Bca protein concentration assay kit

Tumor necrosis factor-beta (TNF-β) and IL-6 enzyme-linked immunosorbent assay (ELISA) kits (Nanjing Jiancheng Biotechnology Co., Ltd.), TRIzol reagent, DEPC-treated water, SuperScript III reverse transcription (RT) kit, SYBR quantitative polymerase chain reaction (qPCR) Mix (ABI), RIPA lysis buffer (Beyotime), loading buffer, protease inhibitor, bicinchoninic acid (BCA) protein concentration assay kit (Biosharp), β-actin, secondary antibody (Boster Bioengineering Co., Ltd.), primary antibody (Santa), tissue homogenizer, electrophoresis system (Bio-Rad), microplate reader (Thermo Fisher Scientific Instruments Co., Ltd.), 2500 gel imager (Bio-Rad, USA), and qPCR instrument (7900 Fast, Applied Biosystems) were the instruments and reagents used.

Total protein was extracted from the liver, kidney, jejunum, and ileum (100 mg, n = 3 chickens for each group) using RIPA cell lysis buffer (BL504A, Biosharp, China) and protein phosphatase inhibitor (P1260, Applygen, China). Protein concentrations were determined using a BCA protein concentration assay kit (BL521A, Biosharp, China). Proteins were electrophoresed using an electrophoresis apparatus (EPS 300, Tanon, China). The following antibodies were used: anti-BCRP (cat. no. bs-0662R, polyclonal, 1:1000, Bioss, China), anti-MRP4 (cat. no. bs-1422R, polyclonal, 1:1000, Bioss, China), anti-β-actin (cat. No. abs137975, monoclonal, 1:1000, Absin, China), and goat anti-rabbit IgG (cat. no. AP132P, 1:1000, Millipore, USA). Immunocomplexes were visualized using a western blotting detection kit (Advansta, USA) and blots were imaged using a ChemiDoc MP Imaging System (Bio-Rad, USA). Band densities were analyzed by Image-Pro Plus 6.0 and were normalized to those of β-actin. The protein relative expression levels were normalized to the average level of the NC group.

Specific pathogen-free (SPF) mice (Shanghai Institutes for Biological Sciences, CSA), TRIzol reagent, DEPC-treated water, SuperScript III reverse transcriptase kit and SYBR quantitative polymerase chain reaction (qPCR) mix (ABI), radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology), loading buffer, protease inhibitor and bicinchoninic acid (BCA) protein concentration assay kit (Biosharp), tumor necrosis factor-α (TNF-α), interleukin- (IL-) 6 and myeloperoxidase (MPO) enzyme-linked immunosorbent assay (ELISA) kit (Nanjing Jiancheng Bioengineering Institute), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and secondary antibodies (Boster Biological Technology Co., Ltd.), primary antibodies (Santa Cruz Biotechnology, Inc.), microplate reader (Thermo Fisher Scientific), 7900HT Fast qPCR instrument (Applied Biosystems) and 2500 gel imaging system, tissue homogenizer, and electrophoresis apparatus (Bio-Rad, USA) are used.

Protein concentration was determined using the BCA protein concentration assay kit (Biosharp, Beijing, China) after lysing cells in RIPA Buffer (Servicebio, Wuhan, China) and adding 100× phosphorylated protease inhibitor A solution, phosphorylated protease inhibitor B solution, 50× protease inhibitor PMSF, 50× protease inhibitor cocktail (Servicebio, Wuhan, China). The protein samples were electrophoresed in SDS Buffer (Omni-Easy One-Step PAGE Gel Rapid Preparation kit, EpiZyme, Shanghai, China), and then, the proteins were transferred to the PVDF membrane for the electrotransfer solution. The membranes were sealed with the sealing liquid for 2 h, and the primary antibody was incubated at 4 °C overnight. Finally, an ECL chemiluminescent substrate kit (Biosharp, Beijing, China) was used to develop the protein bands after the membranes had been treated with the secondary antibody for two hours. After that, it was placed on a Quick Chemi 5200, Monad, Suzhou, China, chemiluminescence detector to scan the band. β-actin protein reference normalization was used to analyze all the protein levels.

The BPTCs treated with glucose with or without the addition of 100 nmol/L rapamycin for 24 h were collected for western blotting analysis. The BPTCs were washed three times with PBS, and 1 mL of cell RIPA solution was added to obtain total protein. The protein concentration was detected by the BCA protein concentration assay kit (Biosharp, Guangzhou, China). Western blot analysis was performed in brief as follows: 20 µg of protein/well was separated on 6–15% separating gel, 2–10% concentrating gel (Servicebio, Wuhan, China) and transferred to PVDF membrane (Absin, Shanghai, China). The membranes were sequentially closed with 5% skimmed milk generated in Tris-buffer, followed by incubation using primary antibodies, overnight at 4 °C. The membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody for 1 h at room temperature. Subsequently, the membranes were incubated with the specific ultrasensitive ECL chemiluminescent substrate (Biosharp, Guangzhou, China) and visualization of the proteins was achieved with the ChemiDOC MP (Bio-Rad). The mean values of protein in 1 mg/mL treatment were set to 1.00. Grayscale analysis was performed using ImageJ Software 1.8.0. The complete details of primary antibodies are listed in Table 2.