Hi guys!
I am doing immunoprecipitation to verify the interaction between two proteins. One protein is His-tagged and the other is HA-tagged. When I use nickel beads to pull down the His-tagged protein, I can detect HA-tagged protein in the precipitation. However, when I use HA-tag primary ab to pull down HA-tag protein, I cannot detect His-protein in the precipitation. And I have identifed that the HA-tagged protein has been pulled down successfully by the beads via WB.
So I am wondering what may cause these differents results in different pull down directions.
Several possibilities, e.g.:
1st: Your HA tag may be burried and is not reachable by the AB when proteins interact (Try tagging the other terminus)
2nd: You may have nonspecific binding of your protein B to ni-coloumn (try to switch the tags of your protein, apply good negative controls (transfect cells with HA-tagged protein only and perform His-Tag purification)
3rd: something in your HA-IP protocol disturbes the interaction (try to switch the tags)
What are the conditions for the nickel beads pull-down? Does the HA-tagged protein have histidyl residues?
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts