Re: [ccp4bb] Interesting DNA contamination

2015-06-30 Thread Chiara Rapisarda
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

Re: [ccp4bb] Interesting DNA contamination

2015-06-28 Thread Guenter Fritz
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

Re: [ccp4bb] Interesting DNA contamination

2015-06-26 Thread Pramod Kumar
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

Re: [ccp4bb] Interesting DNA contamination

2015-06-26 Thread Phoebe A. Rice
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

Re: [ccp4bb] Interesting DNA contamination

2015-06-26 Thread Stefan Gajewski
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

Re: [ccp4bb] Interesting DNA contamination

2015-06-26 Thread Stefan Gajewski
Correction, I meant to say 0.5kb, not 500kb sorry for that. S.

[ccp4bb] Interesting DNA contamination

2015-06-25 Thread Pramod Kumar
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

Re: [ccp4bb] Interesting DNA contamination

2015-06-25 Thread Tim Gruene
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

Re: [ccp4bb] Interesting DNA contamination

2015-06-25 Thread Pius Padayatti
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,

Re: [ccp4bb] Interesting DNA contamination

2015-06-25 Thread Paul Paukstelis
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

Re: [ccp4bb] Interesting DNA contamination

2015-06-25 Thread Bonsor, Daniel
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 lengthy post

Re: [ccp4bb] Interesting DNA contamination

2015-06-25 Thread Tom Peat
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