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His-Tag protein purification:protein eluted with NaOH???


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#1 gabri

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Posted 01 February 2011 - 05:21 AM

Hi everybody!
I am new in the forum...:-D

I am trying to purify a protein of 58 KDa and PI 5,40 with a N-HisTag.
(I expressed my protein in E.coli BL21).

I use the HisTrapHP column from GE Healthcare and buffers at pH 8.

I did obtain clear protein but very less concentrated.
So I though to collect the fraction when at the end I clean the column with NaOH 1M..
And guess what?? My protein is in that fraction (togehter with others protein)..and the band is so fat....

I already increase the Imidazol concentration up to 500 mM...But still my protein is trapped in the column...

Any suggestion??

Thanks...

Gabri

#2 mole

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Posted 01 February 2011 - 06:19 AM

Hello,

Its not common But many protein sticks to the column and can not be eluted with increasing concentration of Imidazolium.

Best option i would like to recommend is Try mannual purification with Ni NTA resin from Novagen or try Ion exchange.

So if you have happy with the protein coming out from column after all sticking then its ok otherwise you have to look other options only.

Regards,

Mole .-

#3 Sunnyside

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Posted 01 February 2011 - 07:36 AM

We use 500 mM Ammonium Hydroxide pH 11.0 for elution from NiNTA columns
It pulls everything off the column.
Ammonium hydroxide is volatile, so I usually do a couple of rounds of "add water, half dry in speed vac" to get rid of it. Or a TCA precipitation also works.
S

#4 gabri

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Posted 02 February 2011 - 12:15 AM

Hi!! Thanks for the answers..
I already tried manual purification with NiNTA but it is not good because I got a lot of protein but it is not pure....:-(

Sunnyside, when you use 500 mM ammonium Hydroxide to eluate, do you have a lot of unspecific protein also?? Do you do a gradient? Do you first use imidazole and then Ammonium?

Thanks a lot!
G




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