I tried different imidazole to elution the his-tag protein from Ni-NTA but failed. Does anyone encounted this problem before and find a solution ?
Dear Qiaoyou Weng
some questions to undestand better:
what do you mean for different imidazole? Different imidazole concentration? in which buffer?
Are you sure that your protein was binded to per coloumn? Did you tried to strip the metal by EDTA? If yes. Was the protein present in this fraction?
Generally in my experience a buffer containg 250-300mM is enough to elute 6X and 10X his tagger protein from Ni-Nta or nickel sepharose.
In my experience only protein precipitation into the coloumn may rersult in no or very slow elution but it is something not so common that may happen for protein with very low solubility or in case there is some mistake in buffer preparation.
in the following video avaialble on my blog (ProteoCool) you can find some example of errors that may happen in buffer preparation
https://www.blogger.com/video.g?token=AD6v5dzP9DkeYt4nwIFl1pNVQuJw7_vnHlvCcbCTzad5M6URrbtpOEJ9Z5MJ-gTcfR6n06A5fXwQmD5qJtKayesvHrQtqzfE0mhrKxtPpHHuJO0u2FMq9kmvLSz60LTG_RLcHgTUWkY
ciao
Manuele
Have you established that the protein was present in the extract applied to the column in the first place? If so, did you observe that the protein bound to the column, rather than flowing through?
After lysis collects the supernatant and pellet.
add the Ni-NTA beads to the supernatant (Include 10mm imidazole during binding ) and after incubation collect the beads, and go for elution with 200mm -300mm imidazole concentration.
Run one SDS PAGE gel load the samples of uninduced and induced samples, eluted fractions and pellet (you'll get to know if it is in inclusion bodies), and beads (after elution) as well and see whether your protein of interest is eluted or not.
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