Quantification of Aldosterone Synthase in H295R Cells
H295R-S2 cells were lysed using RIPA buffer (Bio Basic Canada Inc.) with protease and phosphatase inhibitors mini tablets, EDTA free (Thermo Scientific). Proteins were solubilized for 30 min at 4 °C, under end-over-end rotation, and then centrifuged at 13,000 rpm for 15 min at 4 °C. Protein concentration was determined using Bradford protein assay (Biorad). 15 µg of proteins were submitted to 10% SDS-PAGE and transferred onto nitrocellulose membrane. Membranes were blotted with the following antibodies: mouse anti-aldosterone synthase antibody (1:500, clone CYP11B2-41-13, kindly provided by Dr C Gomez Sanchez92 (link) and mouse anti-tubulin (T9026, 1:2000, Sigma Aldrich). Signals were developed by Clarity Max™ Western ECL substrate (Biorad, Hercules, CA) and detected by Fujifilm Las-4000 mini Luminescent image analyzer (Fujifilm, Tokyo-Japan) and quantified by Multi gauge software (Fujifilm, Tokyo-Japan). Expression of total proteins was normalized to the expression of the housekeeping protein tubulin. Uncropped and unprocessed scans of blots are presented in the Source Data file.
Le Floch E., Cosentino T., Larsen C.K., Beuschlein F., Reincke M., Amar L., Rossi G.P., De Sousa K., Baron S., Chantalat S., Saintpierre B., Lenzini L., Frouin A., Giscos-Douriez I., Ferey M., Abdellatif A.B., Meatchi T., Empana J.P., Jouven X., Gieger C., Waldenberger M., Peters A., Cusi D., Salvi E., Meneton P., Touvier M., Deschasaux M., Druesne-Pecollo N., Boulkroun S., Fernandes-Rosa F.L., Deleuze J.F., Jeunemaitre X, & Zennaro M.C. (2022). Identification of risk loci for primary aldosteronism in genome-wide association studies. Nature Communications, 13, 5198.
Aldosterone synthase Anti antibodyAntibodies Assay Bio basic BufferCells Clone EdtaInhibitors Luminescent Membranes MouseNitrocellulose Phosphatase Protease Protein Ripa Scans Sds pageTubulin
Corresponding Organization :
Other organizations :
Centre National de Recherche en Génomique Humaine, Université Paris-Saclay, Commissariat à l'Énergie Atomique et aux Énergies Alternatives, CEA Paris-Saclay, Université Paris Cité, Inserm, Paris Cardiovascular Research Center, Ludwig-Maximilians-Universität München, University of Padua, Institut Cochin, Centre National de la Recherche Scientifique, Helmholtz Zentrum München, National Research Council, Institute of Biomedical Technologies, Fondazione IRCCS Istituto Neurologico Carlo Besta, Laboratoire d'Informatique Médicale et d'Ingénierie des Connaissances en e-Santé, Université Sorbonne Paris Nord, Sorbonne Université, Centre de Recherche Épidémiologie et Statistique, Conservatoire National des Arts et Métiers, Sorbonne Paris Cité, Hôpital Européen, Assistance Publique – Hôpitaux de Paris, Hôpital Européen Georges-Pompidou
RIPA buffer (Bio Basic Canada Inc.) with protease and phosphatase inhibitors mini tablets, EDTA free (Thermo Scientific)
dependent variables
Protein concentration
Expression of total proteins normalized to the expression of the housekeeping protein tubulin
control variables
Incubation for 30 min at 4 °C, under end-over-end rotation
Centrifugation at 13,000 rpm for 15 min at 4 °C
10% SDS-PAGE
Transfer onto nitrocellulose membrane
Blotting with mouse anti-tubulin (T9026, 1:2000, Sigma Aldrich)
positive controls
Mouse anti-aldosterone synthase antibody (1:500, clone CYP11B2-41-13, kindly provided by Dr C Gomez Sanchez)
negative controls
None mentioned
Annotations
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