how to explain the result of IP and co-IP - (Jan/14/2010 )
Hi everyone,
I try to find out my two protein interaction. My IP and co-IP work very well, but I can not say these two proteins interact directly. Mybe these two proteins interact indirectly through RNA or another protein.
My question is how i can prove these two proteins interaction is not through RNA or another protein.
I try to use RNase to digest the RNA to see it will affcet co-ip, but i have no idea how to use RNase.
Does someone have any ideas about it, please tell me.
Thank you very much.
In vitro experiment is the only thing I see to prove direct interaction. Thus, you would have to purify both protein, put them in a tube and do Co-IP experiment.
Even if you add RNAse and this affects your IP, there could be plenty other reasons why both protein do not longer associate together.
Using RNAse is pretty straight forward : add RNAse to your sample and let it sit for 30 min at 37 C.
madrius1 on Jan 14 2010, 11:04 AM said:
Even if you add RNAse and this affects your IP, there could be plenty other reasons why both protein do not longer associate together.
Using RNAse is pretty straight forward : add RNAse to your sample and let it sit for 30 min at 37 C.
Thank you very much, Madrius1.
i will go to see what happen when i treat the lysate with RNAase.
madrius1 on Jan 14 2010, 08:04 PM said:
Even if you add RNAse and this affects your IP, there could be plenty other reasons why both protein do not longer associate together.
Using RNAse is pretty straight forward : add RNAse to your sample and let it sit for 30 min at 37 C.
Is there any chance that in co-IP conditions the proteins complex could protect a putative RNA from RNAse?
szang on Jan 14 2010, 11:55 AM said:
I try to find out my two protein interaction. My IP and co-IP work very well, but I can not say these two proteins interact directly. Mybe these two proteins interact indirectly through RNA or another protein.
My question is how i can prove these two proteins interaction is not through RNA or another protein.
I try to use RNase to digest the RNA to see it will affcet co-ip, but i have no idea how to use RNase.
Does someone have any ideas about it, please tell me.
Thank you very much.
yeast two hydrid.