One can also add a small amount of DNAse during cell lysis. This can make the lysates less viscous for the early stages of purification and the free nucleotides are then washed away during purification, especially on SEC.<div>
Tony<br><br><div class="gmail_quote">On Tue, Jun 1, 2010 at 10:26 AM, Ewa Folta-Stogniew <span dir="ltr"><<a href="mailto:ef55@email.med.yale.edu">ef55@email.med.yale.edu</a>></span> wrote:<br><blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex;">
How would one identify presence and remove just a single nucleotide rather than nucleic acids? I know one can see by 260 nm absorbance, but differentiate between polymer and a single nucleotide?<br>
<br>
Ewa<br>
<br>
At 12:51 PM 6/1/2010, Jeff Cohlberg wrote:<br>
<blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex">
I can second Lizz Bartlett's recommendation. In purifying recombinant superoxide dismutase from a yeast expression system, we often found PicoGreen-positive DNA in protein that had been purified with phenyl-Sepharose plus size exclusion, but little or none when a DEAE-Sepharose ion exchange step was added.<br>
<br>
Jeff Cohlberg<br>
<br>
<br>
Bartlett, Lizz wrote:<br>
<blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex">
<br>
Hi All,<br>
In industry DNA, etc is removed during the purification steps often using an ion exchange method.<br>
<br>
Cheers!<br>
Lizz Bartlett<br>
<br>
-----Original Message-----<br>
From: <mailto:<a href="mailto:rasmb-bounces@rasmb.bbri.org" target="_blank">rasmb-bounces@rasmb.bbri.org</a>><a href="mailto:rasmb-bounces@rasmb.bbri.org" target="_blank">rasmb-bounces@rasmb.bbri.org</a> [mailto:<a href="mailto:rasmb-bounces@rasmb.bbri.org" target="_blank">rasmb-bounces@rasmb.bbri.org</a>] On Behalf Of Hayes, David<br>
Sent: Tuesday, June 01, 2010 9:26 AM<br>
To: <mailto:<a href="mailto:rasmb@rasmb.bbri.org" target="_blank">rasmb@rasmb.bbri.org</a>><a href="mailto:rasmb@rasmb.bbri.org" target="_blank">rasmb@rasmb.bbri.org</a><br>
Subject: Re: [RASMB] Common contamination in recombinant protein preparation<br>
<br>
Hi Dror Noy,<br>
<br>
I am the AUC guy here at MedImmune, so I am not an expert:<br>
But colleagues here use intercalating dyes like pico green to measure<br>
DNA concentrations. This may be complete overkill (especially the metal<br>
enhanced version my colleagues developed) but you sound like you really<br>
want to be certain if this is DNA and how much is there. I include a<br>
reference to a recent paper. But I also believe that there is a kit and<br>
even a special mini-fluorometer called Quant-It that measures DNA, RNA,<br>
and protein. The kits were not quite good enough for our purposes here,<br>
but may be sufficiently precise for your needs.<br>
<br>
I am too far downstream to know how they get rid of the extra DNA when<br>
they find it.<br>
<br>
1: Dragan AI, Bishop ES, Casas-Finet JR, Strouse RJ, Schenerman MA,<br>
Geddes CD.<br>
Metal-enhanced PicoGreen fluorescence: application for double-stranded<br>
DNA<br>
quantification. Anal Biochem. 2010 Jan 1;396(1):8-12. Epub 2009 Sep 11.<br>
PubMed<br>
PMID: 19748479.<br>
<br>
-----Original Message-----<br>
From: <mailto:<a href="mailto:rasmb-bounces@rasmb.bbri.org" target="_blank">rasmb-bounces@rasmb.bbri.org</a>><a href="mailto:rasmb-bounces@rasmb.bbri.org" target="_blank">rasmb-bounces@rasmb.bbri.org</a> [mailto:<a href="mailto:rasmb-bounces@rasmb.bbri.org" target="_blank">rasmb-bounces@rasmb.bbri.org</a>]<br>
On Behalf Of Dror Noy<br>
Sent: Monday, May 31, 2010 2:52 AM<br>
To: <mailto:<a href="mailto:rasmb@server1.bbri.org" target="_blank">rasmb@server1.bbri.org</a>><a href="mailto:rasmb@server1.bbri.org" target="_blank">rasmb@server1.bbri.org</a><br>
Subject: [RASMB] Common contamination in recombinant protein preparation<br>
<br>
Not exactly AUC related but there's a chance to find here a few<br>
people who work with proteins and do care about their UV absorption<br>
properties:<br>
We make a variety of native and artificial recombinant proteins and<br>
often encounter a situation where the apparently pure fraction<br>
according to SDS-PAGE gels has its UV absorption peak blue shifted<br>
from the expected ~280 nm toward 260 nm. This seems to be a general<br>
problem and usually happens when proteins are purified from inclusion<br>
bodies. We suspect DNA contamination but so far we were unable to get<br>
rid of it. I'd be very grateful for tips from people who experienced<br>
this phenomenon and were able to solve it.<br>
Thanks,<br>
Dror Noy<br>
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--<br>
Jeffrey A. Cohlberg, Professor and Chair<br>
Department of Chemistry and Biochemistry<br>
California State University, Long Beach<br>
Long Beach, CA 90840<br>
562-985-4944 fax 775-248-1263<br>
<mailto:<a href="mailto:cohlberg@csulb.edu" target="_blank">cohlberg@csulb.edu</a>><a href="mailto:cohlberg@csulb.edu" target="_blank">cohlberg@csulb.edu</a><br>
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