Bca assay protein assay kit

MCF7 cells (2 × 10⁶) were seeded onto 100 mm cell culture dishes or (5 × 10⁵) were seeded onto 6-well cell culture plates overnight, before the transient transfection procedure. The cells were harvested 48 h after transient transfection and after centrifuge, the cell pellets were lysed in a RIPA buffer. Protein lysates were placed onto a rotator to rotate at 4 °C for 30 min, and the protein concentrations of the high-speed supernatant were quantified using the BCA™ Protein Assay kit assay (Pierce/Thermo Scientific, Rockford, IL, USA). Immunoblotting was performed, as previously described [23 (link),27 (link),28 (link)]. Equivalent quantities of protein (40–50 µg) were resolved on polyacrylamide-SDS gels, transferred to PVDF membrane (Bio-Rad, Hercules, CA, USA), and immunoblotted with specific antibodies. The immune detection was done with the Supersignal West Dura Extended Duration Substrate kit (Pierce Chemical Co., Rockford, IL, USA). The intensity of the protein band was quantified by the NIH ImageJ program.

The protein content of all experimental groups of RAW264.7 cells was extracted using RIPA lysis buffer that was suitably combined with a mixture of protease inhibitors and phosphatase inhibitors. Then, the cells were scraped and placed in microcentrifuge tubes. The homogenate was placed on ice and sonicated 4 times for 5 s at intervals of 6 s. This was followed by centrifugation at 12,000 × g for 20 min at 4°C. A BCA Assay Protein Assay Kit was used to measure the protein concentration in the supernatant (Thermo Fisher Scientific, Inc., MA, USA). The same amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electro-transferred to a PVDF membrane. After blocking the membranes with 5% nonfat milk for 1 h, they were incubated overnight with the appropriate primary antibodies (1:1000 dilution) at 4°C. This was followed by incubation with HRP-inked anti-rabbit IgG secondary antibody or HRP-linked anti-mouse IgG secondary antibody for 2 h at room temperature. The detection of protein bands was performed using Clarity ECL Western blot substrate (Bio-Rad) and then visualized with a ChemiDoc Touch imaging system (Bio-Rad).

The protein content of all experimental groups of RAW264.7 cells was extracted using RIPA lysis buffer that was suitably combined with a mixture of protease inhibitors and phosphatase inhibitors. Then, the cells were scraped and placed in microcentrifuge tubes. The homogenate was placed on ice and sonicated 4 times for 5 s at intervals of 6 s. This was followed by centrifugation at 12,000 × g for 20 min at 4°C. A BCA Assay Protein Assay Kit was used to measure the protein concentration in the supernatant (Thermo Fisher Scientific, Inc., MA, USA). The same amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electro-transferred to a PVDF membrane. After blocking the membranes with 5% nonfat milk for 1 h, they were incubated overnight with the appropriate primary antibodies (1:1000 dilution) at 4°C. This was followed by incubation with HRP-inked anti-rabbit IgG secondary antibody or HRP-linked anti-mouse IgG secondary antibody for 2 h at room temperature. The detection of protein bands was performed using Clarity ECL Western blot substrate (Bio-Rad) and then visualized with a ChemiDoc Touch imaging system (Bio-Rad).

Nuclear and cytosolic fractions were prepared with a commercial kit according to the manufacturer′s instructions (Thermo Scientific Inc., Rockford, IL, USA). All steps were carried out on ice, unless stated otherwise. Protease and phosphatase inhibitor cocktail tablets (Complete Mini and Phospho STOP, Roche Applied Science, Indianapolis, IN, USA, respectively) were added to each buffer just prior to use. Briefly, cells (5 × 10⁶ cells/well in 100 mm² cell culture dishes) were treated with hyperoside for 0, 1, 2, 4, 6 or 12 h at 37 °C. Protein concentrations in the samples were determined using a bicinchoninic acid assay (BCA assay) protein assay kit (Thermo Scientific Inc., Rockford, IL, USA). Nrf2 levels were assessed using western blot analysis, as described below.