removing DNA

Silke Beismann sbeisma at uni-molgen.gwdg.de
Mon Jan 4 12:25:38 EST 1999




> Achim, to your remarks:
>
> Just a stupid question.
> How do you know from your spectrum that there is DNA present/bound?
> I've worked with DNA-binding proteins and never seen a 'nucleic acid
> spectrum' even when DNA was definitely bound to the protein.

I am not sure but:

The absorption maximum lies at 260 nm, the absorption at 250 nm is still high
and compared with an DNA spectum, e. g. a midi prep, it looks pretty much the
same

The absorption at 260 nm can be reduced by hydroxylapatite chromatography. In
some preparations the fractions with much protein (shown with bradford, SDS gel)
show a "typical protein spectrum" with a maximum at 280 nm and a minimum at 250
nm - in other preparations this was not the case

My protein acts in vitro and probably in vivo in a heterodimeric complex with
another protein, which is highly negatively charged- like DNA.

These are the reasons for which I think that my proteins bind to DNA which I can
not remove. I would be glad for any suggestions what else can cause these
strange absorption behavior and /or how to purify the protein from the
contaminant.

Thanks,


                        Silke

>
>
> Perhaps you should check your buffer/assay matrix composition. Are there any
> free amino acids present, esp. aromatic ones?  Phe in your matrix will
> produce a strong peak at or near  260nm, mimicking DNA?   Do you perhaps use
> a buffer substance that is not fully transparent in the low UV? Is the
> solution composition of your reference the same as the sample-matrix? Did
> you perhaps purify a his-tagged protein and measure the spectrum of a sample
> containing imidazole?
>
> Cheers,
>
> Achim
>
> Bernard P. Murray, PhD wrote in message ...
> >In article <368A090F.767347C0 at uni-molgen.gwdg.de>, Silke Beismann
> ><sbeisma at uni-molgen.gwdg.de> wrote:
> >
> >> Hi,
> >> I am trying to purify a protein from the amino acid biosynthetic
> >> pathway. It should not have anything to do with DNA but it seems that it
> >> binds to it. I tried digestion of the crude extract with DNase I and
> >> RNase A, precipitation with ammonium sulfate or polyehtylene imine,
> >> hydroxyapatite chromatography- but the spectrum still looks like an
> >> nucleic acid spectrum. Does anyone know an efficient method to remove
> >> the
> >> contaminating nucleic acids?
> >> Thanks in advance,
> >>             Silke
> >
> >Just curious.  I don't know how the "spectrum" can pick out
> >the DNA nature of the protein/complex but is there any way
> >that your protein could be modified (eg. ADP-ribosylated
> >or a prosthetic group) that would give the impression of
> >DNA contamination?  Maybe treatment with a DNA kinase
> >would help determine if it is really adsorbed DNA
> >(should only label if it is really "DNA").
> >          Bernard
> >--
> >Bernard P. Murray, PhD
> >Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA
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