Hi,
I am working on purifying alpha-synuclein. In order to purify it, I first precipitated it in ammonium sulfate (the bacteria was sonicated and one of the steps before the ammonium sulfate was to add streptomycin sulfate to remove DNA) and desalted it. Also, in the SDS-PAGE I can see that I have only one band in the right MW.
But, in the UV spectra, I can still see absorbance in 260nm, which means I still have DNA contaminants.
How can I get rid of the DNA? I know for example that using PEI is not useful for this stage as it is supposed to be added to the lysate after the sonication. Can ethanol precipitation help here or it'll precipitate the protein as well as the DNA?
DNA precipitates in ethanol so that's not going to help. Have you tried treating with DNase enzyme?
Aromatic side-groups also absorb at 260 nm, got any tryptophan, tyrosine, or phenylalanine in that protein?
Katie A S Burnette Thank you for the answer!
Actually I would like the DNA to precipitate because then I'll be able to remove it by centrifugation. I didn't want to use DNase because then I would have to remove it as well to purify my protein, and this might be even more complicated.
About the aromatic side groups - the protein does have tyrosine and phenylalanine, but the absorbance I see is different than what my lab members and I usually get for this protein. So that's why I think it comes from DNA.
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