Peroxidase affinipure goat anti mouse igg

Serum binding antibodies against SARS-CoV-2 RBD were determined by ELISA (HIPRA). MaxiSorp plates (Nunc, Roskilde, Denmark) were coated with 100 ng/well RBD protein (Sino Biologicals, Beijing, China) and blocked with 5% non-fat dry milk (Difco Skim Milk, BD, Franklin Lakes, NJ, USA) in PBS. Wells were incubated with serial dilutions of the serum samples and the bound total IgG specific antibodies were detected by peroxidase-conjugated Goat Anti-Mouse IgG (Sigma-Aldrich, St. Louis, MO, USA). Finally, wells were incubated with K-Blue Advanced Substrate (Neogen, Lansing, MI, USA) and the absorbance at 450 nm was measured using a microplate reader (Versamax microplate reader, Molecular Devices, San Jose, CA, USA). The mean value of the absorbance was calculated for each dilution of the serum sample run in duplicate. Isotypes IgG1 and IgG2a were detected using Peroxidase AffiniPure Goat Anti-Mouse IgG, Fcγ subclass 1 specific, and Peroxidase AffiniPure Goat Anti-Mouse IgG, Fcγ subclass 2a specific, (Jackson ImmunoResearch, Cambridge, UK), respectively. The endpoint titer of RBD-specific total IgG binding antibodies was established as the reciprocal of the last serum dilution yielding 3 times the mean optical density of the negative control of the technique (wells without serum added).

Blue native polyacrylamide gel electrophoresis (BN-PAGE) was performed by isolating mitochondrial proteins from mutant and control cell lines, as detailed previously (58 (link),59 (link)). Samples containing 30 μg of total cellular proteins were separated on 3–12% Bis–Tris Native PAGE gel. The total OXPHOS Human WB Antibody Cocktail was applied as primary antibodies for this experiment. Peroxidase AffiniPure goat anti-mouse IgG and goat anti-rabbit IgG (Jackson) were used as secondary antibodies and protein signals were detected using the ECL system (CWBIO).