My protein is not purifying well showing 2-3 bands other than my protein. I have tried many things variable pH, salt concentration, imidazole concentration but still problem is same. What should I do?
You should provide more details. The question is too general for a meaningful answer.
You should provide more details. The question is too general for a meaningful answer.
There could be a number of reasons. One obvious reason is proteolysis during the course of purification. The other reason could be if your protein binds to some other proteins in the cell lysate and elutes out as a complex from the column. The bands then correspond to the individual proteins. Another possible reason could if the protein has post translational modifications, such as, glycosylation. Such protein products are not homogenous.....they usually have multiple bands corresponding to the different glycosylated states. Another possibility is that if you are expressing your protein with N-terminal His tag, due to incomplete translation (can happen if you try expressing eukaryotic proteins in E. coli) there will be truncated products along with the full length of the protein. If you use affinity purification, all these products will bind to the resin and elute out at the same time showing up as multiple bands......having a C-terminal His tag can be helpful in such cases as only the full length protein construct will have the His tag and not the truncated versions. Good luck !!!
It have N-terminal His-tag and I am expressing it in E. coli, its a full length protein not truncated, and as you said that it may be interacting with other E. coli proteins, I have tried high salt concentration in my buffers. I think my protein is degrading so I have used glycerol and EDTA in my buffers but still result is same.
Hi,
by truncation i mean that even if you are trying to express the full length protein, it can so happen due to incomplete translation that truncated forms of the protein are made.......so you will end up with different lengths of the protein each with the N-terminal His tag...when you try to purify, each one will bind to the affinity resin and elute....and will be seen as multiple bands in the gel.
If you think there is degradation due to proteolysis, adding protease inhibitor might be useful. Also, working at 4 deg. conditions will be a good idea if your protein is unstable and susceptible to unfolding and therefore proteolysis. Good luck !!!
Have you analyzed by mass spectrometry the other protein than your protein?
It is not clear whether you have run SDS-PAGE of crude or not. have you tried a native gel too? this may clarify if any problem is there in the purification process. some times more than one protein may co-purified...you may have to check this too.
Hi. If you only did His tag purification (niquel affinity) it is quite likely that you get more than one band, probably due to interactions with other proteins. You could try another step of purification (anion exchage, gel filtration, etc) in order to reach higher purity.
It is also a good idea to check identity of the other bands to discard that it is your protein wich has undergone proteolysis or some post translational processing
Maria Ines Gimenez
After Ni-NTA purification I have done anion exchange chromatography and size exclusion chromatography and still it is showing 2-3 bands.....
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