[RASMB] Common contamination in recombinant protein preparation

[Bartlett, Lizz]

Hi All,
In industry DNA, etc is removed during the purification steps often using an ion exchange method.

Cheers!
Lizz Bartlett

-----Original Message-----
From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On Behalf Of Hayes, David
Sent: Tuesday, June 01, 2010 9:26 AM
To: rasmb at rasmb.bbri.org
Subject: Re: [RASMB] Common contamination in recombinant protein preparation

Hi Dror Noy,

I am the AUC guy here at MedImmune, so I am not an expert:
But colleagues here use intercalating dyes like pico green to measure
DNA concentrations.  This may be complete overkill (especially the metal
enhanced version my colleagues developed) but you sound like you really
want to be certain if this is DNA and how much is there.  I include a
reference to a recent paper.  But I also believe that there is a kit and
even a special mini-fluorometer called Quant-It that measures DNA, RNA,
and protein.  The kits were not quite good enough for our purposes here,
but may be sufficiently precise for your needs.

I am too far downstream to know how they get rid of the extra DNA when
they find it.

1: Dragan AI, Bishop ES, Casas-Finet JR, Strouse RJ, Schenerman MA,
Geddes CD.
Metal-enhanced PicoGreen fluorescence: application for double-stranded
DNA
quantification. Anal Biochem. 2010 Jan 1;396(1):8-12. Epub 2009 Sep 11.
PubMed
PMID: 19748479.

-----Original Message-----
From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org]
On Behalf Of Dror Noy
Sent: Monday, May 31, 2010 2:52 AM
To: rasmb at server1.bbri.org
Subject: [RASMB] Common contamination in recombinant protein preparation

Not exactly AUC related but there's a chance to find here a few  
people who work with proteins and do care about their UV absorption  
properties:
We make a variety of native and artificial recombinant proteins and  
often encounter a situation where the apparently pure fraction  
according to SDS-PAGE gels has its UV absorption peak blue shifted  
from the expected ~280 nm  toward 260 nm. This seems to be a general  
problem and usually happens when proteins are purified from inclusion  
bodies. We suspect DNA contamination but so far we were unable to get  
rid of it. I'd be very grateful for tips from people who experienced  
this phenomenon and were able to solve it.
Thanks,
Dror Noy
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