Sep 15, 2022
Immunoprecipitation protocol
- 1Northwestern university
Protocol Citation: Chuyu Chen 2022. Immunoprecipitation protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l694zzlqe/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: September 15, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 70096
Keywords: ASAPCRN
Abstract
Immunoprecipitation procedure for brain tissue material
Add 750ug tissue in 1ml IP buffer and antibody, incubate 4°C 2hrs on tube rotator
Prepare ProteinA/G beads 30ul/sample. Pool all beads in one tube to perform wash
Centrifuge 4°C 13000g 30sec, remove supernatant
Resuspend beads with 80ul PBS/sample. Centrifuge again at 13000g for 30sec.
Repeat wash 3 times.
Remove supernatant with fine tip
Resuspend beads with 30ul PBS/sample
Add 30ul beads to protein sample
Incubate 4°C overnight on tube rotator
Centrifuge 4°C 4000rpm 4min, remove supernatant
Wash with IP buffer (+ protease inhibitor) 800ul/sample
Repeat 3 times, total 4 wash
Remove supernatant with fine tip
Add 1x loading buffer 25ul and 5ul reducing agent. 75°C 5min
Centrifuge 4°C 4000rpm 4min, collect supernatant for western blotting