Polyclonal mouse igg

We followed our original procedure (Wan et al., 2020 (link)), with modifications. The WBCs were suspended in lysis buffer (40 mM N-octanoyl-N-methylglucamine, 40 mM N-nonanoyl-N-methylglucamine, 1 mM phenylmethylsulfonyl fluoride, 0.2 mM iodoacetamide, 20 µg ml⁻¹ leupeptin, and Roche cOmplete Mini Protease cocktail in PBS) and rocked for 1 h at 4°C. The cell lysate was spun in a centrifuge at 20,000 g for 30 min at 4°C. To eliminate nonspecific binding of peptides, the supernatant was first incubated with polyclonal mouse IgG (1.5 mg antibody per sample; Bio X Cell) bound to sepharose 4B at 4°C for 30 min. The unbound material containing MHC-II–peptide complexes was collected and was then added to a tube containing PBS-washed sepharose conjugated to the anti–I-A^g7 antibody (1.5 mg per sample; AG2.42.7) and incubated at 4°C overnight. The I-A^g7–sepharose was applied to a column and washed four times as follows: 10 ml 150 mM NaCl and 20 mM Tris, pH 7.4; 10 ml 400 mM NaCl and 20 mM Tris, pH 7.4; 10 ml 150 mM NaCl and 20 mM Tris, pH 7.4; and 10 ml 20 mM Tris, pH 8.0. Peptides were eluted with 10% acetic acid and dried with a SpeedVac. Eluted peptides were passed over detergent removal spin columns (Pierce) to remove traces of remaining detergent and were cleaned using C18 Spin Columns from Thermo Fisher Scientific (Pierce).