Hello guys,I am trying to purify a protein with C-terminal HIS tag expressed in insect cells. I have tried 1) buffer of different pH in Ni NTA 2) added urea to the protein and then tried Ni NTA. But nothing seems to work. I want to use this protein for crystallization and purity matters a lot. If anybody has any suggestions I would be glad to try it.
Hi tere,
Are you sure the protein is actually expressed and that it contains the tag? You mentioned that nothing seems to work but no actual result (protein not adsorbed, protein not eluting...) so it's a bit hard to help
@Dominique: protein can be seen on SDS-PAGE but its quite impure. I have done Western blot which again confirms its presence. I have also done activity assay with the substrate which one more time showed its presence. my problem is its not pure enough to set crystal conditions.
Hi again, is the protein actually His tagged (WB anti Histag)? If not NiNTA is useless...
Perhaps the tag is inaccesable to affinity column but it is odd that WB still works.
A final test would be to unravel it with strong uea maybe 5M and if it does not bind to NiA then it must be cleaved and WB result is wrong.
Finally if you are definately certain the sds band is your protein you will have to do it the hard way, ion exchange and size exclusion. You probably needed one of these after NiA to best purity.
@Miroslav . Thanks Miroslav. Even my next plan was to use 5-8 M urea and see the binding on Ni NTA. Let me perform and see how it works.
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