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: M T michelthe {- at -} GMAIL {- dot -} COM
Date: 2007-07-05

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Subject: Re: How to remove nucleic acid contamination for crystallizing zinc finger protein
From: "Benini, Stefano" Stefano {- dot -} Benini {- at -} ASTRAZENECA {- dot -} COM
Date: 2007-07-05

Subject: Re: How to remove nucleic acid contamination for crystallizing zinc finger protein
From: Raji Edayathumangalam redayath {- at -} MAIL {- dot -} ROCKEFELLER {- dot -} EDU
Date: 2007-07-05

Yes, I have seen colleagues get rid of tons of DNA contamination in bacterial RNA polymerase preps
by PolyminP (PEI) precipitation. Seemed like it was the only method that worked (among several
tried) to remove all the non-specific DNA. Worked very well.

As painful as it appeared, PEI precipitation absolutely turned out to be the only solution.

Raji

---------Included Message----------
>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
>_____________________________________
>For mail:
>UCHSC at Fitzsimons
>Mail Stop 8101, PO Box 6511
>Aurora, CO 80045
>
>For courier/packages:
>South Building RC-1, Room 9110
>12801 East 17th Ave.
>Aurora, CO 80010
>
>phone: 303-724-3257
>fax: 303-724-3215
>email: jeffrey.kieft@uchsc.edu
>
>"Open your eyes. You have only to see things clearly, to understand."
> -Leonardo da Vinci
>-----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
>>> >
>>> >
>>> >
>>> > ------------------------------------------------------------------------
>>> > R;Fp @4#,150 Mr HK M, J1 TZ Mf 5D CN ;C Nw SN
>>> >
>>>
>>
>>
>> --
>> Pavan
>> http://umsis.miami.edu/~pvaidyan
>>
>
>
---------End of Included Message----------

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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: M T michelthe {- at -} GMAIL {- dot -} COM
Date: 2007-07-05