Could it be due to the fact that the protein is a histidine kinase - perhaps it is interacting with or even modifying its own his-tag?
Mark J van Raaij Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC calle Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://wwwuser.cnb.csic.es/~mjvanraaij > On 30 Mar 2017, at 21:20, Shubhangi Agarwal <shubhangiagarwal2...@gmail.com> > wrote: > > Hello > > I have a 10kda histidine kinase domain protein with a pI of 9.5. > It has a C-term his tag and despite using different buffers the protein > doesnt bind to the nickel cloumn. it comes out in the flow trhough. > Buffers used- 50mM tris Ph=8, 300mM NaCl > 50mM tris Ph=7, 300mM NaCl > 50mM hepes Ph=7.5, 300mM NaCl > 50mM tris Ph=8, 300mM NaCl, 10% glycerol > Can someone suggest to get ensure binding of the protein to the nickel-nta > column > > Shubhangi > PhD student > under Dr. Jhimli Dasgupta > St. Xavier's College > Kolkata > India
