Hi Shubhangi, As Edward suggested, you can try with N-terminal His tag. For this you can either clone in N-terminal His tagged based vectors or by site directed insertion of 18 nucleotides coding for 6 His at the N-terminus region.
But, before that, I suggest you to try with *CAPS buffer pH 11.0*. The change in net charge on your protein might improve binding to Ni column. Good luck. Regards, Bhanu On Fri, Mar 31, 2017 at 12:50 AM, 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 > -- B4U