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protein purification problem

Paul J. Phelan via methods%40net.bio.net (by Paul.Phelan from tufts.edu)
Fri Sep 4 11:21:29 EST 2009


If I were you, I would answer a couple more questions first:
When you say you express a lot of protein, is it right to assume that 
it is soluble, and you're not losing it (or much of it) in the 
insoluble material?
It should be easy to tell if your protein is binding to your resin or not.
Assay the flow-through from your column, or else take a sample of the 
resin (if you can) before and after adding your extract, boil the 
samples with SDS-PAGE loading buffer and run them on SDS-PAGE.
I think it's critical to know if your protein is either not binding to 
your resin, or not coming off the resin, before trying to solve your 
problem.




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