Hey Praveen There are wonderful advices in all the emails. Having over 8 years experience in protein purification, i still admire the way Antonio Ariza has summarized a work flow. You shall need to devise a strategy to optimzie the purification protocol. I could not resist to ask, why are not using any reducing agent in the eluted protein. is there no cysteine in the protein? Once i had an observation where i always saw the white precipitate in the eluted fractions, turned out later that protein concentration was too high in that particular fraction. Simply pooling all the fractions and keeping protein concentration below 3 mg/ml solved the issue.
Best wishes -Z Zaigham Mahmood Khan, PhD Fox Chase Cancer Center ICR R428 333 Cottman Avenue Philadelphia, 19111 PA Phone: 215 728 3609 On Sat, Dec 24, 2016 at 2:55 PM, Kevin Jin <kevin...@gmail.com> wrote: > > > Likely, the protein is pH sensitive. > > You may chain the anion and cation columns together for protein > separation, then you don't need to use Imidazole. > > P.S. Tris pH 7.5 (RT) is ~ pH 8.2 in cold room > > > > On Sat, Dec 24, 2016 at 2:52 AM, Praveen Tripathi < > tripathipraveen2...@gmail.com> 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 >> > > > >
