CoIP optimization - How to see weak interactions (Jan/13/2006 )
Hi,
Any ideas how to optimize coimmunoprecipitation to see weak protein interactions?
I've been trying to confirm an interaction seen in Y2H with the following CoIP protocol:
-Produce HA- or Myc-tagged proteins by in vitro translation using the Promega TNT system; label with S-35 Met
-Combine proteins and incubate refrigerated for 1 h
-Add antibody (HA or Myc mAb) and incubate refrigerated for 1 h
-Add Protein A/G agarose beads and incubate refrigerated overnight with rotation
-Collect beads by centifugation, wash, elute in SDS-PAGE loading buffer
-Separate by SDS-PAGE and detect with phosphoimaging
I've minimized washing to a single wash in mild conditions (low salt, no detergent) but still I can't get a difference between the experiments and negative controls. (I do get some of my prey protein pulled down, but the band is equally strong in negative controls with no intermediating protein).
What could I try to enhance the true interaction (I hope it's real!) or to reduce unspecific binding?
Jaakko
Hey Jaakko,
I don´t know if I undestood you....
You see the unexpected band in the negative control where the two proteins shouldn´t be present because they don´t interact (perhaps negative control from clontech Y2H).
But if you don´t wash enough that is what you would get becaus although the second protein is not attached to prot A beads it still remains there due to not enaugh washing. Try washing more times (normally protocols mention 5 washing steps) and see then if the undesired protein is still there.
Any ideas how to optimize coimmunoprecipitation to see weak protein interactions?
I've been trying to confirm an interaction seen in Y2H with the following CoIP protocol:
-Produce HA- or Myc-tagged proteins by in vitro translation using the Promega TNT system; label with S-35 Met
-Combine proteins and incubate refrigerated for 1 h
-Add antibody (HA or Myc mAb) and incubate refrigerated for 1 h
-Add Protein A/G agarose beads and incubate refrigerated overnight with rotation
-Collect beads by centifugation, wash, elute in SDS-PAGE loading buffer
-Separate by SDS-PAGE and detect with phosphoimaging
I've minimized washing to a single wash in mild conditions (low salt, no detergent) but still I can't get a difference between the experiments and negative controls. (I do get some of my prey protein pulled down, but the band is equally strong in negative controls with no intermediating protein).
What could I try to enhance the true interaction (I hope it's real!) or to reduce unspecific binding?
Jaakko
Hi, Elisa.
Thanks for the message! You understood just right.
But the problem is that the interaction I'm trying to see seems to be very weak! That is, if I wash as much as recommended in most protocols, I will lose also the interacting protein.
So I would need to find conditions that preserve the interaction but minimize the background due to unbound protein.
Jaakko
I don´t know if I undestood you....
You see the unexpected band in the negative control where the two proteins shouldn´t be present because they don´t interact (perhaps negative control from clontech Y2H).
But if you don´t wash enough that is what you would get becaus although the second protein is not attached to prot A beads it still remains there due to not enaugh washing. Try washing more times (normally protocols mention 5 washing steps) and see then if the undesired protein is still there.
Hi,
Any ideas how to optimize coimmunoprecipitation to see weak protein interactions?
I've been trying to confirm an interaction seen in Y2H with the following CoIP protocol:
-Produce HA- or Myc-tagged proteins by in vitro translation using the Promega TNT system; label with S-35 Met
-Combine proteins and incubate refrigerated for 1 h
-Add antibody (HA or Myc mAb) and incubate refrigerated for 1 h
-Add Protein A/G agarose beads and incubate refrigerated overnight with rotation
-Collect beads by centifugation, wash, elute in SDS-PAGE loading buffer
-Separate by SDS-PAGE and detect with phosphoimaging
I've minimized washing to a single wash in mild conditions (low salt, no detergent) but still I can't get a difference between the experiments and negative controls. (I do get some of my prey protein pulled down, but the band is equally strong in negative controls with no intermediating protein).
What could I try to enhance the true interaction (I hope it's real!) or to reduce unspecific binding?
Jaakko
Hi
I do not think this method will work for weak interactions. I could barely get the protocol to work for a strong interacting pair. I gave up and moved on! A negative result is not really going to tell you that much, and a posistive interaction ultimately needs to be shown to be biological significant in some way.
Its a balance between how much time it is reasonable to spend trouble shooting and determining optimal binding conditions for you proteins and how much time you might waste by jumping ahead with an interaction which does not occur outside of the yeast system!
Good luck!
Thanks,
I've already given up with CoIP. Maybe GST pulldown will do the trick...
Jaakko