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Protein precipitated while kept at 4C over night after elution from a Ni-NTA col

NiNTA precipitation dialysis elution

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#1 Blindspot

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Posted 14 January 2013 - 03:17 AM

I hope i chose the right sub for this question.

I have been doing the same protein purification for the past few months, only with the murine for of the protein.

After eluting the protein from my Ni-NTA column (whole process at 4C in the cold room) using a Tris-HCl (pH 8) and imidazole-based buffer in 10ml fractions, i put the fractions on ice and left them in the cold room for the past two days.

Today i wanted to continue and see that the fractions where i predicted the protein to be had big pellets.

Is this reversible? How? I need to dialyse the stuff for the next two days.

What i did right now was dilute the fractions (added 5ml of elution buffer) and left them to shake at room temperature.

#2 HOYAJM

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Posted 14 January 2013 - 07:58 AM

It should be fine. I make sure that I put my proteins through dialysis immediately after I elute them from the column. I have also had this problem when I left proteins in the imidazole overnight. At this point, though you cant really do more than what you already have. The proteins should be OK, but if you can check their activity in any way, that will be the true test.

#3 Blindspot

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Posted 15 January 2013 - 02:03 AM

I put them through the first dialysis over night (also in the cold room) and the precipitate is still there, but it looks "different".
Do you think i should do the dialysis at room temperature? It seemed to improve the solubility, but this might just be some eye-bias.

If i do that, i am afraid to destroy my protein...

#4 HOYAJM

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Posted 15 January 2013 - 11:50 AM

I would not recommend dialysis at room temperature. Even if it does improve solubility I think the protein will be degrading. If solubility continues to be an issue, you may want to look into protein tags that improve solubiltiy (e.g. thioredoxin Trx)

#5 snowchild

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Posted 28 March 2013 - 02:56 PM

I read somewhere (probably qiagen handbook of His-tag protein purification) said imidazole will help protein precipitation. So I suggest dialyze right after you elute proteins.





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