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
