Bca protein assay kit

Radix isatidis was collected from an officially certified standardized planting base in Tongling city of the Anhui province (China). The botanical origin was authenticated by Professor Jianwei Chen (Nanjing University of Chinese Medicine). A voucher specimen has been deposited in the Herbarium of Nanjing University of Chinese Medicine (voucher No. 120615).
Dialysis bags were purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China). BCA protein assay kits and antioxidant assay kits such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), total antioxidant capability (T-AOC) and malondialdehyde (MDA) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Vitamin C (VC) and other chemical reagents were bought from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All reagents meet the purity required by the experiment.

Lactate dehydrogenase (LDH), sarco/endoplasmic reticulum Ca²⁺‐ATPase, (SERCA), and BCA protein assay kits were supplied with Nanjing Jiancheng Bioengineering Institute (Nanjing, China), creatine kinase (CK) kit purchased from KeyGen Biotechnology Co. Ltd. (Nanjing, China), RIPA lysate buffer obtained from Solarbio Life Science (Beijing, China). Primary antibodies against calmodulin‐dependent protein kinase II (CAMKII), p‐CAMKII, SERCA2a, peroxisome proliferator activator receptor delta (PPARδ), and horseradish peroxidase (HRP)‐conjugated secondary antibody procured from Affinity Biosciences Pty Ltd. (Melbourne, Australia), and β‐Actin procured from Cell Signaling Technology (Beverly, MA, USA). Ginsenosides standards (purity≥95%) used in present work for ginseng composition analysis were purchased from Shanghai Yuanye Biotechnology Co. Ltd. (Shanghai, China). All other reagents used were of analytical grade.

A plant soluble sugar content test kit (Nanjing Jiancheng Bioengineering Institute, China) was used to determine sugar content. The amino acid content was tested using a Total Amino Acid Content Assay Kit (Nanjing Jiancheng Bioengineering Institute, China). Lipid content was measured using a triglyceride content determination kit (Nanjing Jiancheng Bioengineering Institute, China). Succinate dehydrogenase (SDH) was determined using kits (Solarbio, China), according to the manufacturer’s instructions.
The SDH activities, amino acid, and lipid content were measured using BCA protein assay kits (Nanjing Jiancheng Bioengineering Institute, China) and the fresh weight (FW) was used to determine the S. thunbergii soluble sugar content; 0.1 g of frozen macroalgae was homogenized with 1 ml phosphate-buffered (0.1 mol, pH 7.0). The mixture was centrifuged at 4°C. Then, the four indicators in the supernatant were detected using the kits mentioned above.

To evaluate the intracellular triglyceride (TG) content, 3T3-L1 preadipocytes were cultured in 12-well plates as described under Section 2.3. At confluency, cells were washed twice in ice-cold phosphate-buffered saline (PBS) and harvested in ice-cold lysis buffer. Total TG content in the lysates was measured by using TG assay kits, and cellular protein was determined using the bicinchoninic acid (BCA) protein assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

According to our previous methods (22 (link), 23 (link)), the total proteins in liver and ileum tissues were extracted, and proteins were quantified using a bicinchoninic acid (BCA) Protein Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The same amounts of proteins were dissolved on polyacrylamide gels (8–10%) and transferred onto PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 10% non-fat milk in Tris-buffered saline/Tween (TBST) and then incubated with primary antibodies against TLR4, MyD88, IRAK1, TRAF6 (Proteintech Group, Chicago, USA), IκBα, phospho-IκBα (Santa Cruz, CA, USA), NF-κB, ZO-1, claudin 1, occludin, and β-actin (Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. After washing with TBST, the membranes were incubated with the corresponding secondary antibodies (Bioeasy, Beijing, China) for 2 h at 37°C. In the end, the bands of proteins were visualized by the Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE, USA). β-actin served as an internal control.

Total proteins were extracted from the jejunum tissue samples and IPEC-J2 cells using the RIPA lysis buffer (Thermo Scientific, Waltham, MA, USA) containing protease inhibitors. The protein concentrations were determined using the BCA protein assay kit (Nanjing Jiancheng Institute of Bioengineering, Nanjing, China). All of the extracted proteins were diluted with the SDS loading buffer and boiled at 95 °C for 10 min. A total of 40 µg extracted protein were separated by 12% SDS-PAGE gel electrophoresis and then transferred onto the polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). The membranes were blocked with 5% milk for at least 1 h at room temperature, and then were incubated with the primary antibodies against ZO-1, Occludin, Claudin-1, and β-actin (Proteintech, Chicago, IL, USA) overnight at 4 °C. The membranes were washed four times with 1 × Tris-buffered saline-Tween (TBST) for 10 min and incubated with the anti-mouse (or rabbit) IgG HRP-conjugated secondary antibody (1:3000; Cell Signaling Technology, Danvers, USA) for 1 h. After washing with 1 × TBST three times, the target protein bands were visualized with the electrochemiluminescence visualized system (Tanon, Shanghai, China). Band intensities were quantified using the ImageJ version 1.51 software [20 (link)], and all results were expressed as the target protein/β-actin protein ratio.

For physiological index measurements, the free PRO content was measured using a spectrophotometric PRO kit (Solarbio Life Sciences, Beijing, China). The MDA content was measured using a thiobarbituric acid reactive substances assay (Aguilar Díaz de León & Borges, 2020 (link); Hodges, Delong & Prange, 1999 (link)) and the contents of hydrogen peroxide (H₂O₂) and superoxide anions (O₂^.−) were measured using O₂^.− and H₂O₂ kits, respectively (Nanjing Jiancheng Bioengineering Institute, China). Similarly, the total protein contents were determined using a BCA Protein Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) were measured based on the protocols of the corresponding kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) (Bai et al., 2020 (link); Li et al., 2019 (link); Ma et al., 2019 (link)).