Bca protein kit

The cotyledons of 2-week-old watermelon seedlings were sprayed with 0.1 μM flg22^Ac and 0.1 mM SA until run-off was observed, and the total protein was isolated from leaves collected at different time points using a Plant Protein Extraction Kit (Solarbio, Beijing, China). Protein quantification was performed using a BCA protein kit (Takara, Shiga, Japan). Western blotting was performed using anti-ClWRKY6 antibodies (Gene Universal, Inc., Anhui, China; 1:1000 dilution). To test watermelon responses to Ac infection, the WT strain Aac5 was inoculated into the cotyledons of 2-week-old watermelon seedlings at 3 × 10⁸ CFU/mL, and the total protein was isolated from the leaves collected at different time points using a Plant Protein Extraction Kit (Solarbio, Beijing, China). Proteins were quantified using a BCA protein kit (Takara). Western blotting was performed using anti-ClWRKY6 antibodies (Gene Universal, Inc., 1:1000 dilution). Each experiment was independently repeated three times.

The cells (density, 2 × 10⁵/well) were seeded in 6-well plates. The cells stimulated with 10 ng/ml IL-1β or not were incubated with different concentrations of SSD (0, 1, 2, and 4 μmol/L). At the end point, the cells were washed twice with ice-cold PBS and then extracted using RIPA (AMEKO, Shanghai, China) supplemented with phosphatase inhibitor and phenylmethanesulfonyl fluoride (PMSF). The concentration of total protein was normalized using a BCA protein kit (Takara, Dalian). The protein samples were separated using 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to 0.22-μm polyvinylidene difluoride (PVDF) membranes (Millipore, Boston, United States). After incubating with QuickBlock™ Blocking Buffer at room temperature for 30 min, the blocked PVDF membranes were incubated with the primary antibodies overnight at 4°C. On the 2nd day, tris-buffered saline with Tween 20 (TBST) was used to wash the PVDF membranes thrice and the membranes were incubated with the respective secondary antibodies for 1 h on the laboratory shaker at 37°C. Finally, the bands were acquired with an enhanced chemiluminescence reagent (Millipore, Boston, United States). ImageJ software was used to observe the density of these bands.

Using a BCA protein kit (TaKaRa, Shiga, Japan), a working solution (BCA reagent A: B = 100:1) and 0.2 mg μL⁻¹ BCA standard were mixed with the collected proteins in 100 μL 1 × RIPA buffer in a 96-well plate (Matrix Microplate w/lids 96-well blk/clr, flat bottom, tissue culture, PS; Thermo Fisher Scientific). Proteins were incubated at 37°C for 60 min and measured at a wavelength of 562 nm with a Synergy HTX Microplate Reader.