I am working on a cytoplasmic protein(85 KDa) of Mycobacterium tuberculosis which is N and C-terminal His-tagged protein and expressing in it E.coli strain i.e BL21(DE3)Gold. By Ni-NTA column chromatography, I am getting two non-specific bands, one is around 120KDa and one is around 60KDa other than my desired protein. What should i do to remove this?
You could add a second chromatography step using a different property, such as ion exchange chromatography, hydrophobic interaction chromatography or size exclusion chromatography.
Dear Jyoti Rawat
How did you performed the Ni-NTA?
Which buffer did you use and are you pefroming lienar imidazole gradient or step?
You can try ro optimize the washing and elution even if, as Adam suggest, probably is more simple add a second step.
If in the sequence of your clone beetween the His and the GOI, is it present the sequence of a protease, eg TEV, Fatt.Xa you can even perform digestion followed by a 2nd subctactive IMAC. In this case is much probable that you protein will be collected in the flow through of the subctractive imac while the 2 impurities will be retained into the coloumn.
you can find more informations about subctrative IMAC at the following link
https://www.blogger.com/video.g?token=AD6v5dxhCeJUPx5UODBu4Y9hhJZix_l5JZcPb03ZUe7G3qL8GhoPUWZMm6z2wEJJED9OY8gn_ttNXrGNCdVurZ6zkB_oTSCrmRc90-ux7AQAOhaddCkXKREYmgNw87RSh58zQ4uVQVtz
best regards
Manuele
Thank You, Manuele Martinelli. I have been using Tris HCl 50mM, NaCl 300mM, Glycerol 10% and linear gradient of imidazole 20-400mm.
Yes, there is TEV site between His-tag and GOI, but it is present only in one terminal of the protein.
My doubt is whether subtractive IMAC would help in bringing all protein of interest in the flow-through ...
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