coimmunoprecipitation - (Nov/28/2007 )

Dear Friends,
I have a protein-protein interaction that I have originally detected in bacterial two-hybrid system and pull-down experiments with bacterially expressed proteins also show positive interaction. Now I would like to confirm this interaction also in eukaryotic cells by coimmunoprecipitating the two. Since I do not have antibodies for these proteins, I am using GFP -(Bait) and HA-tagged (target) proteins and thus transfected cells. I have performed the experiment couple of times, and it kind of looks promising: I have positive interaction when the two proteins are present and all negative controls are negative. What makes me doubt is that I do not concentrate the target protein in the immunoprecipitation, that is that my band is weaker in the immunoprecipitation sample than in the original cell lysate sample. In order to have positive interaction do I have to be able to concentrate the target protein? So, what do you think, are these two proteins interacting?
Thanks already in advance for your comments!
Mimmi

When you say all negative controls are negative, which negative controls are you doing?
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Thanks for your interest. My negative controls include: co-ip without the antibody, irrelevant bait protein (ab included), and ab included but no bait protein. The immunoprecipitation I made with magnetic Protein A beads, so no centrifugation included. Reaction volume is 200ul, and I wash 5 times 500ul. My buffer includes 0,5% Triton 100. So, what do you think?


Mimmi

When you said that you cannot concentrate your protein in the pull-down, you are comparing to the input? How many percents can you pull-down your protein and how many percent is the protein that you are supposed to co-IP. That is, if you are using anti-GFP for IP, how much GFP-protein you got (compared to input), and how much is the interacting protein (the HA tagged you got), again compared to input? When you pull-dpwn with GFP you get HA, and vice-versa?

I would not much worry about the 'concentrate' thing, the controls are more important. There are many possible reasons for why you don't pu;;-down too much of the supposedly interacting protein. Your IP Ab maynot be able to pull-down all of the first protein, so you don't have much of the second. Or the interacting may be transiently or weak, so the interaction may be reduced due to the procedure of cell lysing or maybe the number of interacting pairs at any given time may be not as much... Do more experiment to confirm instead, such as GST pull down, or may be point mutations. Remember your negative controls in all cases. Good luck.

Thanks for your comments!

Mimmi

Here are a few things u can do to improve ur pull down

1. Use more Ab ( this is probably limiting if u r not able to pull down most of the protein). Guess the beads are enough usually

2. Monoclonal Ab works better for pull down

3. Check ur washing stpes if u losing protein during washes if so use softer washes

4. Increase ur time of incubation - overnight incubation helps, but make sure u have enough protease inhibitors in ur lysate

5. CoIped protein would be for sure less than the Iped one, unless they love each other too much

Hope this helps

praj