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CCP4BB  June 2015

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Subject:

Re: Interesting DNA contamination

From:

Tim Gruene <[log in to unmask]>

Reply-To:

Tim Gruene <[log in to unmask]>

Date:

Thu, 25 Jun 2015 23:49:08 +0200

Content-Type:

multipart/signed

Parts/Attachments:

Parts/Attachments

text/plain (72 lines) , signature.asc (72 lines)

Dear Pramod Kumar,

if the DNA binds to the protein, wouldn't this affect the interpretation
of the Agarose gel?

500 bases should result in a distinct shift during gel filtration. Do
you observe this?

While waiting for suggestions, you might set up crystallisation trials
just in case?

Best wishes,
Tim

On 06/25/2015 11:23 PM, Pramod Kumar wrote:
> Dear all
> 
> Sorry for off topic and lengthy post, but I came across a very unique DNA
> contamination during one membrane protein purification (a microbial
> external environment sensor/response protein)
> 
> Already done
> 
> * DNAse used as stranded protocol during cell break.
> * Membrane extraction to Ni-Affinity and Ovenight TEV dig done with 0.5M
> NaCl
> * Fluorescence size exclusion done with 0.1M NaCl
> * Well stable peak and pure protein profile observed
> * But final purified protein inherently contains specific 0.5 Kb DNA
> stretch visible through out purification (observed by running Agarose gel
> of protien sample @ each step)
> 
> 
> Approach failed
> 
> * Buffer switched from HEPES to Na-PO4
> * O.N. dialysis in presence of DNAse
> * Using Heparin Column purification between Ni-Aff and SE (failed and
> protein starts deterioration)
> * Since protein binds ATP (ATP and MgCl2 added after O.N.
> Dialysis/digestion)
> 
> Now... I need help for
> 
> * How to get rid of DNA without loosing active protein?
> * What are best lipids to dope as -ve DNA replacement?
> * Since protein is pure and ample, how likely I can get crystal hits with
> such big DNA attached?
> * What longest possible DNA can be crystallize/ed with protein?
> * How exclusive are some mem-spanign-proteins to provide anchorage of
> prokaryotic genomic stretch?
> 
> 
> 
> ​Thanks in advance​ :)
> 
> Pramod Kumar
> 

-- 
--
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen
phone: +49 (0)551 39 22149

GPG Key ID = A46BEE1A



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