inactive enzyme

[piefke]

hi
I am purifying a protein with Ni-NTA-columns. Now my protein shows no activity after the purification. Before purifying the protein it is active. So, I fear that the Imidazole in the washing and elution buffer inhibit my enzyme. Did anybody make similar experiences? And do you have any idea how to get rid of the Imidazole afterwards?

[blcr11]

The easiest way to get rid of imidazole is either to dialyze it away, or dilute the Ni column eluate with buffer and do an ion-exchange run as a next step in the purification. It may also be that your protein doesn't like phosphate buffer, or that metal ions are leaching from the column and your protein doesn't like metals.

[Cat]

How do you know it is your protein that functions in the crude extract? What do you know about your enzyme?

[piefke]

I tried to make an exchange with microcons from Miliopore, but by this way I lost my whole protein...
The protein I´m interested in, is a metalloprotein, so it is most likely that imidazole is the compound that inactivates my protein. When I do an enzyme-assay with the lysate before purifying it, there is activity. After purifying there is no activity.