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 16:57, Raji Edayathumangalam escribió: > 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: [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 > >>> > > >>> > > >>> > > >>> > --------------------------------------------------------------------- > >>> >--- R;Fp @4#,150 Mr HK M, J1 TZ Mf 5D CN ;C Nw SN > >>> > <http://event.mail.163.com/chanel/xyq.htm?from=126_NO4> > >> > >> -- > >> Pavan > >> http://umsis.miami.edu/~pvaidyan > > ---------End of Included Message---------- -- 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]
