I am trying to purify RNase A(from Sigma Aldrich), to get rid of DNase, I have been asked to heat the RNase powder mixed in 0.01M Sodium Acetate(ph = 5.2, 10mg/ml ) at 100oC for 15 min, and then adding 0.1 Volume of 0.1 M Tris-HCl(ph =7.4). I have followed the above procedure.
How do I quantitatively find out that Dnase has been removed considering both DNase and RNase A have extinction co-efficient value at 280 nm using UV Spectrophotmeter ? Any ideas and suggestions would be much helpful.
That procedure is not used for purifying RNAseA, but to heat-inactivate contaminating DNAses. The polypeptide chains responsible for DNAse I activity will still be there, albeit misfolded (unless they have precipitated out of solution).
If you want a quantitative determination of residual DNAse I after inactivation you have to set up the corresponding enzyme assay (such as the one described at http://www.worthington-biochem.com/dnase/assay.html).
Thank you Alejandro for your suggestion, if DNAse denatures, it is bound to aggregate and precipitate. Considering DNAse to be a large molecule, it can be filtered out using 220nm filter. I will also look into the link you have given. Thanks again.
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