trouble in his-tagged protein binding with Ni-NTA agarose - (May/02/2013 )

Hi

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!!

Thanks.

Bioscientist

Are you just trying to wash your sample? If so, those imidazole concentrations are very high. I usually wash my resin with 10mM imidazole. So lower the imidazole and you won't lose as much.

I generally elute with 250mM imidazole

No I am not trying to wash it. I am trying to elute using 250mM in the first case and 300mM imidazole in the 2nd case.

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?

Oh OK, I was definitely confused with your question. "so i am losing my protein sample" threw me off. It seems that you have it figured out. The higher NaCl, the presence of tween and or/ tritonX-100 are causing this.

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.