Hi,
1. I have a His-tagged protein and intend to purify it using Ni-NTA resin (in e-tube, not spin column) and I plan to purify it under native condition but also want to purify it under denaturing condition as a control.
However, when I read the protocol providing by Qiagen (the attach file, page 31), the denaturing washing buffer which containing Urea has pH of 6.3, lower than the pI of Histidine (around 7.6). Isn't this acidic buffer will make the His-tagged protein detach from resin and eluted out in the washing buffer?
2. And because after the purification, I intend to conduct another reaction (reaction A) with this His-tagged protein, so I don't elute it out but keep doing my reaction on the resin-linked His-tagged protein. However, this reaction A is not compatible with Cl ion while NaCl is needed in the previous purification step to prevent non-specific binding protein.
So, is the elimination of NaCl in the washing buffer possible? Or would you please suggest a better protocol?
Thank you very much.
There will be some more proteins eluting from the resin with washes at pH 6.3, which is probably the goal of the protocol. This is similar to using small amounts of imidazole (typically 25-50mM) in washes. The real bulk protein elution usually occurs at imidazole concentrations > 150mM and pH
Thank you very much Edward,
I have to ask this question because the amount of my sample is very little (0.5mL) and the concentration of my total protein is also low (1mg/ml) so I have to really careful in every step or I will lose all :)
I will try your suggestion. Thanks again.
Hi there,
from Quiagen ninta handbook, efficient elution of his tagged protein occurs at pH 5.9 or lower (pH 4.5 for strong interactions). Washing at pH 6.3 is just eluting weak bound protein. And yes you can use NiNTA column for buffer exchange as long as the buffer exchange doesn't interfere with protein-matrix interaction.
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