Is Co-IP with natural protein a fantasy? - (Dec/11/2007 )

Hi, I've checked previous posts in this forum and I have not found answer to my questions....

I've worked with co-ip for a while and have not get consistent results...... I do not use any tagged protein so I'm not sure whether it's just really really difficult to work with natural protein.

B.T.W, the antibodies I used worked in chromatin immunoprecipitation in my hands so I'm sure that the antibodies work for immunoprecipitation.

My concern is, my protein interaction occurs with ligand stimulation(interaction of VDR and RXR), so I think maybe after I lyse the cells, the proteins just dissociate with each other?

Many thanks for any input.

Hi,

Yes, I only do interaction with endogenous proteins.
In terms of Co-IP with VDR or other hormone receptors
it is tricky.
#1 The interaction of coactivators is temporary. The ligand binds, coactivators come
and then are released within 1 hr (faster?). Corepressors are easier to find.
#2 You need to use high salt to extract nuclear proteins. This should be diluted when doing the IP.
The high salt may disrupt the interaction you are looking for.
Most people with nuclear receptors use tagged proteins for these reasons.

Thank you for your reply. That sounds reasonable. Would you mind sharing your protocol with co-ip? Thanks a lot!

Hi,

I've tried to upload my protocol as a Word doc. but cannot.
If there is another way to send it let me know.

you can copy and paste the text into a post.

well its just matter of time. make sure that you have ample amount of your protein of interest in the lysate...else you will go nowhere. i have the same problem to find my interaction in primary patient lymphocytes. each patient have a different expression & interaction levels of the proteins and its little hard to be consistent.

i would suggest that you make ample of lysates under similar settings to preserve your interaction and try to repeat co-ip in those samples until you get your protocol standardized.