Pierce protein a g ultralink resin

Immulon 4 plates was coated with OvTrop (10 μg/well) or (KLH) (10 μg/well). Another set of plate for measuring the Der p 10-specific IgE response was coated with Der p 10 (5 μg/well). Both sets were incubated overnight at 4C, then blocked with 200 μl of blocking buffer (PBS 1X, 0.05% Tween-20 containing 5% BSA). The IgE detection ELISA plate was placed at 4C until later use. Three pools of 50 μl of undiluted IgG-depleted (using Pierce Protein A/G Ultralink Resin (ThermoFisher, Waltham, USA) sera was added to both OvTrop and KLH antigen-containing wells and incubated overnight at 4°C. 50uL of each pool were transferred to the Der p 10-coated plate, following incubation for 1 hour at RT. After washing, 50 μl of 1:250 (diluted in PBS 1X, 0.05% Tween-20 containing 1% BSA) biotinylated anti-mouse IgE (Invitrogen, Carlsbad, USA) were added to the appropriate wells and incubated 1 hour at RT. Following an additional wash step, 50μl of streptavidin-conjugated to HRP (R&D Systems, Minneapolis, USA) was added diluted 1:100 and incubated for 30 minutes at RT. A final wash step was performed and 50μl of 1Step Ultra TMB-ELISA Substrate (ThermoFisher) was added to each well and the colorimetric reaction was stopped after 10 minutes 30 minutes by adding 25 μl of H₂SO₄. The plate was then read at 450 nm.

Co-immunoprecipitation (Co-IP) analyses were conducted according to a previous report [26 (link)]. To exclude the contaminating effect of DNA attached to tested proteins, 20 U/ml of DNase I (Roche) was added in lysis buffer. In brief, the cell lysates were subsequently sonicated, washed, and incubated with anti-MGMT antibodies (Abcam) or negative control IgGs (Santa Cruz Biotechnology). After incubation for 24 h, Pierce Protein A/G UltraLink Resin (Thermo Fisher Scientific) was added to capture immune complexes. After washing, the precipitated proteins were resuspended in nonreducing loading buffer and heated at 95 °C for 5 min before Western blot analyses.