Bca 100 protein quantitative analysis kit

Total proteins were extracted from HepG2 cells and liver tissues with RIPA buffer (Cowin Biotech, Beijing, China). The protein concentrations were determined using a BCA-100 Protein Quantitative Analysis kit (Beyotime, Shanghai, China). Then, the proteins were subjected to SDS-PAGE and transferred to PVDF membranes (Millipore). The membranes were blocked and incubated with primary antibodies overnight at 4°C. Primary antibodies included monoclonal antibodies against ApoB100, ApoE, ApoC3, MTTP, LC3, and p62 (Abcam, Cambridge, USA), monoclonal antibodies against HK1 (Millipore), and antibody against β-actin (Beyotime). Then membranes were incubated with HRP-labelled secondary antibodies (Bio-Rad, Hercules, USA) for 1 h. Finally, the protein bands were visualized using ECL reagent (Bio-Rad).

Protein extraction of cells and liver tissue was performed with RIPA in the presence of protease inhibitors. A BCA-100 Protein Quantitative Analysis Kit (Beyotime Biotechnology, China) was used to measure the protein concentration. A total of 20 μg of protein lysates was loaded onto 10% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore) and subjected to immunoblot analysis using antibodies against proteins.

After exposure to different treatments, cells were collected and lysed in ice-cold buffer (tris-(hydroxymethyl) aminomethane 50 mmol/l, pH 7.4, NaCl 150 mmol/l, 0.5% Triton X-100, edetic acid 1 mmol/l, phenylmethylsulfony fluoride 1 mol/l, and aprotinin 5 mg/l), and then centrifuged at 12,000 g for 5 min at 4°C. The supernatant was collected and the protein concentration was determined using the BCA-100 Protein Quantitative Analysis Kit (Beyotime, Haimen, China). Equal amounts of proteins were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA) followed by blocking in 5% non-fat milk for 1 h. The membranes were then incubated overnight at 4°C with rabbit polyclonal anti-Bcl-2, rabbit polyclonal anti-Bax, rabbit monoclonal anti-PARP, rabbit monoclonal anti-caspase-3 or rabbit polyclonal anti-iNOS antibodies (1∶1000, Cell Signaling Technology, Beverly, MA). After washing with TBS/T (TBS with 0.05% Tween 20), membranes were incubated in IRDye 680-conjugated affinity purified goat anti-rabbit IgG (1∶5000, Bioworld Technology, Inc., Minneapolis, MN, USA)) at room temperature for 1 h. The images were scanned with the Odyssey infrared imaging system (LI-COR) and quantified with Multi Gauge. GAPDH (Anti-GAPDH antibody, Bioworld, Minneapolis, MN, USA), was used as an internal reference.