nuclease contamination in protein prep - (Jun/28/2011 )

Hi all,

I am having trouble with nuclease contamination in my protein prep. I need to get rid of this, cos I intend to use the RNA binding protein prep for RNA degradation studies.
Pls suggest some ways to do this.
I use a 4 step purification procedure that includes HIs-purification, an anion exchange step, a FLAG purification step and a final gel exclusion step.
None of these steps have helped me get rid of the nucleases.
Suggestions required..

~Bump~

Maybe a contamination on your size exclusion column?

Do you add DNase to the breakage buffer?

@Gaai: I am not too sure, but I do an activity assay after every step and it seems to present throughout..

@papaver: No I do not add Dnase to the lysis buffer

But Thanks for the 2 replies after so loong..i am desperate for a solution..been working on this for too long...