size.
Hope to help.
Nicolas
De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Chiara Rapisarda
[chiara.rapisa...@pasteur.fr]
Envoyé : lundi 29 juin 2015 10:48
À : CCP4BB@JISCMAIL.AC.UK
Objet : Re: [ccp4bb] Interesting DNA contamination
Hi,
> Hi,
I found that my protein bound ethidium bromide in an agarose gel. I tested that
by treating my protein with protease and DNAse in two different tubes and
running a gel. The band in the agarose gel disappeared only when the
protein was treated with protease. It is worth trying.
I hope that
Dear Stefan,
just saw this after reading post:
http://www.nature.com/nature/journal/v522/n7557/full/nature14559.html
Best, Guenter
Pramod,
You already got good suggestions on how to handle DNA contamination in protein
preparations.
Let me point out briefly that you haven't demonstrated yet tha
Correction,
I meant to say 0.5kb, not 500kb
sorry for that.
S.
Pramod,
You already got good suggestions on how to handle DNA contamination in protein
preparations.
Let me point out briefly that you haven't demonstrated yet that your
contamination is DNA.
I had the same observation when purifying UvsX. A very persistent and strong
contamination in all my
That an entire 500bp would be protected from DNase seems very very strange -
but very interesting if true!
Could that band on the agarose gel be something else? Protein will stain a bit
with ethidium as well as with coomassie. Did you add SDS before running the
agarose gel or is it native (in
Hi
Thanks all, I appreciate all the valuable inputs..
Piush..
I ll be trying Benzonase up next.. but since the DNA appears so secluded
for DNAses, it makes me little skeptical as mg and DNAse already been there
for ON dialysis.
Tim
if the DNA binds to the protein, wouldn't this affe
Stepwise addition to 1% PEI (polyethylenimine) following cell lysis
(before dialysis) should do the trick.
--paul
On 06/25/2015 05:23 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 purificati
with what Nature gives you.
Cheers, tom
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bonsor,
Daniel
Sent: Friday, 26 June 2015 11:20 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Interesting DNA contamination
Several different approaches may help you to separate DNA
ltimore
MD 21201
From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Pramod Kumar
[pramod...@gmail.com]
Sent: Thursday, June 25, 2015 5:23 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Interesting DNA contamination
Dear all
Sorry for off topic and le
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 wishe
pramod
Do a spin after bacterial cell breaking at 8K to get rid of a nuclear pellet
Also there is a nuclease preparation available called benzonase
it is most effective when you also add magnesium in your buffer
so in your homogenization buffer add magnesium and should avoid any EDTA
Pius
On Thu,
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-Affin
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