trouble in his-tagged protein binding with Ni-NTA agarose
Posted 02 May 2013 - 10:22 AM
When I use 100mM NaCl and 50mM Tris in my binding buffer, I get complete elution using 250mM imidazole just 2 times with 1 ml each.
When I use 300mM NaCl, 50mM tris, 15mM beta-mercaptoethanol and 1% tween-20 and tritonX-100 using 300mM imidazole and 6 times 1 ml elution I can still see my bands even in the 6th sample. So I am losing my protein sample.
Any suggestion on this!!
Posted 02 May 2013 - 10:35 AM
I generally elute with 250mM imidazole
Posted 02 May 2013 - 10:42 AM
Even after using 300mM imidazole, I still find my proteins attached to the agarose. I am asking if due to the binding buffer this happens?
Posted 02 May 2013 - 10:47 AM
If you need to store your protein in that binding buffer with the extras, you can elute with the first buffer, and then dialyze with the second.