Immunoprecipitation With Agilent Bravo AssayMAP Liquid Handling Robot

Anti-HA (12CA5) antibody (Roche) was immobilized on Protein A (PA-W) cartridges (Agilent, G5496-60000), using the Immobilization App (Agilent Sample Prep Workbench v3.0.0). All steps use PBS buffer (Sigma-Aldrich). Cartridges were primed with 100 μL (at 300 μL/min) and equilibrated with 50 μL (at 10 μL/min) followed by loading of 100 μg antibody (or otherwise as stated) in a final volume of 50 μL PBS buffer at 3 μL/min. A cup wash with 50 μL and an internal cartridge wash step (100 μL at 10 μL/min) were performed before re-equilibrating the cartridges with 50 μL at 10 μL/min).

The Affinity Purification App was used for pull-downs. Briefly, Protein A cartridges with immobilized anti-HA antibody were primed (100 μL at 300 μL/min) and equilibrated (50 μL at 10 uL/min) with NP-40 buffer, which was also used for all following steps. The sample was loaded at a flow-rate of 1 μL/min. After sample loading the cup was washed (50 μL) and an internal cartridge wash step (100 uL at 10 μL/min) performed to remove unbound lysate. Peptides were eluted using 50 μL at 5 μL/min 6M Urea or 0.15% TFA or 5% SDS.