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nuclease contamination in protein prep


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4 replies to this topic

#1 sukan

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Posted 28 June 2011 - 01:37 PM

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..

#2 sukan

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Posted 23 July 2011 - 09:25 AM

~Bump~

#3 Gaai

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Posted 10 August 2011 - 07:11 AM

Maybe a contamination on your size exclusion column?

#4 Papaver

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Posted 10 August 2011 - 07:54 AM

Do you add DNase to the breakage buffer?

#5 sukan

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Posted 15 August 2011 - 05:58 PM

@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...




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