Apr 03, 2023
Endogenous coimmunoprecipitation
- michela.deleidi1,
- Pascale Baden1
- 1German Center for Neurodegenerative Diseases (DZNE), Tübingen, 72076 Germany
Protocol Citation: michela.deleidi, Pascale Baden 2023. Endogenous coimmunoprecipitation. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly7bqmlx9/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: 79871
Keywords: ASAPCRN
Abstract
Protocol used for immunoprecipitation of HSP60 and LONP1 in HEK cells to show the interaction with V5-Flag-tagged WT-GCase
Endogenous coimmunoprecipitation
Endogenous coimmunoprecipitation
Wash Protein G agarose fast-flow beads in TBS 1X + 0.05% NP40.To this
end, the beads were incubated with lysis buffer on the spinning wheel (25 RPM) for 2
–5 min at 4 °C, followed by a 1 min centrifugation at 500 RPM 4 °C.
Repeat step 1 for a total of 3 washes.
Incubate 20ul of pure beads with 5ug of anti-LONP1 antibody or 5ug of normal rabbit IgG as control (or 3ug of anti-Hsp60 and 3ug of mouse IgG as control) and add 300ul of washing buffer to each tube.
Incubate 2h at 20RPM on a rotating wheel at 4°C
Meanwhile, detach HEK cells using Accutase for 5 minutes at 37°C and collect them.
Detach 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 7 and 8 for a total of 2 washes.
Lyse cells in 1% TBS + 0.5% NP40 + PI/PHI (Pierce #A32959)
After 2h incubation, wash beads again 3 times in washing buffer.
Incubate antibody-coated beads with 3.7 ug of lysate on the spinning wheel for 2h.
After 2h incubation, wash beads again 3 times in washing buffer.
Elute by boiling the beads twice with 2x Laemmli buffer at 95°C for 8min at 300rpm in a thermoblock.
Spin beads for 1 minute at 10000 RCF.
Collect supernatant and proceed with western blot analysis.