topoisomerase problem - (Dec/08/2009 )

I have got some problems with topoisomerase IA activity assay.

I observe to many nicked form of plasmid with higher amount of enzyme.. Concentration of plasmid is constant. From left to right topoisomerase concentration elevates. Topoisomerase is purified with NiNTA resin followed by Sephadex G200 chromatography.

I see both topoisomers and supercoiled form, but i dont know why nicked form elevates too. I suppose it could be some kind of DNase or other nuclease contamination but i dont see nothing more than my enzyme on SDS-PAGE gel and after two steps of purification probability of contamination is rather low, isn't it?

Thanks for any suggestion

hi martin,

i saw the gel posted by you regarding the topoisomerase I assay. I felt that there might be nuclease contamination because of which nicked band is increasing with increasing enzyme. The reason why i am thinking that way is that if you see the amount of DNA in the middle lanes compared to the DNA in the last few lanes, the DNA is becoming lesser which is possible only if there is nuclease contamination. It's rather unexpected that after NiNTA and gel filtration u still get nucleases, but it might happen. Try to make the washes for NiNTA more stringent or give a gradient elution with imidazole.

regards

Leelaram