Anti mouse igg igm hrp conjugated secondary antibodies

Protein extraction and western blot analysis were performed as described previously (Azzag et al., 2020 (link)). Briefly, cells were washed with PBS, scraped, and total protein was extracted via homogenization in Tris-Buffer Saline (TBS, 50 mM Tris-Cl, pH 7.5, 150 mM NaCl) with 1% Triton X-100 supplemented with protease inhibitor cocktail (Complete – Millipore Sigma) at 4°C for 30 min. The supernatant was collected by centrifugation for 30 min at 14000 g and concentration was determined by Bradford assay (Millipore-Sigma). 50 μg of the total protein was then electrophoresed on a 4%–20% then transferred to a PVDF membrane. The PVDF membrane was blocked with 5% nonfat dry milk in PBS with Tween 20 (PBST) for 1 hr, and then were incubated with primary antibodies IIH6C4 (1:1000), β-DG (1:500) and MF-20 (1:100) overnight at 4°C. Anti-mouse IgG/IgM HRP conjugated secondary antibodies (Abcam) were applied at a dilution of (1:10000) for 1hr and the protein detection was performed using the Supersignal West Femto chemiluminescent substrate (Thermo Fisher Scientific) and was imaged in chemidoc imager (Bio-Rad).