Cyp11a1 antibody

Cell or adrenocortical tissue lysates in RIPA buffer (Santa Cruz Biotechnology) were pretreated in sample buffer (Life Technologies / Invitrogen) for 5 min at 90°C, and 30 – 40 g protein per lane was loaded on 10% SDS-PAGE gel (Life Technologies/Invitrogen). After electrophoresis, proteins were transferred to nitrocellulose membranes (Life Technologies / Invitrogen). Membranes were blocked for 1 hour with 5% nonfat dry milk in PBS (Phosphate-Buffered Saline) with 0.1% Tween-20, and subsequently incubated with a 1:250 rabbit polyclonal CYP27A1 antibody (Abcam, Cambridge, MA) or with a 1:500 rabbit polyclonal CYP11A1 antibody (Abcam), following by the incubation with an anti-rabbit-HRP antibody (1:1000, Fisher Scientific, Pittsburgh, PA) for JEG-3 cells. For rat adrenocortical cells and adrenocortical tissue, proteins were detected with CYP11A1 and CYP27A1 goat polyclonal antibodies followed by the secondary anti-goat-HRP antibodies (Santa-Cruz Biotechnology). The gel loading control was performed by anti-GAPDH mouse antibody (Santa-Cruz Biotechnology). Protein bands were visualized with Pierce SuperSignal West Pico Chemiluminescent Substrate or with ECL Western Blotting System (Fisher Scientific), and protein amounts were measured by densitometric analysis, using Kodak molecular imaging software, Version 5.0 (Molecular Imaging Systems, Carestream Health, Inc., Rochester, NY).