I purified an membrane protein ( DDM stablilized ) with a C-terminal 6his-tag.The purification is based on affinity column. However, the purified protein couldn't bind to a Ni-NTA modified Au electrode. I tried to elute the protein from column by lowing the pH of buffer or elute with imidazole and remove imidazole by de-salting column. However neither of them showed an good binding ability to Ni-NTA modified Au electrode. Other soluble proteins work well with the Ni-NTA electrode. I would be grateful if someone could help me with this.
As far as I know it is generally difficult to have a membrane protein binding to a NTA-modified surface. It is important that the protein is properly stabilized. You could also try to spread an appropriate membran including NTA groups at the electrode but properly that will change your system too much.
Dear Andreas Henkel and Xunhai Zheng
The protein was expressed in membrane of E.coli. I collected the membrane fraction by ultra speed centrifugation and re-solubilized in DDM, then purified by Ni-NTA column. The protein shows identical peak in UV-VIS as reported in published paper. Could this indicate that the protein is in soluble state and folded well?
In the case the protein could not bind to Ni-NTA modified electrode, it still can re-bind to the Ni-NTA column which is the most weird part.
Another issue with Ni-NTA is the so-called metal ion transfer (MIT) process.
Modified electrode involve only low loading in Ni-NTA compared to the one in a Ni-NTA column.
Most His-tags are able to partially dissociate the Ni from the surface bound NTA. When the Ni-NTA modified electrode is incubated in a His-tagged protein solution, the His-tag is in large excess compared to the Ni-NTA. As a result the metal is transferred from the NTA to the HIs-tag (MIT).
This is a lesser issue for Ni-NTA column since in this case Ni-NTA is in excess compared to the amount of His-tagged protein.
For electrode modification via NTA-Ni-His-tag, the trick is to adjust the incubation time and enzyme concentration to limit the MIT process.
Some more details can be found in "W. H. Campbell, J. Henig, N. Plumeré, Affinity binding via Zinc(II) for controlled orientation and electrochemistry of Histidine-tagged nitrate reductase in self-assembled monolayers, Bioelectrochemistry, 2013, 93, 46-50."
Best regards,
Nicolas.
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