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Protein contamination with DNA

Protein DNA contami

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#1 milojet

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Posted 14 October 2011 - 11:24 AM

Dear all! Have to deal with a strong DNA-binnding protein and cannot get rid of DNA-contamination of protein prep. DNase-digestion is not helpful, 260/280 ratio is still around 2, and moreover DNAse treatment is not desirable due-to further DNA-binding activity assays. After Ni-NTA purification protein solution was equilibrated with 2M NaCl and applied to anion-exchanger (Resource Q), that didn't help, all the DNA coeluted with protein. Would appreciate any help, suggestions, comments!!!Posted Image

#2 bob1

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Posted 15 October 2011 - 01:29 PM

Have you tried sonication?

#3 JHFBio

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Posted 15 October 2011 - 04:18 PM

can you try to lyse cell in higher salt concentration as lysis buffer if your protein wouldn't precipitate in high salt buffer? High salt buffer may help to separate DNA from protein.
Cheers.

#4 milojet

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Posted 15 October 2011 - 10:34 PM

Thank you guys, to bob1: normally, we lyse our cells with french press, but on early stages we used sonication as well, which gave us still high 260/280 ratio. Do you think sonication ruins nuclein acids more, that french press?

To JHFBio: I though about this, up to 2 M NaCl I could add in lysis buffer. I also heard about Nuclein Acids precipitating agent (streptomycine sulfate, protamine etc.) to be added in lysis buffer, but I do not have an experience with all this stuff yet

What bothers me is that when I try Ethidium Bromide staining of my gel with this protein, I see nothing at all, what else could give 260/280 ration around 2???Posted Image

#5 bob1

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Posted 17 October 2011 - 03:48 PM

There are all sorts of things that could give you a 260/280 ratio of two. Are the samples aggregating or forming a sticky mass that plugs pipette tips? If not, then you probably don't actually have a problem with DNA. Sonication will break up DNA usually, whereas a french press doesn't (I think). You can also pass the solution through a narrow gague needle several times, which should break DNA strands.

As you are running a Ni-NTA system, I take it you aren't denaturing your protein?

#6 milojet

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Posted 18 October 2011 - 12:50 AM

There are all sorts of things that could give you a 260/280 ratio of two. Are the samples aggregating or forming a sticky mass that plugs pipette tips? If not, then you probably don't actually have a problem with DNA. Sonication will break up DNA usually, whereas a french press doesn't (I think). You can also pass the solution through a narrow gague needle several times, which should break DNA strands.

As you are running a Ni-NTA system, I take it you aren't denaturing your protein?



Yes, it is Ni-NTA, non denaturating conditions, I don't observe any stickyness, not the lysate, neither the protein solution after Ni-NTA. Protein sequence is saturated with His and Phe, which absorb around 260 nm. And it is a metall binding protein, Zink and probaly iron (may be some other instead of iron), what can also theoretcally shift 280 nm peak. I will try do put it through size exclusion columne and than to stain protein gel with Ethidium Bromide, if i do not see anythig, means, there is no DNA bound. Somehow, ratio 260/280 is not a criterium in this particular case.




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