Universität zu Köln

Koch, Peter, Herzig, Stefan and Matthes, Jan ORCID: 0000-0003-2754-1555 (2016). An expert protocol for immunofluorescent detection of calcium channels in tsA-201 cells. J. Pharmacol. Toxicol. Methods, 82. S. 20 - 26. NEW YORK: ELSEVIER SCIENCE INC. ISSN 1873-488X

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Abstract

Introduction: Pore-forming subunits of voltage gated calcium channels (VGCC) are large membrane proteins (260 kDa) containing 24 transmembrane domains. Despite transfection with viral promoter driven vectors, biochemical analysis of VGCC is often hampered by rather low expression levels in heterologous systems rendering VGCC challenging targets. Especially in immunofluorescent detection, calcium channels are demanding proteins. Methods: We provide an expert step-by-step protocol with adapted conditions for handling procedures (tsA-201 cell culture, transient transfection, incubation time and temperature at 28 degrees C or 37 degrees C and immunostaining) to address the L-type calcium-channel pore Ca-v 1.2 in an immunofluorescent approach. Results: We performed immunocytochemical analysis of Ca-v 1.2 expression at single-cell level in combination with detection of different markers for cellular organelles. We show confluency levels and shapes of tsA-201 cells at different time points during an experiment. Our experiments reveal sufficient levels of Ca-v 1.2 protein and a correct Ca-v 1.2 expression pattern in polygonal shaped cells already 12 h after transfection. Discussion: A sequence of elaborated protocol modifications allows subcellular localization analysis of Ca-v 1.2 in an immunocytochemical approach. We provide a protocol that may be used to achieve insights into physiological and pathophysiological processes involving voltage gated calcium channels. Our protocol may be used for expression analysis of other challenging proteins and efficient overexpression may be exploited in related biochemical techniques requiring immunolabels. (C) 2016 Elsevier Inc. All rights reserved.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Koch, Peter UNSPECIFIED UNSPECIFIED UNSPECIFIED Herzig, Stefan UNSPECIFIED UNSPECIFIED UNSPECIFIED Matthes, Jan UNSPECIFIED orcid.org/0000-0003-2754-1555 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-256463
DOI:	10.1016/j.vascn.2016.07.001
Journal or Publication Title:	J. Pharmacol. Toxicol. Methods
Volume:	82
Page Range:	S. 20 - 26
Date:	2016
Publisher:	ELSEVIER SCIENCE INC
Place of Publication:	NEW YORK
ISSN:	1873-488X
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language Pharmacology & Pharmacy; Toxicology Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/25646