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Which buffer to use?


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#1 SStfter

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Posted 29 March 2009 - 07:23 PM

Hey guys,

I've been trying to express and purify this protein for quite a while now and it looks like I finally found an expression system that gives me enough protein to use IF I could purify it. Some details of the protein:

~20kda
pI of 8 (with 6xhis tag, ~9.2 without)
high % of Gln and Leu (11 and 12% respectively)

I'm expressing in Hi5 insect cells and everything is fine until I try to dialyse after IMAC to remove imidazole. The protein precipitates no matter what. I've tried buffers mostly around pH 7.0 so maybe that's too close to the pI?

I have a few questions about dialysis/buffer exchange:

1. I load my sample onto Ni resin in 20mM Hepes, 150mM NaCl pH7. Is it detrimental to elute with let's say pH 5.5 Na-Cit?
2. I tried dialysing my insect culture sup directly into 20mM NaCit ph5.5 and got massive precipitation. What else can I try?
3. What buffers are people on here using for proteins with a pI of around 8?

Thanks a lot

#2 MaggieRoara

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Posted 29 March 2009 - 09:26 PM

why dont you try changing the pH to elute the protein insteas of using imadazole?
What system are you using for IMAC

Edited by MaggieRoara, 29 March 2009 - 09:30 PM.


#3 SStfter

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Posted 29 March 2009 - 09:47 PM

why dont you try changing the pH to elute the protein insteas of using imadazole?
What system are you using for IMAC


Could you tell me more about eluting with pH?

I'm using GE Ni Sepharose 6 Fast Flow.

#4 MaggieRoara

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Posted 29 March 2009 - 11:53 PM

It really depends on your protein of interest. My logic is like this; in more acidic environments, negative ions will compete with your protein for afiinity to the column. Lower your Elution buffer and binding buffer pH and see how it goes. Also trying lowering the amount of imadazole. Try combinations of lowered pHs and imadizole concetrations. U might not need to omit imadazole fully. In the correct acidic pH, you might be able to dialyze out the imadazole without precipitation of the protein.

Good luck and do keep me in the loop

Edited by MaggieRoara, 29 March 2009 - 11:54 PM.





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