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Problem with his tag protein purification


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#1 lcuby

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Posted 13 October 2004 - 05:46 AM

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!

#2 Oleksii

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Posted 15 October 2004 - 06:47 AM

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.

Alexei

#3 celvas

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Posted 15 October 2004 - 07:18 AM

Hi,

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.

Good luck




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