Dynabeads Antibody Coupling and Immunoprecipitation

Monoclonal antibodies were coupled to superparamagnetic Dynabeads using Dynabeads Antibody Coupling Kit (ThermoFisher, catalog #14311D). In total, 5 mg of Dynabeads were coupled with 0.75 mg of the 7B8D7 or D3H55 monoclonal antibody as per the instructions in the kit. Once the beads were coupled and re-suspended, the antibody-coupled Dynabeads were stored at 4°C until use. Ten microliter of anti-IL-1RAcP antibody conjugated Dynabeads were incubated with 0, 50, or 100 ng of IL-1RAcP in 50 μL of PBS for 1 h prior to elution by boiling in 20 μL of elution buffer for 5 min. Samples were then run on a SDS-PAGE gel, transferred to a PVDF membrane using western blotting techniques.