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Re: [ccp4bb] How to remove nucleic acid contamination for crystallizing zinc finger protein
 

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CCP4bb <-- 2007 <-- July 2007 <-- 05 July 2007
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Subject: Re: How to remove nucleic acid contamination for crystallizing zinc finger protein
From: William Scott wgscott {- at -} CHEMISTRY {- dot -} UCSC {- dot -} EDU
Date: 2007-07-05 		Next message:
Subject: Re: How to remove nucleic acid contamination for crystallizing zinc finger protein
From: M T michelthe {- at -} GMAIL {- dot -} COM
Date: 2007-07-05


Subject: Re: How to remove nucleic acid contamination for crystallizing zinc finger protein
From: Jeffrey {- dot -} Kieft {- at -} UCHSC {- dot -} EDU Jeffrey {- dot -} Kieft {- at -} UCHSC {- dot -} EDU
Date: 2007-07-05
We have used the PEI precipitation described by Pavan, very high salt (2M or even higher) as alluded to by Ana, or heparin columns with success. In some cases, a denaturing purification protocol can be very useful, but of course this assumes you can refold the protein. In the case of a student whose thesis committee I am on, the RNase and DNase has not worked, maybe because the NAcid is protected by being bound to the protein, although I know others have used it with success. For that student, the ONLY thing that worked was PEI.

Jeff
_______________________________________
Jeffrey S. Kieft, Ph.D.
Assistant Professor
Dept. of Biochemistry and Molecular Genetics
University of Colorado School of Medicine

http://www.uchsc.edu/sm/bbgn/kieftj.htm
http://www.evolutionarygenomics.com/CERT/CERT.html
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-----Original Message-----
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of William Scott
Sent: Thursday, July 05, 2007 8:04 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] How to remove nucleic acid contamination for crystallizing zinc finger protein

It can also ppt out your protein, if it is bound to the DNA.

You could also DNase and RNase the bejesus out of it, and temporarily
unfold the protein to aid in release of the nucleic acids. Then you need
to get rid of these evil enzymes before you put back your nucleic acid of
choice.

Pavan wrote:
> You could also try polyethyleneimine (Polymin P) precipitation to
> precipitate out your DNA. Add Polymin P dropwise to a final
> concentration of 0.24% to your cell lysate while stirring in the cold
> for an hour. This should precipitate out your DNA. Then centrifuge
> your lysate and use the relevant fraction (supernatant or pellet) for
> further purification.
>
> Pavan
>
> On 7/5/07, Ana Silva wrote:
>> Hi,
>>
>> How much salt do you have in your protein buffer?
>> I would try to increase the salt concetration, during purification.
>>
>> Hope it helps.
>> Ana
>>
>>
>>
>> tony_htc@126.com wrote:
>> >
>> > Dear all,
>> >
>> > Sorry for the off-topic question.
>> >
>> > I am purifying a zinc finger transcription factor for crystallization.
>> > The protein appeared as a single band on SDS-PAGE (MW 44KD) after NTA
>> > chelating column, but its OD280/OD260 ratio is as high as 1.0. So I
>> > doubt the protein is nucleic acid contaminated, probably because of
>> > the zinc finger. I tried to remove the nucleic acid by Mono Q and
>> > Superdex 75 pg, but failed. So could any one recommend some method to
>> > remove the nucleic acid during protein crystallization, esp. zinc
>> > finger protein? Any experience or references will be appreciated.
>> >
>> > Thanks a lot!
>> >
>> > Tiancen Hu
>> >
>> > Shanghai Institute of Materia Medica
>> >
>> >
>> >
>> > ------------------------------------------------------------------------
>> > 一起 来，150 万 人 同 时 在 玩 的 梦 幻 西 游
>> >
>>
>
>
> --
> Pavan
> http://umsis.miami.edu/~pvaidyan
>

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CCP4bb <-- 2007 <-- July 2007 <-- 05 July 2007
Previous message:
Subject: Re: How to remove nucleic acid contamination for crystallizing zinc finger protein
From: William Scott wgscott {- at -} CHEMISTRY {- dot -} UCSC {- dot -} EDU
Date: 2007-07-05 		Next message:
Subject: Re: How to remove nucleic acid contamination for crystallizing zinc finger protein
From: M T michelthe {- at -} GMAIL {- dot -} COM
Date: 2007-07-05


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