p53 protein - design experiments to illustrate p53 protein character. (Sep/13/2008 )
i have 2 questions. hopefully some kind souls can give me some hints on how should i go about doing it or provide me with some references..
a novel protein Y through preliminary experiment is found to interact with p53. (that's all the info i have for this study)
1) what experiment can be done to identify the domain of p53 that is important for this interaction?
2) when p53 protein has a mutation in the DNA binding domain, it still retains its ability to interact with protein Y and induce apoptosis. How is it possible? what experiments can be done to find out the mechanism of p53-protein Y mediated apoptosis?
a novel protein Y through preliminary experiment is found to interact with p53. (that's all the info i have for this study)
1) what experiment can be done to identify the domain of p53 that is important for this interaction?
2) when p53 protein has a mutation in the DNA binding domain, it still retains its ability to interact with protein Y and induce apoptosis. How is it possible? what experiments can be done to find out the mechanism of p53-protein Y mediated apoptosis?
I think one way would be immunoprecipitation (or in your case co-immunoprecipitation). you would need to get an antibody specific for a region of p53. then using protein A/G precipitate p53. this way p53 along with the Y protein will precipitate. then you will need to denature the proteins by heating up at 95 degrees. then run on gel. you must see different bands. each band corresponds to a protein (except the light and heavy chain of your anti-p53). then you may need to purify the protein from the gel. and sequence it.....I've never done this, so I can not guarantee it. I remembering having such a case 5 years ago and I read a paper that mentioned IP.
However the majority of proteins which interact with p53 are already known. just search different databases. p53 is famous and many teams around the world work on it.
Hmm, looks like homework to me.
1) think about how you might get different regions of p53 expressed individually and what experiments you could do to detect an interaction in vitro. Otherwise you may want to think about mutations of p53 and what they would do to an interaction?
2)Maybe, just maybe, protein Y doesn't bind the DNA binding domain! For part two, are you looking at pathways or direct interactions inducing apoptosis? p53 dependent apoptosis is well known, you may want to google it for papers.