Authors: Alexander Fabian Le Blanc Christiane Albrecht Tomas Bonn Peter Fechner Günther Proll Florian Pröll Mats Carlquist Günter Gauglitz
Publish Date: 2009/08/26
Volume: 395, Issue: 6, Pages: 1769-
Abstract
A novel combined procedure for estrogenaffinity purification and labelling of estrogen receptor α ligandbinding domain with Cy™ 55 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 ligandcoated affinity materials are available The labelled receptor was used in a total internal reflection fluorescencebased 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 0139 nM of estradiol equivalents The assay also provides a highly sensitive tool for pharmaceutical research and can be adapted to diagnostic applicationsSchematic illustration of the combined affinity purification and labelling procedure for ERαLBD a E2derivatised sepharose gel b ERαLBD bound to E2–sepharose other components from the bacterial lysate are removed in the following washing steps c Labelling with Cy™ 55 followed by carboxymethylation Excess of reagents as well as denatured protein material are removed by washing d Elution of the receptor without addition of ligandWe kindly acknowledge the financial support by the Analytics Section of the German chemical society GDCh in the form of the “Publikationsstipendium ABC” and the European Union project “CASCADE” FOODCT2004506319 We are grateful to Dr Ram Abuknesha King’s College London UK for kindly providing E117CMO
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