Co-Ip mystery - Part II - How to explain and solve this Co-IP problem ? (May/01/2008 )
Hello everyone,
I send a message again because I experience problems with Co-IP and I'm not able to find any explanation.
I precipitate one protein to see the interactions with other proteins following (or not) the treatment of the cells. I precipitate the protein with one antibody and detect the correct band with another antibody, the signal is even much stronger in what has been precipitated compared to what's remaining in the lysate after precipitation. On the other hand, I don't get proteins which have not been precipitated and should not interact with my protein (whereas I see this protein in what has not been precipitated). So until then, the technique is working fine. Now when I try to detect any protein supposed to interact with the one I precipitate, I observe only one band corrsponding to .... the protein I precipitated and not the one the antibody is directed against ! I tried several proteins and I always get the same result => a band corresponding to the protein I precipitate.
- I precipitate with a mouse antibody, I detect the partners with mouse or rabbit antibodies, it doesn't change anything
- I elute the proteins from the G-sepharose beads by heating at 37°C for 20 min, because boiling seems to destroy the protein I precipitate.
If someone does have an idea ...
are you sure that the antibodies to the other proteins work?
have you tried ip of the other protein(s) and then try to detect protein 1?
maybe the proteins don't interact in the conditions that you are using.
have you tried ip of the other protein(s) and then try to detect protein 1?
maybe the proteins don't interact in the conditions that you are using.
I am sure that the antibodies against the other proteins do work very good. Actually, I load each time on the same gel what has been precipitated and what has not been precipitated. I detect the proteins supposed to interact with the protein in what has not been precipitated.
I tried IP with other protein and try to detect protein 1 indeed, without success. No detection of protein 1 ....
I am sure these proteins interact from the litterature, I think I am facing a technical problem which I don't understand ...
have you tried ip of the other protein(s) and then try to detect protein 1?
maybe the proteins don't interact in the conditions that you are using.
I am sure that the antibodies against the other proteins do work very good. Actually, I load each time on the same gel what has been precipitated and what has not been precipitated. I detect the proteins supposed to interact with the protein in what has not been precipitated.
I tried IP with other protein and try to detect protein 1 indeed, without success. No detection of protein 1 ....
I am sure these proteins interact from the literature, I think I am facing a technical problem which I don't understand ...
have you matched the conditions from the literature?
maybe your buffers are not prepared identically (different balances, pH meters, chemical grade and supplier, and water supply can make a difference).