Apr 03, 2023
Flag co-immunoprecipitation
- michela.deleidi1,
- Pascale Baden1
- 1German Center for Neurodegenerative Diseases (DZNE), Tübingen, 72076 Germany
Protocol Citation: michela.deleidi, Pascale Baden 2023. Flag co-immunoprecipitation. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l27rpjg1y/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 01, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 79867
Keywords: ASAPCRN
Abstract
Protocol used to pull down V5-FLAG-Gcase interacting proteins in HEK cells
Flag co-immunoprecipitation
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.