For IgGs, 20 µL of Urea 8M and 0.4 M NH4HCO3 and 2 µL DTT 1M were added to the reaction mixtures and samples were incubated at 50°C for 30 minutes. Then, 1 µL of PNGaseF (0.5 µg/ µL) was added and the mixture was further incubated at 37°C for 3 hours.