Bca protein quantitative kit

Blood was collected from neonatal mice (postnatal day 7; P7) and centrifuged at 3000× g for 10 min at 4 °C to isolate plasma, then 10,000× g for 20 min at 4 °C to remove residual blood cells. A Hieff ^® Quick exosome isolation kit (for serum/plasma) was used for the isolation of plasma exosomes based on the manufacturer’s instructions (Yeasen Biotech, Shanghai, China). Each exosome sample was purified from 200 μL plasma (collected from 5 neonatal mice and EDTA-Na2 was used as an anticoagulant at the working concentration of 1.5 mg/mL).The isolated exosomes used for subsequent experiments and proteomic sequencing were further purified by 100 kD ultrafiltration tubes (Millipore, Boston, MA, USA) to centrifuge at 14,000× g for 20 min at 4 °C, then sterilized by 0.22 μM filters and diluted in sterile PBS to the final concentration of 400 μg/mL after being quantified by a BCA protein quantitative kit (TransGen Biotech, Beijing, China).

The purified protein was quantified using a Protein Quantitative Kit (BCA; Trans Gen Biotech, Beijing, China). For the chalcone isomerase assay, the reaction was in a volume of 200 μl, containing 50 mM Tris-HCl (pH 7.5), 2 mM DTT, 10 μg purified protein, and 100 μM naringenin chalcone. The substrate is finally added to the reaction and the reaction incubated for 1 min at room temperature. For chalcone synthase assay, the reaction was in a volume of 250 μl, containing 100 mM HEPES-KOH buffer (pH 7.0), 2 mM DTT, 10 μg purified protein, 200 μM malonyl CoA and p-Coumaryl CoA at a series of concentrations (20–200 μM). The reaction was carried out at 35°C for 30 min. For CHS/CHIL complex analysis, the reaction was carried out according to the procedure described by Ban (Ban et al., 2018 (link)). After extraction, the product detected at 280 nm. Three biological replicates were analyzed of each enzyme reaction.

According to a previous description, the levels of Bax, B‐cell lymphoma‐2 (Bcl‐2), TLR4, MyD88, p‐p65, and p65 were detected.
⁴⁴ (link) Total protein was extracted from H9c2 cells and myocardial tissues with the help of Total protein extraction kit (BB‐3101; BestBio), the concentration of which was quantified using the Protein quantitative kit (BCA; DQ111‐01; TransGen Biotech). Afterwards, sodium dodecyl sulfate–polyacrylamide gel (SDS‐PAGE) was prepared by SDS‐PAGE gel preparation kit (BB‐3702; BestBio), and 20 μL of protein samples were electrophoresed. The separated proteins were transferred onto the polyvinylidene fluoride membrane (LC2002; Thermo Fisher Scientific) with Western Transfer Buffer (BB‐35112; BestBio). Subsequently, the membrane was blocked with Western Blocking Buffer (BB‐3512; BestBio) at room temperature for 1 h, and incubated with the primary antibody working dilution at 4°C overnight. Post washing with Western Wash Buffer (P0023C3; Beyotime), the membrane was incubated with secondary antibodies at room temperature for 1 h, rinsed with Western Wash Buffer, and visualized by ECL working solution (32209; Thermo Fisher Scientific). Finally, the Western blot imaging system (FluorChem M; Alpha Innotech) and ImageJ software (Version. 5.0; Bio‐Rad) were used to analyze the results of Western blot. In this test, all information of antibodies was listed in Table 2.