title: An expert protocol for immunofluorescent detection of calcium channels in tsA-201 cells.
abstract: BACKGROUND
  Pore-forming subunits of voltage gated calcium channels (VGCC) are large membrane proteins (260kDa) 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 Cav1.2 in an immunofluorescent approach.
  
  
  RESULTS
  We performed immunocytochemical analysis of Cav1.2 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 Cav1.2 protein and a correct Cav1.2 expression pattern in polygonal shaped cells already 12h after transfection.
  
  
  CONCLUSIONS
  A sequence of elaborated protocol modifications allows subcellular localization analysis of Cav1.2 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.
authors.affiliation: Department of Pharmacology, University Clinic Cologne, Gleuelerstrasse 24, D-50931 Cologne, Germany. Electronic address: peter.koch@uk-koeln.de.
authors.forename: Peter
authors.initials: P
authors.lastname: Koch
authors.affiliation: Department of Pharmacology, University Clinic Cologne, Gleuelerstrasse 24, D-50931 Cologne, Germany. Electronic address: stefan.herzig@uni-koeln.de.
authors.forename: Stefan
authors.initials: S
authors.lastname: Herzig
authors.affiliation: Department of Pharmacology, University Clinic Cologne, Gleuelerstrasse 24, D-50931 Cologne, Germany. Electronic address: jan.matthes@uk-koeln.de.
authors.forename: Jan
authors.initials: J
authors.lastname: Matthes
mesh_terms: D020746:Calcium Channels, L-Type / Q000235:genetics* / Q000378:metabolism*; D018929:Cell Culture Techniques; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D005455:Fluorescent Antibody Technique / Q000379:methods*; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D021122:Protein Subunits; D059010:Single-Cell Analysis / Q000379:methods*; D014162:Transfection
chemical_list: D020746:Calcium Channels, L-Type; C419385:L-type calcium channel alpha(1C); D021122:Protein Subunits; D049452:Green Fluorescent Proteins
keywords: Calcium channel; Immunocytochemistry; Membrane protein; Methods; Protein expression; tsA-201