Anti mouse igg conjugated to horseradish peroxidase

For protein detection, cells grown for 3 days in a 25 cm² flask were washed with PBS and extracted with 150 µl 1% SDS in TE prior to protein quantification by bicinchoninic acid (BCA) (Pierce). Fifty micrograms of total protein were loaded per gel, and a western blot was performed using M2 anti-Flag mouse monoclonal antibody (Sigma, Cat No. F3165, 1:2000 dilution) or anti-GFP monoclonal antibody (Roche, Cat No. 11814460001, 1:2000 dilution) and detected with anti-mouse IgG conjugated to horseradish peroxidase (Jackson, Cat No. 515035003, 1:10,000 dilution). Blots were developed using high-sensitivity electrochemiluminescence (ECL) reagent (Thermo Fisher Scientific) and visualized using the Fujifilm LAS-3000 developer. Intensities of immunoreactive bands on western blots were quantified using Quantity One (Bio-Rad) software. Protein levels were normalized using a ribosomal protein (RL7) antibody as a loading control^{30 (link)} (1:500 dilution).

Rabbit polyclonal anti-H2a carboxy-terminal antibodies were the ones used in earlier studies^{24 (link)} at 1:1000 for immunoblot (IB) and 1:100 for immunoprecipitation. Rabbit polyclonal anti-EDEM1 (1:1000), mouse monoclonal anti-FLAG (1:1000), mouse monoclonal anti-HA (1:1000) and mouse monoclonal anti-GAPDH (1:2000) were from Sigma, anti-S-tag (1:500) from Novagen (Gibbstown, NJ). Goat anti–rabbit, and anti-mouse IgG conjugated to horseradish peroxidase (1:10,000) were from Jackson Labs (West Grove, PA).

Enzyme linked immunosorbent assay was carried out based on published techniques (52 (link), 56 (link)). Briefly, 96-well maxisorp plates were coated with SARS-CoV-2 RBD overnight at 1 µg/ml and 50 µl per well. Plates were blocked with 200 µl of 3% non-fat milk with shaking, at room temperature for 1 h. Serum sample dilution series in 1% non-fat milk were performed in dilution plates, and transferred to blocked plates, then incubated for 2h at room temperature with shaking. Plates were then washed three times with PBST and anti-mouse IgG conjugated to horseradish peroxidase (Jackson Immuno, USA) added at a 1:3000 dilution. Plates were incubated for 1h at room temperature, in the dark with shaking. Plates were then washed three times with PBST and 50 µl TMB solution added per well. Reactions were quenched after 2-3 minutes in the dark using 2N H₂SO₄, and plates read at 450 nm with a Biorad plate reader. AUC values were generated using Prism GraphPad 9.3.1.