Problem with his tag protein purification - (Oct/13/2004 )
Dear all, I am quite fresh in protein studies and therefore looking forward to any of your favorable suggestions.
I am working with a his-tagged protein (~18KDa with tag) which is a protease inhibitor. The protein is quite soluable and can be produced in a big amount after induction. But I got a headache when I tried to purify it under native condition(I need to test the activity). I eluted the protein in the buffers with different imidazole concentrations. It comes out with washing buffer (no imidazole)and keep leaking until 100mM imidazole. It is pretty hard to find a proper point to seperate it from the other proteins.
I have read some of the related posts from this forum, but since I have too few knowledge in this area, I could not deduce anything for my case. So here comes my silly questions:
What is the purpose of increasing salt concentration? May I use DTT? Does it work to purify the protein in a cold room? Etc.
Thank you so much in advance for your help!
I think, you overloaded your column with your protein. First, try reduce amount of protein which you load on the column. Increasing a salt concentration make binding of your protein more difficult. And DTT I would not reccomend to use at all. Make analitical chromatography with reduced amount of total lysate and see what will happend.
NaCl is used to avoid undesired ionic interactions. Tipical concentration is 0.5 M in washing buffer.
I agree with Oleksii, if you overload the column, your protein will elute as soon as possible. Try to charge a lower amount of total protein and run the chromatography.