if you can figure out what is making your protein precipitate, you could put something in the collection tubes of the NiNTA column to prevent precipitation. For example a bit of concentrated buffer to change the pH, or EDTA. Another thing, you say your protein is pure after NiNTA, but it may still contain some nucleic acids or other impurities not visible on an SDS-PAGE gel (a UV spectrum of the protein might give you more information). I would recommend dialysis and an ion exchange chromatography second purification step. Then you can buffer exchange and concentrate the protein afterwards and hopefully it would not precipitate because the non-protein impurities are removed.
Mark J van Raaij Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij <http://www.cnb.csic.es/~mjvanraaij> > On 24 Dec 2016, at 11:52, 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
