Bca protein quantitative detection kit

Streptozotocin (STZ) was purchased from Sigma (No. 101764603); RAPA was purchased from Shanghai Baoman Biotechnology Co., Ltd. (No. 53123–88-9); Anti-nephrin and anti-podocin primary antibodies were purchased from Beijing Boao Sen (bs-0513R and bs-6597R). RNA extract was purchased from Servicebio (Cat. No. G3013); The RevertAid First Strand cDNA Synthesis Kit was purchased from Thermo (No.K1622). The FastStart Universal SYBR Green Master (Rox) was purchased from Roche (No. 04913914001). The BCA Protein Quantitative Detection Kit was purchased from Servicebio (NO.2026). The Protein Marker was purchased from Thermo (NO.26616). The horseradish peroxide labeled goat anti-rat antibodies were purchased from Servicebio Co., Ltd. (GB23301 and GB23302).

Tibial growth plates were homogenized in ice-cold buffer and incubated at 4 °C for 2 h. The samples were centrifuged at 12,000 rpm for 10 min to collect the supernatants (total protein), and the total protein concentrations were determined using the BCA protein quantitative detection kit (Servicebio technology, Wuhan, China). All samples were cryo-preserved at −70 °C for subsequent use. Protein samples were separated by SDS-PAGE on 12% polyacrylamide gels until the dye band reached the end of the gel and were then transferred to PVDF membranes, which were incubated in 5% skim milk at room temperature (1 h). The membranes were incubated overnight at 4 °C with the rabbit monoclonal anti-HIF-1α, anti-VEGFA and anti-VEGFR1 primary antibodies (1:1000 dilution; A11945, A5708 and A1277, respectively) (ABclonal technology, Wuhan, China). The membranes were washed 3 times with PBS-TWEEN 20 for 5 min each and were then incubated with the secondary antibody (1:3000 dilution) (HRP-labeled rabbit anti-goat secondary antibodies) for 1 h at room temperature. After washing, the bands were visualized by chemiluminescence and exposed radiography film. The images were obtained using an imaging system (EPSON, China, V300).

The protein concentration in hepatic tissue was measured using a Bicinchoninic Acid (BCA) protein quantitative detection kit (Wuhan, China, Servicebio). Total proteins were separated by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the proteins were transferred from the gel to polyvinylidene difluoride (PVDF) membranes (Wuhan, China, Servicebio). The membranes were blocked for 30 min with 5% defatted milk solution at room temperature and then incubated with primary antibodies overnight at 4 °C. Following PCR washing, the membranes were incubated for 30 min with secondary antibodies at room temperature. After washing with Tris-buffered saline containing 0.05% Tween 20 (TBST), membranes were exposed to ECL reagents (Wuhan, China, Servicebio). Finally, the blots were visualized by the chemiluminescence apparatus (Shanghai, China, CLINX).

To identify physiological interactions between EMC6 and APAF1 in AP acinar cells, immunoprecipitation was carried out according to the manufacturer’s instructions. Briefly, tissue homogenate was incubated with immunoprecipitation lysate (Servicebio) overnight at 4°C, followed by centrifuging for 10 min at 14,000 × g. After the supernatant was collected and the protein concentration was measured by BCA protein quantitative detection kit (G2026; Servicebio, China), a small amount of tissue lysate was collected for input experiments. The tissue lysate was added with 1.0 μg of rabbit IgG (Servicebio) and 20 μl of A /G beads (Millipore) and incubated at 4 °C for 1 h. After centrifugation, the supernatant was incubated with respective primary antibodies against EMC6 (Omnimabs; 2 μg), APAF1 (Proteintech; 2 μg) overnight at 4 °C and rabbit IgG (Servicebio) served as negative control. Similarly, the protein complex was incubated with 80 μl A/G beads (Millipore) at 4 °C for 2 h, then, centrifuged at 1000 × g for 5 min at 4 °C to collect the immunoprecipitation complex. After washing several times, boiled in boiling water for 10 min and centrifuged at 4 °C at 1000 × g for 5 min to collect the supernatant. Finally, Supernatant was analyzed by immunoblotting with EMC6 (Omnimabs; diluted 1:1000) and APAF1 (Proteintech; diluted 1:1000) antibody.