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 <[email protected]> > 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
