Hi,
Check pI vs pH and try different pH. Can also try room temp purification.
Try lower salt conc. Is it a DNA-binding protein? Also consider adding a ligand or partner if known. Your 10% glycerol should help to improve solubility but you can also try 15% glycerol, which should also help in storage/freezing.
Can try adding a little detergent/non-ionic detergent.
Consider different tag (N- vs C-term), construct optimization, engineer mutations (happy to elaborate if desired).
Best,
Debanu
> On Dec 24, 2016, at 4:22 PM, Praveen Tripathi <[log in to unmask]> wrote:
>
> Dear all,
> I am graduate student working on a functional protein which i have cloned in pET-28a vector for recombinant protein production in E.coli expression system.
> The expressed protein is purified on Ni-NTA resins with Imidazole gradient. Surprisingly, i am getting distinct visible white precipitate in pure fractions in eluted fractions itself.
> Please suggest how to make it soluble or how to prevent the precipitation.
> On concentrator the precipitate ration is very much increasing. The protein is pure in soluble as well as precipitate.
> Buffer condition- 50mM Tris(7.5), 500mM NaCl, 10% Glycerol. Elution buffer has varying concentration of imidazole varying from 10mM to 300mM.
> Any kind of suggestion will be highly appreciated.
> My project requires structure determination.
>
> Thanks in advance.
>
> Regards
> Praveen
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