Horseradish peroxidase hrp conjugated anti mouse secondary antibody incubation

Proteins from LIΔ-Rv3875 and LIΔ-Rv0129c were obtained by trichloroacetic acid precipitation as described previously^{17 (link)}. Proteins were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Following incubation with Tris-buffered saline containing 0.1% Tween-20 with 5% skim milk, the blots were probed with anti-ESAT6 (Abcam, USA) at a dilution of 1: 5 000 and anti-Mycobacterium tuberculosis Ag85 (Abcam, USA) at a dilution of 1: 500 overnight at 4 °C. Following horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody incubation (1:1000) (Beyotime Institute of Biotechnology, China), protein bands were visualized using Super Signal West Pico (Thermo Scientific, USA).

To assess the heterologous protein expression by the recombinant strains in broth, we collected total intracellular and extracellular proteins by trichloroacetic acid precipitation (10 (link)).
To assess the heterologous protein expression by both recombinant strains within mouse macrophage-like cells, we infected monolayers of RAW264.7 cells with Listeria strains at a MOI of 100:1 for 1 h. Cells were washed and treated with DMEM containing 200 μg/mL gentamicin for 1 h to kill the extracellular bacteria, and further incubated in DMEM medium containing 20 μg/mL gentamicin for 6 h before washing with PBS and lysing cells with RIPA Buffer (Solarbio, China). Total proteins were collected.
Proteins were separated on an 8% SDS gel, transferred to a PVDF membrane, incubated with HA monoclonal antibody (1:5,000) (Sigma-Aldrich, USA). Following the step of horseradish peroxidase (HRP) conjugated anti-mouse secondary antibody incubation (1:1,000) (Beyotime Institute of Biotechnology, China), the signals were developed using Super Signal West Pico (Thermo Scientific, USA).