Contamination with Nucleic Acids

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Thu Feb 17 10:39:37 EST 2000


Carmelo wrote:

> Hello,
>
> How to get rid of nucleic acids which seem to strongly bind to a protein
> we try to get pure. This protein naturally binds nucleic acids.
>
>

Hi Carmelo,
It depends on what you want to do with the protein. If you want to study
its DNA or RNA binding properties, nuclease treatment is not an option,
otherwise it will be a good thing to test.
I´d go for high salt treatment, because protein:DNA interactions are
usually ionic. Salt usually disrupts the bonds between DNA and protein.
As to the purification, I can think of two ways to try:
1. treat with high salt (1-2M NaCl, or harsher, up to 3M MgCl2), then
purify by hydrophobic interaction chromatography (where you can load your
protein to the column with high salt, and elute with decreasing salt) or
hydroxyapatite chromatography (where you can also load at high salt, then
elute with increasing potassium phosphate)
2. precipitate with ammonium sulfate. The high concentration of ammonuim
sulfate needed to bring down the protein will most likely disrupt
interactions with DNA or RNA. Then dissolve protein in buffer of choice and
proceed with purification.

Hope this helps,
Frank








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