I'd agree with Pascal as well, and can add that if you have a protein
for which you use an affinity tag, often high salt (>500mM) will strip
the DNA off your protein, and might even keep it stable.
Cheers -
Miles
On Aug 10, 2009, at 3:59 PM, Pascal Egea wrote:
Hi Neeraj,
An absorption spectra between 220 and 400 nm (for example) should
show you if there is DNA coming along with your protein. In theory
A280 is about 1.7 times A260 for a pure protein sample. This is a
rough estimate. If your peak is shifted towards 260 instead of 280
then you can suspect the presence of contaminating DNA or RNA.
As to get rid of the DNA, I can suggest several ways to do it.
1-An heparin affinity column could out-compete your contaminating
DNA while binding your protein. They work really well.
2-Ion exchange is worth trying. It has worked for me with an
extremely basic archeal RNA binding protein purified in E.coli and
bringing along some endogenous RNA/DNA. An ion exchange ( Mono-S
type) got rid of the contaminating nucleic acid, however with some
loss of protein.
3-DNAse treatment may be worth trying but the presence of traces of
DNase may be a problem with subsequent crystallization trials in
presence of the bona-fide target DNA sequence.
4-Precipitation of the DNA with streptomycine sulfate. You could
also loss some protein.
1 and 2 are in my opinion the less invasive soutions.
Hope this helps.
Best regards
Pascal Egea
Miles Pufall
Postdoctoral Scholar
Yamamoto Lab
UC San Francisco
Cellular and Molecular Pharmacology
Mail Stop 2280
600 16th Street, Genentech Hall S-574
San Francisco, California 94158-2517
(415)476-4480
