From monicamittal41 from yahoo.com Fri Sep 4 09:59:26 2009 From: monicamittal41 from yahoo.com (Monica Mittal) Date: Fri Sep 4 11:33:41 2009 Subject: [Protein-analysis] QUERY Message-ID: <305107.6670.qm@web94906.mail.in2.yahoo.com> HI I'M FACING PROBLEM IN PURIFYING A protein which is a dna binding protein but its showing a kind of hydrophobic behaviour as its max part is going into pellet after lysis. moreover its not responding to ni2+ nta column also and the lysis sol. is not so clear that i go for gel filteration as it may probably clog it so can u suggest any solution? monica Love Cricket? Check out live scores, photos, video highlights and more. Click here http://cricket.yahoo.com From wajih76 from hotmail.com Fri Sep 4 22:25:02 2009 From: wajih76 from hotmail.com (wajahat mahmood) Date: Sat Sep 5 10:42:38 2009 Subject: [Protein-analysis] QUERY In-Reply-To: <305107.6670.qm@web94906.mail.in2.yahoo.com> References: <305107.6670.qm@web94906.mail.in2.yahoo.com> Message-ID: Hi Monica=2C If your protein is in the pellets I hope you are using denaturing condition= s to solubilize it before purification over Ni-NTA. solubilize the protein in urea or guanidine for one hour and spin down to g= et a clear lysis solution before you proceed to further purification steps = Ni-NTa or gel. =20 cheers wajahat > Date: Fri=2C 4 Sep 2009 20:29:26 +0530 > From: monicamittal41@yahoo.com > To: proteins@magpie.bio.indiana.edu > CC:=20 > Subject: [Protein-analysis] QUERY >=20 > HI > I'M FACING PROBLEM IN PURIFYING A protein which is a dna binding protein = but its showing a kind of hydrophobic behaviour as its max part is going in= to pellet after lysis. > moreover its not responding to ni2+ nta column also and the lysis sol. is= not so clear that i go for gel filteration as it may probably clog it > so can u suggest any solution? >=20 > monica >=20 >=20 >=20 > Love Cricket? Check out live scores=2C photos=2C video highlights and mor= e. Click here http://cricket.yahoo.com > _______________________________________________ > Proteins mailing list > Proteins@net.bio.net > http://www.bio.net/biomail/listinfo/proteins _________________________________________________________________ Drag n=92 drop=97Get easy photo sharing with Windows Live=99 Photos. http://www.microsoft.com/windows/windowslive/products/photos.aspx= From yogendra from ccmb.res.in Fri Sep 4 23:01:06 2009 From: yogendra from ccmb.res.in (yogendra) Date: Sat Sep 5 10:42:45 2009 Subject: [Protein-analysis] QUERY In-Reply-To: <305107.6670.qm@web94906.mail.in2.yahoo.com> Message-ID: <567489252.578451252123266751.JavaMail.root@127.0.0.1> its not going into pellet due to hydrophobicity. It is forming inclusion bodies during overexpression. You are required to dissolve the protein and refold it. Please fiollow the procedure "how to refold protein from inclusion bodies" Good luck with your work Yogendra -- Original Message -- From: Monica Mittal To: proteins@magpie.bio.indiana.edu Date: Fri, 4 Sep 2009 20:29:26 +0530 (IST) Subject: [Protein-analysis] QUERY HI I'M FACING PROBLEM IN PURIFYING A protein which is a dna binding protein but its showing a kind of hydrophobic behaviour as its max part is going into pellet after lysis. moreover its not responding to ni2+ nta column also and the lysis sol. is not so clear that i go for gel filteration as it may probably clog it so can u suggest any solution? monica Love Cricket? Check out live scores, photos, video highlights and more. Click here http://cricket.yahoo.com _______________________________________________ Proteins mailing list Proteins@net.bio.net http://www.bio.net/biomail/listinfo/proteins From engelbert_buxbaum from hotmail.com Tue Sep 8 08:58:37 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Tue Sep 8 13:07:12 2009 Subject: [Protein-analysis] Re: QUERY References: Message-ID: Am 04.09.2009, 10:59 Uhr, schrieb Monica Mittal : > HI > I'M FACING PROBLEM IN PURIFYING A protein which is a dna binding protein > but its showing a kind of hydrophobic behaviour as its max part is going > into pellet after lysis. > moreover its not responding to ni2+ nta column also and the lysis sol. > is not so clear that i go for gel filteration as it may probably clog it > so can u suggest any solution? Does that mean you are expressing a poly-His tagged protein in bacteria? If so, have you checked that your protein is not in inclusion bodies? How did you lyse? How long and at what g did you try to clarify your lysate (1 h at 100000 g should do the trick)? Have you fragmented the bacterial DNA, and are you sure that your protein is not binding to the fragments? Are you protecting against oxidation and proteolysis? From winlight from mail.ustc.edu.cn Sun Sep 13 20:22:25 2009 From: winlight from mail.ustc.edu.cn (winlight@mail.ustc.edu.cn) Date: Mon Sep 14 10:25:22 2009 Subject: [Protein-analysis] Re: Proteins Digest, Vol 52, Issue 3 In-Reply-To: <200909091704.n89H48p12365@net.bio.net> References: <200909091704.n89H48p12365@net.bio.net> Message-ID: <30108713.171221252891345504.JavaMail.coremail@mailweb> Are you sure the aggregation is not caused by dna interweaving between protein molecules? If that's the cause, Benzonase will be a good choice for DNA clearance. > -----Original