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protein with good soulbility but doesnt bind to Ni-NTA coloumn

His-tag protein purification solubility

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4 replies to this topic

#1 shivu11

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Posted 01 September 2012 - 02:57 AM

Hi All,

i have cloned one of the bacterial protein in Pet28c vector with a His-tag at N-terminal which i have confirmed by sequencing.

I have tried overexpressing it using IPTG. and it gives excellent expression and solubilty at 37, 25 and 18 degrees. But when i try purify it using Ni-NTA coloumn, all the protein comes in flowthru and further washing with 40mM immidazole with other impurities and no protein in elution at 250mM immidazole. I have added 50mM tris buffer with 50mM NaCl and 5% glycerol.

Can anyone please suggest what could be done to increase the binding of the protein with coloumn.

thanks in advance,

#2 Papaver

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Posted 01 September 2012 - 04:23 AM

The problem might be that the His tag is hidden by protein folding so no binding occurs. You can try purify your protein under denaturing conditions.

Or, try to tag it C-terminally, it might bind better.

#3 metionina

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Posted 05 September 2012 - 01:57 PM

Yes, probably the tag is hidden by the protein. You can try a C-terminal tag or a longer N-terminal His-tag. How long is your tag now?
Maybe you can try also to lower your imidazole concentration (~20 mM) during the wash step.

#4 Missle

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Posted 10 September 2012 - 10:04 AM

Hi. What is in your binding/sample buffer?

#5 shivu11

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Posted 04 January 2013 - 05:51 AM

hi, thanks to the reply. Sorry I cudnt reply sooner. I tried using 1M NaCl in the binding buffer and increased incubation of beads with supernatant for 1hr. It helped binding. Now, almost all of the protein gets bind to the coloumn.
Thanks again to all, for their suggestion !!





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