Hello all,
I am working with a protein domain which hypothetically can go into nucleus (as it contains two NLS). I wanted to do IP-Mass spec to see what kind of proteins it is interacting with. But I am facing some problem in planning the strategy.
The problem is:
1. This domain is very short-lived and there is no antibody to detect the endogenous protein. However, I put a 3X-flag tag in the c-terminal of the domain and generally overexpress it. In western blot, it works okay.
2. But even the cell line (glial cell line) I am working with (the cells which endogenously express this protein) very hard to transfect (30-40% efficiency max.) and I do not usually get enough protein to do IP.
So, while I was optimizing the transfection condition and pull down strategies; I parallely overexpressed my Flag-tagged domain in HEK 293 cells and got excellent amount of IPed POI domain. After washing 3 times, I incubated these pulled down domain with either the cytosolic and nuclear fraction of the cells which actually express this protein (its own environment).
After running a coomassie gel, I found 2 unique bands every time.
My question is, I am not sure how good this experimental plan is. I know it is not ideal, but do you think I can make something useful out of it? Should I do Mass spec or should I try to get things done from my actual protein expressing cell line only?
Any advice would be appreciated!
We do this kind of thing (your second approach) all the time. I would expect it is quite a common approach in the literature. If you really are seeing 2 unique bands then it also sounds quite specific. I would recommend you analyse the bands by MS. Depending on your situation, this really is quite simple and will give you a good indication if the approach is working.
Peter... Thank you for your reply! My supervisor was a bit skeptical about this approach and i failed to come up with some nice literature with these kind of method to convince her. Anyway, i went with MS and got some nice indication. But if you have any parricular literature in your mind, can you kindly send me the link?
Thanks in advance.
Some examples from our lab and collaborators follow. I would think putting "pulldown" and "binding partners" into Medline would get hits as well.
http://pubs.rsc.org/en/Content/ArticleLanding/2012/OB/c2ob07139h#!divAbstract
http://www.jbc.org/content/286/35/30295.long
http://www.blackwell-synergy.com/doi/full/10.1111/j.1471-4159.2007.04574.x
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