Hi! With PEI precipitation, the systematic approach will save you time in the end: do a PEI titration as it is described in the chapter "Use of Polyethyleneminie in Purification of DNA-Binding Proteins" by Richard R. Burgess of Methods in Enzymology, Vol. 208. It takes into account PEI % and salt concentration. In my case, adding DNAase in a first step, and then using 0.15% PEI and incubating for 10 minutes was the only thing that worked, my protein was sticking very much to non-specific nucleic acids until I tried this. Good Luck! Dominique
El Thursday, 5 de July de 2007 16:58, Benini, Stefano escribió: > Dear Tiancen, > > a friend of mine succesfully used a partial denaturation approach using > urea to get rid of bound DNA, see following citation: > > > Acta Cryst. (1998). D54, 1043-1045 [ doi:10.1107/S0907444998000341 ] > > Cloning, overproduction, purification and crystallization of the DNA > binding protein HU from the hyperthermophilic eubacterium Thermotoga > maritima > > may be it works on zinc finger proteins as well > > ciao > > Stefano > > > > -----Original Message----- > From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] Behalf Of > [EMAIL PROTECTED] > Sent: 05 July 2007 15:43 > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] How to remove nucleic acid contamination for > crystallizing zinc finger protein > > > 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: [EMAIL PROTECTED] > > "Open your eyes. You have only to see things clearly, to understand." > -Leonardo da Vinci > -----Original Message----- > From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] 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 <[EMAIL PROTECTED]> 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 > >> > >> [EMAIL PROTECTED] 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 > >> > > >> > > >> > > >> > ---------------------------------------------------------------------- > >> >-- Ò»Æð À´£¬150 Íò ÈË Í¬ ʱ ÔÚ Íæ µÄ ÃÎ »Ã Î÷ ÓÎ > >> > <http://event.mail.163.com/chanel/xyq.htm?from=126_NO4> > > > > -- > > Pavan > > http://umsis.miami.edu/~pvaidyan -- Dominique Monferrer Institut de Biologia Molecular de Barcelona (IBMB-CSIC) c/Jordi Girona, 18-26 Barcelona 08034 SPAIN Tel. + 34 93 400 61 00 (ext.332) Fax. + 34 93 204 59 04 email: [EMAIL PROTECTED]
