Sds page gel preparation kit

First the total cell protein was extracted and its concentration was measured. The gel was prepared according to the instructions of Solarbio’s SDS-PAGE gel preparation kit, and protocols for (i) loading (15 μL per well), (ii) electrophoresis (in the concentrated gel, apply constant voltage modulation 80V, run for 30 minutes; once the marker is separated, adjust the voltage to 120V for 60 minutes), (iii) transferring of the membrane (a sponge pad, 3 layers of filter paper, gel plate, PVDF membrane, 3 layers of filter paper, a sponge pad from the negative plate should be put on the positive plate; the constant current of 300 mA at 4 °C for 150 min should be applied), (iv) blocking (TBST solution containing 5% skim milk), (v) incubation of primary antibody (Anti-B4GALNT2-Sigma-HPA015721; Anti-GAPDH-Santa-Cruz-sc-32233; dilute 1:1000, incubate for 2 h at room temperature) and (vi) incubation of secondary antibody (mouse IgG-CST-#7076; rabbit IgG-CST-#7074; dilute 1:10000, incubate for 2h at room temperature) were followed.

The egg sample was bought from local supermarkets. Pv standard from egg yolk was obtained from Sigma Chemical Company (St. Louis, MO, USA). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel preparation kit (Solarbio, Beijing, China) was used for characterization of the protein. All other chemicals obtained from Aladdin (Shanghai, China) were of analytical grade. All the solutions were prepared using ultrapure water obtained from Northeast Agricultural University.

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.

The protein was characterized by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel preparation kit (Solarbio, Beijing, China). A PHS-3C pH meter (Shanghai Instrument Electric Scientific Instrument Co., Ltd., Shanghai, China) was used to measure the pH of the solution. Centrifugation was performed on a CT14D desktop high speed centrifuge (Shanghai Techcomp Scientific Instrument Co., Ltd., Shanghai, China) and a SC-3610 low speed centrifuge (Hefei Zhongke Zhongjia Scientific Instrument Co., Ltd., Hefei, China). An AL-04 electronic analytical balance (Mettler Toledo Instruments Co., Ltd., Shanghai, China) was used to weigh the sample. The sample was dialyzed with an 8000 Da MWCO filter (Spectrum Labs, Los Angeles, CA, USA). The lyophilizer (LyoQuest-85 Plus, Telstar, Terrassa, Spain) was used to lyophilize the sample.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to analyze the cellular proteins according to a previously described method [33 (link)]. The collected samples were consistent with those described in Section 3.4.4. The samples were mixed with 5X protein loading buffer, whereafter the mixtures were boiled for 5 min, then cooled and centrifuged. The supernatants of the S. aureus cells were collected for SDS-PAGE analysis. An SDS-PAGE gel preparation kit (Solarbio, Beijing, China) was used to prepare the gel (5% stacking gel and 12% separating gel). After electrophoresis, the gel was stained with Coomassie brilliant blue R-250 and decolorized for analysis.