title: Generation of an antibody toolbox to characterize hERG.
abstract: The human ether-a-go-go related gene (hERG) potassium channel plays a major role in the repolarization of the cardiac action potential. Inhibition of the hERG function by mutations or a wide variety of pharmaceutical compounds cause long QT syndrome and lead to potentially lethal arrhythmias. For detailed insights into the structural and biochemical background of hERG function and drug binding, the purification of recombinant protein is essential. Because the hERG channel is a challenging protein to purify, fast and easy techniques to evaluate different expression, solubilization and purification conditions are of primary importance. Here, we describe the generation of a set of 12 monoclonal antibodies against hERG. Beside their suitability in western blot, immunoprecipitation and immunostaining, these antibodies were used to establish a sandwich ELISA for the detection and relative quantification of hERG in different expression systems. Furthermore, a Fab fragment was used in fluorescence size exclusion chromatography to determine the oligomeric state of hERG after solubilization. These new tools can be used for a fast and efficient screening of expression, solubilization and purification conditions.
authors.affiliation: Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland.
authors.forename: Georg J
authors.initials: GJ
authors.lastname: Hausammann
authors.forename: Thomas
authors.initials: T
authors.lastname: Heitkamp
authors.forename: Hugues
authors.initials: H
authors.lastname: Matile
authors.forename: Bernard
authors.initials: B
authors.lastname: Gsell
authors.forename: Ralf
authors.initials: R
authors.lastname: Thoma
authors.forename: Georg
authors.initials: G
authors.lastname: Schmid
authors.forename: David
authors.initials: D
authors.lastname: Frasson
authors.forename: Martin
authors.initials: M
authors.lastname: Sievers
authors.forename: Michael
authors.initials: M
authors.lastname: Hennig
authors.forename: Markus G
authors.initials: MG
authors.lastname: Grütter
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D002850:Chromatography, Gel / Q000379:methods; D000072237:ERG1 Potassium Channel; D004797:Enzyme-Linked Immunosorbent Assay; D051638:Ether-A-Go-Go Potassium Channels / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D057809:HEK293 Cells; D006801:Humans; D007140:Immunoglobulin Fab Fragments; D051379:Mice
chemical_list: D000911:Antibodies, Monoclonal; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; D007140:Immunoglobulin Fab Fragments; C000606913:KCNH2 protein, human