When purifying my his-tagged protein much of it was eluting at only 50mM imidazole. The protein was binding to the charged beads as shown by samples from the beads and supernatant after incubation with the cell samples.
My protein consisted of 2 subunits of pI 4.6 and 7.8 and it's been suggested to me that the contrast here could be responsible but I can't find support for this in any literature so am struggling to understand. Is this true and could anybody shed some light as to why?
The most likely reasons that a His-tagged protein does not bind to a Ni column are: (1) the protein is highly aggregated, (2) the His tag is inaccessible, (3) the His tag is absent due to proteolysis. I don't think the pI of the subunits is an issue for binding to the column. The pH should be buffered within the normal working range of the column.
The most likely reasons that a His-tagged protein does not bind to a Ni column are: (1) the protein is highly aggregated, (2) the His tag is inaccessible, (3) the His tag is absent due to proteolysis. I don't think the pI of the subunits is an issue for binding to the column. The pH should be buffered within the normal working range of the column.
Completely agree with Adam's answer.
Elution at 50 mM imidazole is also not that unusual - depending on the nature of the protein. For a large protein (complex) where there will be only one His-tag connecting it to the column, it is quite reasonable that it might elute at this concentration without needing to invoke any other effects. High concentrations of imidazole are usually required for elution when a multimer with multiple His-tags is binding (e.g. a homotetramer where each protomer is HIs-tagged). There is then a very strong avidity effect that drives the tighter binding.
If your protein is binding to the beads as expected, allowing you to separate it from the bulk of cellular components, then your experiment is fine. If the protein elutes at a low concentration of imidazole, don't worry about it unless this presents you with a problem: high imidazole concentrations can denature proteins, and so your sample will be spared this too!
Thank you both so much for your replies. I did manage to get enough pure protein to use but was baffled why it was coming off so soon!
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