contamination of DNA in protein sample - (Apr/09/2008 )
I find that my protein has abnormally high OD 260nm reading. Usually 260/280 nearly 1.5. I checked my protein sample on DNA gel electrophoresis and found that there are high molecular weight DNA contamination.
Since my protein is refolded from inclusion bodies so I suggested that the DNAs are genomic DNA which are heavy to be pelleted down with inclusion bodies during preparation.
So I did a DNaseI treatment in the initial cell lysis step. Adding 20ug/ml DNaseI after cells(e.coli) lysed by sonication and incubate for 1hr at 37'C. I took time points and run the samples on agarose gel. I found that only plasmid size(3-10kDa) DNA are digested. I can still see a bright band on the top of the gel and nearly identical to DNaseI untreated sample. For samples without lysed cells, such high molecular weight DNA does not exist.
My questions is: how can I get rid of the high molecular weight DNA during my preparation so that I can get pure protein sample without nucleic acid contamination?
Add between 0.5% and 1% (v/v) of 10% polyethyleneimine (PEI) to your protein solution.
Stir on ice for 30mins
Centrifuge at 8k or 10k rpm for 30mins, @ 4 degrees if necessary
Pellet = DNA
Supernatant = protein
I make up 10% PEI in tris-Cl pH 8.8
Works 4 me