I am trying to get a protein extract of A, platensis in order to measure some nuclease activities, but I get a highly acid nucleic contaminated sample. How can I remove all the contamination?.
I already tried with 2% precipitation with strptomicine sulfate, should I ultracentifuge the precipitacion or just a regular centrifugation works?
Thanks inm advance
I always add DNaseI to my lysed cells that I then purify the protein of interest for DNA metabolism characterisation. This can be purchased from any of the big suppliers and just follow their recommended conditions - should remove all that contaminating DNA.
I generally use one of the two methods to get rid of DNA bound to my protein sample.
I am not sure if DNase can get rid of nucleic acid bound to your target protein. If you use high salt to displace the nucleic acid, most DNases are not compatible with high salt, so it may not work. Although I am happy if someone can provide evidence on the contrary.
Yes Arthur you are right DNaseI will not degrade DNA that is bound by proteins, but it will clean up the unbound DNA in the sample sample. When I have gone on to purify proteins I have always included a heparin column in the purification process of DNA interacting proteins.
Another useful step is an Ammonium Sulphate cut that "salts out" proteins dependent on their solubility.
Simon: adding DNAse to an extract in which you want to assay for DNAses seems odd. How will you specifically eliminate DNaseI without affecting the platensis DNAse?
Amaya, et al.,: high salt + protamine SO4 or streptomycin should work to precipitate DNA provided that the salt concentration required for dissociation of the platensis DNAse is known to allow this. But in the past, we have had real problems with this approach as our protein always co-precipitated, even though on DNA cellulose columns we could elute it at about 0.2M NaCl. So DNA cellulose (available as SS or DS from sigma) might be worth trying.
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