My protein of interest has 6His tag. When it is bound to Ni beads it cannot be eluted with imidazole.
PI of my protein is 7.04
Lysis buffer pH is 7.4
I used up to 1M imidazole in the imidazole elution and reduced the pH up to 4 when I am trying to elute with pH gradient.
Then I tried stripe the Ni with EDTA too. Non of them didn't work.
Can anyone please explain what needs to be done to get my protein out of the beads?
It might have been degraded by proteases and so is not there anymore. Alternatively, it may have precipitated into an insoluble mass. You might be able to retrieve it by boiling the beads in SDS, or by soaking them in 6M guanidine HCl, but your protein will be completely denatured.
Most likely, your protein didnt bind to the column at all or as adam said it is degraded. Did you check your elute, and wash solution and quantified them ?
To troubleshoot Ni-NTA purification problems, always check an aliquot of the initial sample, the flow-through, wash, eluate, and high-imidazol column cleaning step as well as a small aliquot of the column material after elution on an SDS gel.
This allows you to track where your protein ended up.
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