in-vivo method of identifying 2 protein binding? - (Jun/02/2005 )
is there any in-vivo method that i can use to id whether if 2 proteins bind to each other?
immunocytochemistry? if they co-localize in the cell, it will support their binding. Or gel filtration, if they co-fractionate, it will support the binding. but these are not giving you a absolute answer. they should be combined with in vitro IPs, I think.
Though I never tried, for in-vivo identification of protein binding, you should try first the Yeast-two hybrid technique, you can find more information on Google or Pubmed.
There are several other methods which you can try, I recommend you to read the paper Curr. Opin. Struc. Biol, 2005, 15:4-14. A comprehensive review on current techniques monitoring protein interaction in-vivo.
While immunocyto. can be used to support binding, this doesn't address the poster's question. Immunocyto experiments, when used to colocalize two proteins, only tell you if two proteins occupy the same space. Seeing a 3rd color as in yellow for example, (red/green fluorophores), never means that two proteins are binding. In fact even if you see the 3rd color, it doesn't definitively mean that two proteins occupy the same space. Seeing yellow may depend upon how, or rather from what perspective you are looking at the cell (en face as opposed to an XZ reconstruction)
So do not use immunocyto experiments as a means to confirm protein binding, because it doesn't answer that question.
Second with regard to coIPs. CoIps by themselves do not answer the question of "are my two proteins binding" Many people, students and faculty, have often said that coIPs demonstrate protein binding. It doesn't, coIPS may only tell you if two proteins exist as a complex, not if they are binding.
I personally have not tried in vivo protein interaction techniques, however I have seen people use photoaffinity labeling compounds, and cross linking agents as a means to answer the question you ask. Pierce makes some of these reagents.