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I've been working as the computer guy for a microbiology lab for the past few months. I've always been interested in bench work, but my wet lab experience is rather limited and thus so is my understanding of wet lab techniques.

The PI has expressed his support and given me the opportunity to conduct my own project from start to finish (generation of wet lab data and computational analysis) and write a paper in preparation for my Ph.D school applications.

I realize that coming up with an actual question is a long and thought out process so I want to do my homework before approaching him with a question. I've read quite a bit of literature and have come up with a question i'd like to answer, the computational stuff I can handle and explain in detail, but I'm having a hard time with the wet lab techniques I could use to generate some of the data i'm interested in.

Effectively I am looking to obtain a list of potential cellular transcription factors that are physically associated (bound) with/by a specific viral protein. Is there a lab technique that would allow me to generate such a list?

Links to papers, wikis, your own theories or anything will be greatly appreciated. Also any advice, or "this isn't possible" is all appreciated, as long as you can give me a bit of "this is why".

Thank you! (PS I'm not sure if this type of post is allowed here).

EDIT: It is important to note that I am looking for new binding partners.

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  • $\begingroup$ Co-immunoprecipitation followed by a western blot comes to mind; you pull out a known TF from lysate with antibodies and then try to detect what you suspect is complexed with it in a western blot with another antibody for the viral protein, or vice versa. $\endgroup$ – CKM Feb 8 '16 at 20:56
  • $\begingroup$ @Kendall this can only be done for TF's that are known or that we believe to be associated with the viral protein correct? What if there is no research on TF's that bind with this viral protein, so I have to actually identify new binding partners (I know this is non-trival and probably an entire paper on its own). $\endgroup$ – System Feb 8 '16 at 21:05
  • $\begingroup$ Basically you need to have antibodies to what you are studying, and when you do the actual pull down the hope is your TF is complexed in some way with your viral protein, so that during the western blot if you get detection with the second antibody, you can logically conclude that there is a physical interaction between the two. Co-IP cant discriminate direct/indirect association, however, so if you need to know the direct binding partners, a more complex technique is necessary $\endgroup$ – CKM Feb 8 '16 at 21:08
  • $\begingroup$ If you need to identify new binding partners for some reason, you probably need to do something like tandem affinity purification (basically somehow purify the unknown binding partners) followed by separation and biophysical methods like mass spec. $\endgroup$ – CKM Feb 8 '16 at 21:17
  • $\begingroup$ That second answer is what I was looking for, the viral protein hasn't been studied in the context of binding to host cell chromatin so i'm interested in determining at least one of it's binding factors. Some computational analysis done on another paper my lab has written leads me to believe that the viral protein recruits some regulatory transcription factors in order to bind to chromatin, but there isn't much or anything known about who these TF's are. $\endgroup$ – System Feb 8 '16 at 21:31

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