An expert protocol for immunofluorescent detection of calcium channels in tsA-201 cells

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• 作者：Peter Koch ; ^{peter.koch@uk-koeln.de" class="auth_mail" title="E-mail the corresponding author} ; Stefan Herzig ^{stefan.herzig@uni-koeln.de" class="auth_mail" title="E-mail the corresponding author} ; Jan Matthes ^{jan.matthes@uk-koeln.de" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Calcium channel ; Immunocytochemistry ; Membrane protein ; Methods ; Protein expression ; tsA-201
• 刊名：Journal of Pharmacological and Toxicological Methods
• 出版年：2016
• 出版时间：November-December 2016
• 年：2016
• 卷：82
• 期：Complete
• 页码：20-25
• 全文大小：818 K

文摘

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 °C or 37 °C and immunostaining) to address the L-type calcium-channel pore Ca_v1.2 in an immunofluorescent approach.

Results

We performed immunocytochemical analysis of Ca_v1.2 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_v1.2 protein and a correct Ca_v1.2 expression pattern in polygonal shaped cells already 12 h after transfection.

Discussion

A sequence of elaborated protocol modifications allows subcellular localization analysis of Ca_v1.2 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.