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
