Hybond enhanced chemiluminescence ecl nitrocellulose membrane

Western blot assay was performed as previously described (Yu et al., 2021a (link)). Cells were collected, rinsed twice with cold phosphate-buffered saline (PBS), centrifuged, and lysed in cold lysis buffer containing a protease inhibitor cocktail (Sigma) at 4°C for 50 min. The cell lysates were subsequently separated with 8%-12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto Hybond enhanced chemiluminescence (ECL) nitrocellulose membrane (GE Healthcare, Little Chalfont, Buckinghamshire, UK). After blocking, the membranes were incubated with primary antibodies at 4°C overnight. The membranes were rinsed thrice with Tris-buffered saline and Tween 20 (TBST) wash buffer and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling). Immunoreactive bands were developed using an ECL detection system (GE Healthcare).

To confirm specificity of the anti‐CYP17 antibody, Western blot was performed using the immunoprecipitate from the previous paragraph (0.39 μg/μL), a normal adrenal homogenate (4 μg/μL), and a negative control containing only RIPA buffer base. All samples were diluted 1 : 1 with sample buffer with dithiothreitol and heated at 95°C for 2 minutes. A 10% acryl/bisacryl running gel was used; 20 μL of the diluted samples or 12 μL of the Precision plus Protein Standard (BioRad) was loaded onto the gel. After gel‐electrophoreses, the gel was blotted onto a Hybond Enhanced Chemiluminescence (ECL) nitrocellulose membrane (Amersham, GE Healthcare, Diegem, Belgium). The membrane was blocked for 60 minutes in TBST0.1% with 4% ECL Blocking Agent, after which it was incubated overnight at 4°C in the anti‐CYP17 antibody in a 1 : 2,500 concentration, diluted in TBST0.1% in 4% BSA. The next day, the membranes were incubated for 60 minutes with the secondary antibody (anti‐rabbit, horseradish peroxidase conjugated, 1 : 20,000). TBST0.1% was used for all washing steps. An ECL advanced Western blotting detection kit (Amersham RPN2135, GE Healthcare) was used for protein visualization, and chemiluminescence was detected using a ChemiDoc XRS Chemi Luminescent Image Capture (BioRad).

For WB analysis, the protein concentrations of the samples were equalized and the proteins were separated by electrophoresis using an 8% polyacrylamide gel with 0.1% sodium dodecyl sulfate according to the Laemmli method. Protein transfer, control of the quality and uniformity of transfer, blocking with 5% nonfat milk, blotting with primary rabbit antibodies against rat ceruloplasmin (Cp) that cross-reacted with murine Cp, and visualization of the immune complexes were performed as described previously.6 (link) Hybond enhanced chemiluminescence (ECL) nitrocellulose membrane, ECL reagent, ECL hyperfilm (GE Healthcare, Piscataway, NJ, USA), and horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (Abcam, Cambridge, UK) were used. The film was processed using the method reported by Aldridge et al.7 (link) Protein markers with molecular masses ranging from 14.4 to 116 kDa were purchased from Thermo Scientific (Swedesboro, NJ, USA).