Aug 26, 2023
Co-immunoprecipitation
- 1Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States
Protocol Citation: wusj , schekman 2023. Co-immunoprecipitation. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn32zzl5d/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: August 03, 2023
Last Modified: August 26, 2023
Protocol Integer ID: 85899
Keywords: immunoprecipitation, common procedure, procedure, protocol
Funders Acknowledgements:
Randy Schekman
Grant ID: Howard Hughes Medical Institute
Abstract
This protocol describes a common procedure to perform co-immunoprecipitation
Co-immunoprecipitation
1h 10m
Media fractions were collected and centrifuged at 1000×g for 00:10:00
10m
The supernatant fractions were collected and concentrated (20-fold) using a 10 kDa Amicon filter.
Concentrated media fractions (1 ml) were incubated with 20 μl of anti-FLAG M2 affinity gel for 01:00:00 at 4 °C
1h
After washing 5 times with lysis buffer , SDS-PAGE sample loading buffer was added to the beads (twice the beads volume) and samples were processed for SDS-PAGE and immunoblot.