Generation of an antibody toolbox to characterize hERG

• Published in: Biochemical and biophysical research communications Vol. 431; no. 1; pp. 70 - 75
• Main Authors: Hausammann, Georg J., Heitkamp, Thomas, Matile, Hugues, Gsell, Bernard, Thoma, Ralf, Schmid, Georg, Frasson, David, Sievers, Martin, Hennig, Michael, Grütter, Markus G.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.02.2013
• Subjects: Animals; Antibodies, Monoclonal - biosynthesis; Antibodies, Monoclonal - immunology; Antibodies, Monoclonal - isolation & purification; Chromatography, Gel - methods; ELISA; Enzyme-Linked Immunosorbent Assay; ERG1 Potassium Channel; Ether-A-Go-Go Potassium Channels - analysis; Ether-A-Go-Go Potassium Channels - immunology; Ether-A-Go-Go Potassium Channels - isolation & purification; Fluorescence size exclusion; HEK293 Cells; hERG; Humans; Immunoglobulin Fab Fragments; mAB; Mice; hERG; RPC; Fab; Fluorescence size exclusion; FSEC; mAB; ELISA
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2012.12.089

► Recombinant hERG was purified to immunize mice. ► Monoclonal antibodies were generated; they recognized native, linear epitopes. ► A sensitive and specific ELISA was set up. ► Fluorescently labeled Fab was used for FSEC of hERG-expressing membranes. 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.