Question about the strategy of protein purification - (Aug/17/2006 )

Hi,everyone:
I want to overexpress my protein and then purify my it from mammalian cells, such as 293T. My purpose is to use this purified protein to do ubiqutination assay. What I have on my hand is Ni-NTA beads. So I tried once to express my protein and Ni-NTA pull down it. As I want to see whether the beads bind enough of the protein for further elution, I added the SDS-loading buffer into beads and do western then stain the gel. The band representing my protein is clear enough but there are many other band also showed up on the gel and they are as strong as my protein band. So I have several questions:
1. Can I estimate whether my protein is enough for my following assay by looking at the intense of the band?
2. How to reduce the background bands?
3. If after elution, I still got several bands besides my protein bands, how to further purify it?
4. Is any other better strategy to purify protein from mammalian cells without affectting its activity?


Thank you very much!

Hi,

If I understood well you expressed your protein and purifyed it using Niquel beads. Do you also overexpress ubiquitin in your assay? Cause if so, then what your are seeing (many bands above your protein and not below it, right?) might be polyubiquitination of your protein.

regards,

Adriana

Thanks for your reply! I did not overexpress ubiqutin in the assay. this is just preliminary experiment to see whether I can purify the protein. if I can, i will use the purified protein in the following in vitro experiment.
any good ideas?

good that your protein binds to the beads.. now you can optimize the specificity by playong with imidazole concentration in washings