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My protein doesn't bind to the Ni-NTA column - (Nov/07/2012 )

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Hi everyone, I need your help to solve the problem with the purification of my His-tagged protein.
I'm trying to purify a small protein (16 kDa) that has 6XHis tag at the C-terminus by the Ni-NTA column of QIAgen. This is membrane protein but highly soluble and expressed well in E. coli BL21(DE3). The pI of this protein is 6.4
I need to purify this protein under native condition.
The protocol that I used:
- Lyse the cell by sonication in the Lysis buffer (TE buffer: 10 mM Tris HCl, 1 mM EDTA, pH7.8), centrifuge down the cell debris
- Equilibrate the Ni-NTA column with Buffer 1 (50 mM Imidazole, 300 mM NaCl, 50 mM Tris-Cl, pH7.8)
- Load the cell lysate twice at room temperature, collect the unbound fraction
- Wash with Buffer 1
- Elute with Buffer 2 (250 mM Imidazole, 300 mM NaCl, 50 mM Tris-Cl, pH7.8)
- Wash with Buffer 3 (350 mM Imidazole, 300 mM NaCl, 50 mM Tris-Cl, pH7.8)

After checking on the SDS-PAGE, most of my protein showed in the unbound fraction, along with other proteins, and the rest showed in the first washing fraction (also with a lot of E. coli proteins that bound unspecifically to the column). Nothing was eluted from the column with neither Buffer 2 nor Buffer 3.
I cloned this gene again with 8XHis tag but the situation didn't change.
Now I guess that probably the pH of the buffer, that is much higher than the pI of this protein, may be the problem, or the worst case is this His tag can't be exposed. This protocol worked well with other 6XHis-tagged proteins (pI of 7.8 to 8.4) and they can be eluted with Buffer 2.
I want to reduce the pH of these buffers to pH6.2 but since the pI of the His tag is 7.2, could it cause problem with the binding?
Any suggestion from you is highly appreciated, thank you so much in advance.

-Qundo12-

50mM imidazole is fairly high to start with as the equilibration or wash buffer. I would try 10mM or even no imidazole when loading your sample, then when washing you can slowly increase the amount of imidazole to remove any non-specific binding before your protein should elute.

-PhDinAcronyms-

Oh and I just saw that you used EDTA in your lysis buffer- EDTA will chelate and remove the nickel from your column so nothing will bind. Is there any particular reason you added EDTA?

-PhDinAcronyms-

Oops, thank you for your suggestion, first I will repeat it without using EDTA, I used it just as a habit but it didn't cause any problem with other proteins so I didn't notice it.

-Qundo12-

it probably isn't necessary to say but... don't forget to recharge your ni-nta with nickel before you try loading your sample.

-mdfenko-

The same problem bothering me in the process of protein purification! So i nieed your help!

My fushion protein, 44KDa, with 7x his-tag in upstream, was extracted from BL-21(DE3) Eco.li, then was purified by Roche cOmplete His-Tag Purification Resin (Catalog Number: 05 893 682 001). The protocol are as follows:

- Lyse the bacterium by sonication in Buffer A ( 50 mM NaH2PO4, 300 mM NaCl, pH8.0), centrifuge down the cell debris
- Equilibrate the Ni-NTA column with Buffer A
- Load the cell lysate twice at 4°, collect the unbound fraction
- Wash with Buffer A
- Elute with Buffer B (5--250 mM Imidazole,  50 mM NaH2PO4, 300 mM NaCl, pH8.0)

The radiant  concentration of imidazole in Buffer B is 5mM, 25mM, 50mM, 100mM,150mM, 250mM.

The results was validated by SDS-PAGE, showed that the amount of protein that binding to the resin is low and hybridprotein was not washed away. And the figures were presented in attach files.

It wound be appreciated no matter what suggestion is.
 


Attached Image

Attached Image

-yingquliang-

Could you provide some more details?  Is the big blob in lane 10 your starting material (lysate)?  Have you analyzed your flow thru?  How big of a culture is your lysate from?

-Missle-

was the column fresh or used? if used was it regenerated?

 

was the protein of interest eluted in the buffer a wash? if so then you should ensure that you are not overloading the column. if not then you should slow the flow rate when loading and/or increase the number of times you reload the sample.

 

do you ensure that the wash with buffer a is complete? you want to avoid carry-over.

 

your protein apparently elutes at a relatively low imidazole concentration. you may want to elute with just one high concentration.

-mdfenko-

was the column fresh or used? if used was it regenerated?

 

was the protein of interest eluted in the buffer a wash? if so then you should ensure that you are not overloading the column. if not then you should slow the flow rate when loading and/or increase the number of times you reload the sample.

 

do you ensure that the wash with buffer a is complete? you want to avoid carry-over.

 

your protein apparently elutes at a relatively low imidazole concentration. you may want to elute with just one high concentration.

first, thank you for your attention! the column was fresh, and the flow rate is below 0.5ml/min, the loading samples was 5ml, come from 500ml culture. before elutate, enough wash was done(by 20ml buffer A)

-yingquliang-

Could you provide some more details?  Is the big blob in lane 10 your starting material (lysate)?  Have you analyzed your flow thru?  How big of a culture is your lysate from?

first, thank you for your attention. the big blob in lane 10 is input lystate (before washing). the loading samples is 5ml, come from 500ml culture. and the flow rate is below 0.5ml/min

-yingquliang-
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