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Can I label a protein with a single His-tag with Ni-NTA-Atto conjugates? Papers generally use this technique to label 6His-tag.

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  • $\begingroup$ Please don't substantially change your question after it has been answered. The changes you made completely invalidated my answer. If you have a new question, ask it. $\endgroup$
    – MattDMo
    Jun 23, 2021 at 17:51

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According to the unreferenced introduction to Wikipedia's article on poly-histidine tags, the number of His residues can vary from two to ten, with six being the most common. Since histidine is a naturally-occurring amino acid in many (most?) proteins, adding just a single one at either end likely won't do much of anything. In my work with His-Tags (20-something years ago), I seem to remember that 3-4XHis was about the minimum that produced any kind of differential binding in the purification system we were using (Ni2+-agarose, elution with imidazole). However, for our purposes (scaling up protein purification), the quality of the end product wasn't great, and there was a lot of our target protein in the flow-through, so we ended up going with either five or six His residues, I forget which one.

As always, your mileage may vary, so I strongly suggest testing out a range for your application, if for some reason using the full 6X-His isn't an option.

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  • $\begingroup$ Thank you for your answer. I am doing single molecule and I want to count how many 6His Tags I have in my molecular construct. Do you think I will have one or multiple binding of Ni-NTA-Atto conjugates by 6His Tag ? $\endgroup$ Jun 23, 2021 at 15:55
  • $\begingroup$ If you have a standard, and are able to reproducibly load exactly the same amount of protein in each lane, you should be able to. I haven't used this particular system before, so it'll be interesting to see how it turns out. $\endgroup$
    – MattDMo
    Jun 23, 2021 at 17:33

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