E-mail----- > From: proteins-request@oat.bio.indiana.edu > Sent Time: 2009-9-10 1:04:09 > To: proteins@magpie.bio.indiana.edu > Cc: > Subject: Proteins Digest, Vol 52, Issue 3 > > Send Proteins mailing list submissions to > proteins@net.bio.net > > To subscribe or unsubscribe via the World Wide Web, visit > http://www.bio.net/biomail/listinfo/proteins > or, via email, send a message with subject or body 'help' to > proteins-request@net.bio.net > > You can reach the person managing the list at > proteins-owner@net.bio.net > > When replying, please edit your Subject line so it is more specific > than "Re: Contents of Proteins digest..." > > > Today's Topics: > > 1. Re: QUERY (Dr Engelbert Buxbaum) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Tue, 08 Sep 2009 09:58:37 -0400 > From: "Dr Engelbert Buxbaum" > Subject: [Protein-analysis] Re: QUERY > To: proteins@net.bio.net > Message-ID: > Content-Type: text/plain; format=flowed; delsp=yes; > charset=iso-8859-15 > > Am 04.09.2009, 10:59 Uhr, schrieb Monica Mittal : > > > HI > > I'M FACING PROBLEM IN PURIFYING A protein which is a dna binding protein > > but its showing a kind of hydrophobic behaviour as its max part is going > > into pellet after lysis. > > moreover its not responding to ni2+ nta column also and the lysis sol. > > is not so clear that i go for gel filteration as it may probably clog it > > so can u suggest any solution? > > Does that mean you are expressing a poly-His tagged protein in bacteria? > If so, have you checked that your protein is not in inclusion bodies? How > did you lyse? How long and at what g did you try to clarify your lysate (1 > h at 100000 g should do the trick)? Have you fragmented the bacterial DNA, > and are you sure that your protein is not binding to the fragments? Are > you protecting against oxidation and proteolysis? > > > ------------------------------ > > _______________________________________________ > Proteins mailing list > Proteins@net.bio.net > http://www.bio.net/biomail/listinfo/proteins > > End of Proteins Digest, Vol 52, Issue 3 > *************************************** From james.herbert from flinders.edu.au Mon Sep 21 07:42:13 2009 From: james.herbert from flinders.edu.au (James Herbert) Date: Mon Sep 21 10:03:59 2009 Subject: [Protein-analysis] Hettich centrifuge schematics Message-ID: <1253536933.4ab774a5a23de@imp.flinders.edu.au> Does anybody have a Hettich centrifuge schematics. The Hettich 30RF Universal centrifuge repair manual would be best but any Hettich should do as we just want to limit a broken centrifuge to a set speed. The expense to buy the circuit diagram is too much. So if anyone could help it would be greatly appreciated. Thanks James. James Herbert Lab Manager School of Biological Sciences Flinders University Adelaide South Australia From elham.rastegar from gmail.com Wed Sep 23 09:10:06 2009 From: elham.rastegar from gmail.com (elham rastegari) Date: Wed Sep 23 10:00:41 2009 Subject: [Protein-analysis] SDS-PAGE Message-ID: Dear all, I perform *TCA-acetone, methanol washes and phenol precipitation* to extract protein from fruit sample. To see clear bands in SDS-PAGE, I need to remove polysaccharide. Does anybody know how to remove extracellular polysaccharide? Thanks in advance Eli From sticher from bioc.uzh.ch Fri Sep 25 07:50:33 2009 From: sticher from bioc.uzh.ch (Patrick Sticher) Date: Fri Sep 25 10:03:03 2009 Subject: [Protein-analysis] Practical Course in Biomolecular Modelling Message-ID: <4ABCBC99.1090205@bioc.uzh.ch> Dear colleagues, please be informed that online applications are still accepted for the following course until October 16, 2009: 8TH NCCR PRACTICAL COURSE IN BIOMOLECULAR MODELLING January 10 - 15, 2010 Kandersteg, Switzerland http://www.structuralbiology.uzh.ch/course2010.asp Course topics include Simulation techniques, force-field development, conformational search, computation of free energy and entropy, treatment of electrostatic forces, simulation of folding, comparison of simulation with experiment This course is primarily directed to PhD students and postdocs from experimental structural biology groups wishing to learn more on biomolecular modelling. The course format will include morning lectures and late-afternoon/early evening tutorials, and provide ample opportunities for discussions with experts and fellow participants. Participants will be invited to bring own problems for tutorials and/or discussion. The course will be organized as a winter retreat in the Swiss Alps offering a stimulating learning atmosphere with the afternoons available for informal participation in discussions, reading and self-study or recreational activities in the area. Interested candidates are encouraged to apply online on http://www.structuralbiology.uzh.ch/course2010_application.asp. Application deadline will be October 16, 2010. We will be able to accept 20 participants to this course. Best regards, Patrick Sticher -- _________________________________ Visit the NCCR on the Internet www.structuralbiology.uzh.ch Dr. Patrick Sticher Moser NCCR Scientific Officer Institute of Biochemistry University of Z?rich Winterthurerstrasse 190 CH - 8057 Z?rich Phone +41 / (0)44 / 635 54 84 Fax +41 / (0)44 / 635 59 08 Mail sticher@bioc.uzh.ch