How to get rid of nuclease in protein preparation? - (Dec/04/2006 )
Hi everyone,
I just purified a fusion protein, which is very pure as visualized by SDS-PAGE. Unfortunately, it has nuclease contamination. I have tried Heparin and ionic exchange columns to remove nuclease, neither is successful. As I have to use nuclease-free protein in my assay, could anyone give me some advice? Thanks a lot.
Is this a DNase or RNase nuclease contaminant you're talking about? RNase contamination is pretty horrible, but many DNases have an absolute requirement for divalent cations e.g. Ca2+, Mg2+ for activity, do you have any of these in your assay? If so, would it be possible to omit them?
Can you try gel filtration? You should be able to get some separatio, unless the nuclease is sticking to your protein, (and unless, of course, they are the same size...).
Thanks a lot for the reply. Gel filtration could be a very good idea. I need to try it.
It is RNase contamination. I need Mg2+ in my assay though. Thanks.
It is RNase contamination. I need Mg2+ in my assay though. Thanks.
Standard method for inactivating RNase is to use DEPC, which I think chemically modifies histidine. However, you need to ensure that it does not inactivate your protein. It appear you are using you preparation in a RNA based assay and thats why you have problem with RNAse. You must realise that this is the major problem for working with RNA as copious amounts of it is secreted in you sweat. Consequently you need to work with gloves from the start of you purification. Further, all the material (buffers, matrices and containers) need to be treated with DEPC-water beaofre use. DEPC can then be removed by thermolysis (autoclave the sample)