Goat anti mouse igg hrp antibody

The expression of recombinant SCARB2 was conducted as described with some modifications (Seidmoradei et al. 2020 ). pET-SCARB2 obtained from previous steps was transformed into competent E. coli BL21 (DE3) strain. Positive colonies were inoculated in LB media shaking tubes supplemented with ampicillin and allowed to grow at 37 °C in 16 h. The cultures were then sub-cultured at 1:10 (v/v) and inoculated at 37 °C until OD₆₀₀ reached 0.8–1.0. At this point, the cultures were induced with the final concentration of 0.1 mM IPTG, and the protein expressions were performed at 37 °C in 4 h. Harvested cells proceeded for sonication on ice in PBS buffer (pH 7.4) to obtain proteins in total, soluble and insoluble fractions. A sample of the bacterial culture was taken as negative control with non-induced E. coli BL21 (DE3)/pET-SCARB2. Recombinant protein expression was analyzed by SDS-PAGE and Coomassie Brilliant Blue stain, followed by Western blot and probed with His-probe (Santa Cruz) and goat anti-mouse IgG-HRP antibody (Proteintech). The membrane plot was developed using 100 µL of ready-to-use 3, 3′, 5, 5′-tetra methyl benzidine (TMB) (Thermo Scientific, 37,574) and incubated at room temperature. The reaction was stopped with PBS buffer and digitized documented.