Bacterial protein extraction kit

Refer to the experimental method of Rhayour et al. [32 (link)]. P. syringae suspension (approximately 10⁷ CFU/mL) was prepared and treated with juglone at the 0 (control), 1/2MIC, MIC and MBC levels. A series of total mixture of 1 mL (obtained at 0, 4, 8 and 12 h, respectively.) was centrifuged to get the supernatant. The obtained supernatant was measured with a protein detection kit (Beyotime Biotechnology, Nanjing, China). The sediment was washed by PBS, and the bacterial protein extraction kit (Solarbio, Beijing, China) was used to process the sediment to obtain bacterial intracellular proteins. Similarly, protein content in the extracted precipitate was determined with a protein detection kit.

First, 10 mL of P. aeruginosa (approximately 10⁷ CFU/mL) cells treated with juglone (at a final concentration of 0 (control), 1/2MIC, MIC, and MBC) for 12 h were collected and resuspended in 1 mL of PBS, before treating with ultrasound for 5–10 min in an ice bath. The cleaned sediment was treated with a bacterial protein extraction kit (Solarbio, Beijing, China) to obtain bacterial intracellular proteins. The extracted protein was mixed with the protein dye in equal amounts, and then the mixture was boiled for 5 min, before being centrifuged at 10,000 rpm for 5 min. According to the protocol of the SDS-PAGE gel preparation kit (SolarBio, Beijing, China), 5% stacking and 12% separation gels were used for electrophoresis, before dyeing with Coomassie Bright Blue for 2 h. Finally, the gel was rinsed with eluent for another 10 h.
At 0, 2, 4, 6, 8, 10, and 12 h, 1 mL of bacterial suspension was centrifuged to obtain supernatant. The protein content in the supernatant was determined using the protein detection kit (Beyotime Biotechnology, Shanghai, China) to determine the damage to the P. aeruginosa membrane.