Pierce bca protein assay kit

Renal cortex proteins were lysated in a dissecting buffer (0.3 M sucrose, 25 mM imidazole, and 1 mM (EDTA), pH 7.2) including protease inhibitors Complete Mini Protease Inhibitor Cocktail Tablets (Roche Diagnostics, Hvidovre, Denmark) and Phosphatase Inhibitor Cocktail 2 and 3 (Sigma-Aldrich, Brøndby, Denmark) using a tissue homogenizer (Qiagen, Hilden, Germany) followed by centrifugation. The total protein concentration was determined using a Pierce BCA protein assay kit (Roche Diagnostics, Hvidovre, Denmark) following the manufacturer’s instructions.
Proteins were separated on a 12% Criterion TGX Precast Gel (Bio-Rad Laboratories, Copenhagen, Denmark) and transferred to a Hybond ECL nitrocellulose membrane (GE Healthcare, Hatfield, UK). The membrane was then blocked in 5% non-fat dry milk in PBS-T (80 mM Na₂HPO₄, 20 mM NaH₂PO₄, 100 mM NaCl, and 0.1 Tween 20, pH 7.4), washed in PBS-T, and incubated with primary antibodies overnight at 4 °C. Subsequently, the membrane was again washed and incubated with HRP-conjugated secondary antibody for one hour at room temperature. Antigen-antibody reactions were visualized using a chemiluminescence system (Amersham ECL Plus, GE Healthcare). All western blots were normalized to total protein content measured with Stain-Free technology [23 (link)]. Primary and secondary antibodies are listed in Table 1.

Renal inner medulla tissue was homogenized in dissecting buffer (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, pH 7.2) containing protease inhibitors: phosphatase inhibitor cocktails 2 and 3 (Sigma Aldrich, St. Louis, MO, USA) and complete mini protease inhibitor cocktail tablets (serine, cysteine and metalloprotease inhibitor, Roche, Hvidovre, Denmark), using a TissueLyser LT (Qiagen, Hilden, Germany). Afterwards, samples were centrifuged and 2% SDS sample buffer was added to the supernatant. The protein concentration of the homogenate was measured using a Pierce BCA protein assay kit (Roche). Total protein was separated by SDS/PAGE using 12% Criterion TGX stain-free gels (Bio-Rad Laboratories, Copenhagen, Denmark) and subsequently blotted onto a nitrocellulose membrane (Hybond ECL, GE Healthcare, Hatfield, UK). Afterwards, the blots were blocked with nonfat dry milk in PBS-T (80 mM Na₂HPO₄, 20 mM NaH₂PO₄, 100 mM NaCl, 0.1 Tween 20, adjusted to pH 7.4). The blots were then incubated with primary antibodies against AQP3 (1591AP) [16 (link)] overnight at 4 °C. Subsequently, secondary horseradish peroxidase (HRP)-conjugated antibodies were added, and the antigen-antibody complex was visualized using an enhanced chemiluminescence system (ECLPlus, GE Healthcare). All Western blots were normalized to total protein, as measured using stain-free technology [17 (link)].

Nuclear and cytoplasmic extracts were harvested from renal cortical tissue using a Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) according to manufacturer's protocol. The extracts were used for protein concentration determination and immunoblot analysis, as previously described [13 (link)]. Protein concentrations of the extracts were determined using a Pierce BCA protein assay kit (Roche). Briefly, equal amounts of proteins were separated on a 12% Criterion TGX Precast Gel (Bio-Rad Laboratories, København, Denmark) followed by transfer to a Hybond ECL nitrocellulose membrane (GE Healthcare, Hatfield, UK). The membrane was blocked in nonfat dry milk and incubated with primary antibodies (described below) overnight at 4°C. Afterwards, the membrane was incubated with a HRP-conjugated secondary antibody (P448, goat anti-rabbit immunoglobulin, Dako A/S, Glostrup, Denmark). Bound antibody was detected using an enhanced chemiluminescence system (Amersham ECL Plus, GE Healthcare) and visualized on the ChemiDoc MP Imaging System (Bio-Rad). All western blots were normalized to total protein, as measured using Stain-Free technology [14 (link)]. GAPDH was examined in parallel and shown as an additional control of equal loading.