Elute in batch and remove imidazole immediately :) half the time imidazole is the enemy.
Artem www.harkerbio.com "From gene to sausages." Artem On Dec 24, 2016 6:04 AM, "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 >
