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DNA contamination in protein sample


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#1 Margaret

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Posted 10 July 2002 - 06:51 AM

I have a problem with DNA contamination in my protein samples.  My protein (expressed in ecoli)is very small, hydrophillic and negatively charged, ie similar in properties to DNA.  I've done ammonium sulphate precipitation, heat precipitation, anion exchange and gel filtration chromatography, and I still have DNA there.   Adding DNAse before lysing the cells makes no difference, and I just tried adding DNAse to the GF fractions and that didn't work either.  Any ideas?

#2 milanoj

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Posted 10 July 2002 - 09:21 AM

How are you initially lysing the cells? Try something really harsh like french press with DNAse1 and RNase A. Are you sure the nucleic acid contamination IS DNA and not RNA? Is the protein heterologously expressed or are you starting from scratch? Is your protein known to bind DNA? How are you determining the presence of nucleic acid?
By PCR? If so, that's way too sensitive.

#3 Margaret

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Posted 11 July 2002 - 01:00 AM

Hi,

Thanks for the reply.  I'm lysing by sonication.  I've tried adding DNAse and RNase before sonication, but it hasn't made any difference.  I know I have nucleic acid contamination because when I look at the UV absorbance spectra, I have a maximum at 260nm, or at best an A280/A260 ratio of 1.  The 280nm extinction co-efficient for my protein is very small, so I don't think I have a huge amount of nucleic acid contamination, but I want to do spectroscopic studies on the protein which this problem is stopping.  Your idea about it being RNA is good - I had pretty much assumed it was DNA.  Yesterday, I tried adding DNAseI with magnesium to my sample, and it didn't make any difference.  I was going to try a nucleic acid precipitating agent now, but I think I'll try adding RNAse to my sample and see if that makes a difference.  The protein is not thought to be a DNA binding protein and it is not a native E coli protein so I think thats unlikely.  Also, after gel filtration, I did get one fraction out of the four which contained my protein, that was fairly free of nucleic acid, so I'm pretty sure its contamination instead of binding.

Any other ideas?

Thanks





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