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no binding to Ni-NTA coloumn

no binding to Ni-NTA coloumn - Biology Forum

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Old 08-22-2012, 04:13 PM
Pipette Filler
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Default no binding to Ni-NTA coloumn



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,
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Old 09-18-2012, 05:06 AM
Pipette Filler
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Default Re: no binding to Ni-NTA coloumn

are you sure your resin is charged with Ni? It will fall off the column over time if this is an oldish column.
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binding , coloumn , excellent solubility , his tag , ninta , no binding


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