From: Theresa H. Hsu
Date: 15 January 2012 18:23
Hi all
I have a His-tagged soluble protein (8 His residues added to 90 kDa protein) that do not bind to IMAC column based on flowthrough showing up with Western blott. Do you have suggestions to improve the binding?
Binding condition is 50 mM Tris-HCl 8.0, 300 mM NaCl, 10 mM imidazole pH to 8.0.
Thank you.
Theresa
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From: Edward A. Berry
Maybe the His Tag is blocked by the folded protein.
Try using 6M Guanidine-HCL and see if it sticks.
Then you will need to find some way of refolding your protein,
if you want to crystallize it.
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From: Artem Evdokimov
Your protein is either misfolded/aggregated or the his tag is chewed off/never translated. You could try detergents etc. To improve the state of the protein but I would also doubl check sequence for early termination (assuming cterm) or protelysis.
Artem
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From: Cécile Breyton
I addition to the suggestions of checking folding/aggregation/proteolysis, you might also want to try lowering the NaCl concentration. Whereas I like having some to prevent ion exchange effects on the IMAC column, I have had the case of a protein that does not bind if NaCl is present. Binds perfectly well without, is eluted fine with 200 mM Imidazol. This protein does not bind at low Imidazol concentration either by the way.
hth,
Cécile
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From: Artem Evdokimov
This likely means that your imac column is acting as an ion exchanger :-)
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From: Xiaodi Yu
Hi Theresa:
If you can make sure that your target protein is expressed.
You can first use 6 M urea to denature the protein and then try to bind it to the column. If the denatured protein can bind to the column, it seems the histag is hided inside of the protein. It is not exposed enough to interact with the column. In this case, you can design a new construct, for example, put the tag to the other end of the sequence, or introduce a flexible linker between the tag and the protein. Another thing you can try is using Cu ion instead of Ni ion. It will fasten the binding.
Good luck
Yu Xiaodi
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From: Jodie Johnston
Hi Theresa,
I am right in thinking the protein construct you are using is just 8 His residues added to 90kDa ?
It could be (if this is the case) that the tag is not accessible for binding
to the Ni column. Sometimes you need a linker sequence between the protein for the
His-tag to coordinate well to the Ni2+.
Hope that helps
Cheers
Jodie
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From: Theresa H. Hsu
Can I know what is difference in binding chemistry of Ni, Cu, Co and Fe? Is there specific rule for binding affinity versus purity?
>Good luck
>Yu Xiaodi
>
>
Theresa
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From: Artem Evdokimov
Typically affinity goes down in order cu ni co zn mg
Artem
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From: Tim Gruene
Dear Theresa,
you might also try changing the resin. I have worked on a protein which
would no bind to Ni-NTA (Qiagen) at all but could greatly be purified
when using Ni-IDA (then Pharmacia) instead.
The protein expressed to about 60mg/l LB in E. coli and was well folded.
Best wishes,
Tim
- --
- --
Dr Tim Gruene
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From: Gregory T Costakes
Hi Theresa,
You could try lowering the pH down to 7.4 or 7. I have found that some proteins bind very poorly at pH8 and higher. Hope this helps. Cheers!
From: Metlitzky, Moritz, Dr.
Hi Theresa,
did you try to change the position of the His-Tag?
Perhaps it is not exposed in your current construct and would work better at the N- or C-terminus.
Greetings
Moritz Metlitzky,
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From: Luca Jovine
…alternatively, depending on what is the source of your protein, you might have to dialyze the (concentrated) sample before applying it to IMAC. Insect cell media, in particular, can be pretty good at stripping off Ni2+ from IMAC supports.
HTH, Luca
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