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I´m trying to purify a his-tagged protein with nickel-columns.
When I use imidazol in the elution buffer my enzyme is inactive after purification. I showed that imidazol is the reason for this loss of function. Because before purification I can clearly detect activity. When I use the same assay buffer with same components and pH, but with imidazole, there is no activity detectable any more.
So my idea is now to elute the protein by decreasing pH-values or to elute the protein with EDTA.
Has anybody any experiences with the elution of protein from nickel-collumns in this ways?
I fear that my enzyme will denaturate when I decrease the pH. Or is it possible that the pH has not to be decreased as much?
After elution with EDTA it must be possible to get rid of the EDTA with dialysis, right?
Thanks for comments or ideas!!
Ni columns have been eluted by either low pH or with EDTA. I have no direct experience with it, but it has been known to work.
When you assayed your protein in the presence of imidazole you checked to make sure the pH was what you thought it was after adding imidazole, I presume. Imidazole, especially 250 mM, can change the pH of your buffer. Imidazole can also carry in contaminants like zinc. If you have a metalloenzyme inhibited by zinc, that could contribute to loss of activity in the presence of imidazole which wouldn't necessarily be due to imidazole. You can't dialyze away the imidazole and restore activity?
Your protein is inactive due to the high concentration of imidazole. Try to use a cobalt based resin and that should do the trick. You will extract and wash in absence of imidazole and then purify in low concentration s of imidazole.
1、the high concentration of imidazole can change the pH of your buffer.
2、It may be the toxic effect of nickel ions and you can use a non-toxic Ni column. when protein is eluted, nickel ions is still remained in solution to maintain recombinant protein production activity to be best to look at the dialysis concentrate.
3、Low temperature operation throughout the process.
4、Preservation with low temperature, Aliquoting with small bottles
5、Avoid repeated freezing and thawing
Don't choose high concentration of imidazole, just at the concentration you could wash the protein down. High temp. could also lead to activity losing, so keep the process at low temp.
Genius only means hard-working all one's life.
CreativeBiomart, Recombinant protein expert
5 posts • Page 1 of 1
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