Flag co-immunoprecipitation
michela.deleidi, Pascale Baden
Abstract
Protocol used to pull down V5-FLAG-Gcase interacting proteins in HEK cells
Steps
Flag co-immunoprecipitation
Detach HEK cells using Accutase for 5 minutes at 37°C and collect them.
Spin cells in a centrifuge at 250g for 5 minutes at room temperature.
Remove the supernatant and wash cells in PBS.
Repeat steps 2 and 3 for a total of 2 washes.
Lyse cells in 1% TBS + 0.5% NP40 + PI/PHI (Pierce #A32959)
Filter lysates through a 0.22uM PES syringe filter (Millipore).
Wash 50ul of M2 antiflag agarose resin (Sigma–Aldrich, #A2220) according to the manufacturer's instructions for 10 mg of total protein.
Incubate 10 mg of lysates with prewashed M2 anti-flag agarose resin for 2h at 4°C on a rotating wheel.
Elute Flag with Flag Peptide (Sigma-Aldrich, #F3290) by moving on a wheel for 20 min at 4°C followed by a spin for 1 min at 1000 g.
Collect the flow-through and proceed with TMT labeling or Western Blot analysis.
