Primary rabbit anti hla antibody

Staphylococcus aureus cultures were treated with various echinatin concentrations (0 to 32 μg/mL) and incubated with shaking until the late logarithmic growth period was reached. The supernatant was then collected and mixed with protein loading buffer for 10% SDS–PAGE. Proteins were then transferred onto PVDF membranes by a semidry transfer device. The membranes were blocked with 5% bovine serum albumin (BSA) for 2 h at room temperature and then washed three times with PBST. The membrane was then incubated with the primary rabbit anti-Hla antibody (1:3000, Sigma–Aldrich) followed by incubation with the HRP-labeled goat anti-rabbit IgG (1:5000, Sigma–Aldrich) secondary antibody. After washing the membrane three times, the bands were observed by a chemiluminescence detection device after adding BeyoECL Star reagent (Beyotime, Beijing, China).

An equal volume of the bacterial culture supernatants that were harvested in the hemolysis assay was resolved using SDS-PAGE, and the proteins were transferred onto PVDF membranes. After blocking in 5% non-fat milk for 2 h, the membranes were incubated with a primary rabbit anti-Hla antibody (Sigma-Aldrich) diluted 1: 8,000 for 2 h and a horseradish peroxidase-conjugated secondary antibody (Proteintech) diluted 1: 4,000 for 2 h. The signals were visualized on a Tanon-4200 imager using Amersham ECL Western blotting detection reagents (GE Healthcare, Buckinghamshire, UK).

Bacterial culture supernatants as described above were subjected to 12% SDS-PAGE and the Hla proteins were then transferred to polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA, USA). Membranes blocked by 5% non-fat dry milk in TBST were probed with a primary rabbit anti-Hla antibody (Sigma-Aldrich) diluted to 1:8000 and then with a horseradish peroxidase-conjugated secondary antibody (Sigma-Aldrich) diluted to 1:4000. The blots were developed with chemiluminescence detection Kit (ECL) (GE Healthcare, Buckinghamshire, UK).