingen, Auf der Morgenstelle 18, 72076, Tübingen, Germany. alexander.leblanc@ipc.uni-tuebingen.de
authors.forename: Alexander Fabian
authors.initials: AF
authors.lastname: Le Blanc
authors.forename: Christiane
authors.initials: C
authors.lastname: Albrecht
authors.forename: Tomas
authors.initials: T
authors.lastname: Bonn
authors.forename: Peter
authors.initials: P
authors.lastname: Fechner
authors.forename: Günther
authors.initials: G
authors.lastname: Proll
authors.forename: Florian
authors.initials: F
authors.lastname: Pröll
authors.forename: Mats
authors.initials: M
authors.lastname: Carlquist
authors.forename: Günter
authors.initials: G
authors.lastname: Gauglitz
mesh_terms: D002623:Chemistry Techniques, Analytical / Q000379:methods*; D004784:Environmental Monitoring / Q000379:methods*; D047628:Estrogen Receptor alpha / Q000737:chemistry* / Q000302:isolation & purification; D004967:Estrogens / Q000032:analysis*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008024:Ligands; D011485:Protein Binding; D013194:Staining and Labeling; D014874:Water Pollutants, Chemical / Q000032:analysis*
chemical_list: D047628:Estrogen Receptor alpha; D004967:Estrogens; D005456:Fluorescent Dyes; D008024:Ligands; D014874:Water Pollutants, Chemical