This short paper in Nature Methods is probably worth a read and may
help with your Ni-NTA issues:
http://www.nature.com/nmeth/journal/v6/n7/full/nmeth0709-477.html
Nature Methods 6, 477 - 478 (2009)
doi:10.1038/nmeth0709-477
Enabling IMAC purification of low abundance recombinant proteins from
E. coli lysates
In short, they detail that removing E. coli periplasm prior to cell
lysis, or spiking lysates with 2 mM FeSO4, may improve purification of
low abundance proteins on IMAC resins - their theory being that
removal or saturation of metallphores (metal chelators) will prevent
Ni2+ leaching from the IMAC resin. If possible avoid using an NaCl
concentration as low as 20 mM, as there will be lots of non-specific
interactions at such a low ionic strength.
Roger
On 26 August 2010 13:24, ganesh pathare <[log in to unmask]> wrote:
>
> Dear all,
>
> I have problems in purifying a protein. The protein is 38,000 daltons and has a N-ter His-Tag. The protein expression levels are low and as a result I have a limit for the purification steps.
> Initially I used NiNTA columns with 50 mM sodium phosphate buffer pH8, 300 mM NaCl, 20 to 250 mM Immidazole for the affinity purification, but it contains lot of impurities. I varied the salt concentrations out of which I could get optimal results at 20 mM NaCl concentration but still the amount of impurities was more.
> After affinity purifications I used Ion exchange chromatography using MonoQ column (25 mM tris pH 7.5, NaCl 0 to 1M) which could not seperate the protein from the impurities. I also tried using Hydrophobic interaction chromatography (Resource Ether, Phenyl sepharose, Resource Isopropyl) instead of ionexchange chromatography, which resulted in better purification of the protein, but the problem is I get very less protein after this step and there are still two major impurities. The buffer conditions for HIC was (1.5 M ammonium sulphate, 25 mM Phosphate buffer pH 7).
>
>
> I would be very greatful if someone could help me in this concern.
> Thanks in advance.
>
> Regards,
> Ganesh
--
Roger B. Dodd
Cambridge Institute for Medical Research
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Hills Road
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CB2 0XY
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