4 replies to this topic

[szang]

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.

[madrius1]

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.

[szang]

madrius1, on Jan 14 2010, 11:04 AM, said:

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.

Thank you very much, Madrius1.

i will go to see what happen when i treat the lysate with RNAase.

[doglo]

madrius1, on Jan 14 2010, 08:04 PM, said:

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.

Is there any chance that in co-IP conditions the proteins complex could protect a putative RNA from RNAse?

[eldon]

szang, on Jan 14 2010, 11:55 AM, said:

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.

yeast two hydrid.