Goat anti mouse igg hrp antibody

In the case of AML12 cells, total protein was extracted with 200 µL of lysis buffer as previously reported [27 (link)]. Protein concentration was measured by BCA protein assay kit (Thermo Scientific, Wilmington, DE, USA).
For FAS protein, 65 µg of cell protein extract were used to perform the immunoblotting. Protein were separated by electrophoresis in a 7.5% SDS-polyacrylamide gel and transferred to PVDF membranes. The membranes were blocked with casein PBS-Tween buffer for 2 h. These membranes were incubated overnight at 4 °C with mouse origin FAS IgG (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Afterwards, new incubation with goat- anti-mouse IgG-HRP antibody (1:5000) (Sigma, St. Louis, MO, USA) was carried out for 2 h at room temperature. Antibodies were visualized by using a chemiluminescent substrate (Thermo Scientific, Wilmington, DE, USA) and quantified by a ChemiDoc MP imaging system (BioRad, Hercules, CA, USA). Coomassie Blue staining of membranes was used as protein loading control. In case of CPT1a and SREBP1, immunoblotting after immunoprecipitation was performed. A total of 40 μg for CPT1a and 70 μg for SREBP1 of cell extracts were immunoprecipitated. The total amount of protein was used for immunoblotting in both cases and following the same conditions as described above.

This immunogenicity analysis referred to the previous study (21 (link)). The rTgBFD1 was separated by 12% SDS-PAGE gel and transferred to the nitrocellulose membranes (Millipore, Shanghai, China). The blots were blocked with 5% skim milk in phosphate-buffered saline (PBS) with 0.5% Tween 20 (PBST) for 1 h at 37°C and then incubated with the serum collected from normal or T. gondii-infected mice (1:100 dilution) for 1 h at 37°C. After three washes, 5 min each using PBST buffer, the blots were incubated with goat anti-mouse IgG-HRP antibody (Sigma, Shanghai, China) (1:5000 dilution) for 1 h at 37°C. After three washes, 5 min each using PBST buffer, the blots were finally visualized by the enhanced chemiluminescence kit (Vazyme, Nanjing, China).