I am trying to purify a fusion protein using the E.Coli expression system. I have successfully expressed my protein and verified this using an anti-His antibody and an antibody that recognises my protein. My protein is insoluble and thus formed inclusion bodies, as such I resuspended my protein in 8M Urea. I then attempted to purify my protein using Ni-NTA beads with no such luck. My protein doesn't bind to the beads and instead comes out all at once during the flowthrough. I have again tested to see if the His tag is present in the protein via western blot and have confirmed this. Does anybody have any ideas on how I can bind my protein?
8M urea is not always enough to dis-aggregate the protein. Consider adding reducing agent (TCEP), detergent, or heat.
Also, if you wash inclusion bodies well you should be able to experiment with refolding w/o having to resort to denatured purification. Although, it sounds like you need to try some other expression system :)
Dear Dr Edwards,
Is it possible that your thiols have formed S-S bonds during the purification process ? You could perhaps test this possibility using also an excess of DTT in the 8M urea. You may also contact Drs Steingrimur Stefansson and Ziguo Zhang who have commented on this problem in other Q and A requests.
All the best
Sture F.
May be the 6his tag is not available to interact with the Ni-NTA column, try to change position of the 6his on the protein and retry the purification. As using reducing agent like b-Me or DTT might work.
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