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How exactly do i show two proteins interacting?

protein:protein interaction co-ip

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#1 cm13

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Posted 14 June 2012 - 06:29 AM

Hi there, im not sure if this is in the right part of the forum, but here goes!

I've looked at a protein in cells in the lab (i'm not allowed to say which one)
I have been able to blot it on a Western, do immunofluorescence, and importantly Co-immunoprecipitate it.
Following mass spectrometry analysis of the co-ip sample, i got back another result as well that seemed like an interesting protein also.
I've since shown this one on western blots too, and immunofluorescence.


I want to be able to show how these two proteins interact with each other.
I've a feeling that when i knock down one of them, the other will be more up-regulated.

What is the best way to go about showing a protein:protein interaction?
Do i do yeast-2-hybrids, or a computational simulation? If so how do i go about them?
If it's not those methods, then what do i do?

Thanks!

#2 Anexum

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Posted 14 June 2012 - 10:25 AM

yeast two hybrid would be the more convential. However co-immunoprecipitation/immunoflourescene is convincing enough.

#3 cm13

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Posted 15 June 2012 - 02:02 AM

But would co-ip / co-immunofluorescence not just show the two proteins are there?
I want to show that one has an effect on another?
Can i do this with yeast two hybrids?
And what sort of kit/protocol do i need for this if that's the case?





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