From owner-proteins@net.bio.net Sun Aug 02 23:00:00 1998 Path: biosci!kemi.dtu.dk!hemc From: hemc@kemi.dtu.dk ("Hans E. M. Christensen") Newsgroups: bionet.molbio.proteins Subject: Postdoc position in metalloprotein research Date: 3 Aug 1998 00:27:03 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 51 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Research Assistant Professor (Postdoctoral Fellowship) in Metalloprotein Research. A Postdoctoral position in the area of design and characterization of artificial metalloproteins is available in the Department of Chemistry, Technical University of Denmark. The position is for 1 year with a possible extension for an additional year. We are seeking a highly motivated individual to join a team working on characterization of artificial metalloproteins based on rational design. A recent Ph.D degree and a strong background in purification and handling of metalloproteins is required. The preferred candidate should have a broad expertise and interest in metalloprotein characterization, e.g. structural and spectroscopic techniques such as mass spectrometry, EXAFS, crystal structure determination, NMR and ESR, and development of catalytic assays. Candidates must send a list of their publications and the work they wish to be included in the evaluation. The evaluation committee may request further information or documentation. We request that the application be written in English, in view of the possibility of foreigner being member of the evaluation committee. The Technical University of Denmark seeks an improved gender balance and therefore invites both women and men to apply. The appointment is salaried in accordance with a collective agreement with the relevant trade union and there is granted an annual bonus, which at present amounts to DKK. 37.600,00. Applications will be considered according to the regulations of the Ministry of Education, 9 September 1993 and will be submitted to an evaluation committee. The recommendations of the committee will be forwarded in full to all applicants. For further information, please contact Hans E. M. Christensen, telephone +45 45 25 23 47 or email hemc@kemi.dtu.dk. Applicants should send their application, and enclosures, all in triplicate, to: Head of Department Inger Sotofte Department of Chemistry Building 207 Technical University of Denmark 2800 Lyngby, Denmark. The closing date for application is 28 August 1998 at noon local time. From owner-proteins@net.bio.net Sun Aug 02 23:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!howland.erols.net!rill.news.pipex.net!pipex!server1.netnews.ja.net!news.nott.ac.uk!pdxkgs From: pdxkgs@pdn1.gene.nott.ac.uk (Karen spink) Newsgroups: bionet.molbio.proteins Subject: Blotting onto PVDF Date: Mon, 03 Aug 1998 09:58:46 +0100 Organization: University of Nottingham Lines: 9 Message-ID: NNTP-Posting-Host: ppdirg.nottingham.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=ISO-8859-1 Content-Transfer-Encoding: 8bit X-Newsreader: Yet Another NewsWatcher 2.4.0 Has anyone had any problems Western blotting onto PVDF in CAPS buffer? I am trying to get a sequence on my protein and it appears that the efficiency of transfer is letting me down. Could it be the buffer, (10mM CAPS, 10% methanol, pH 11) ? I have had no problems blotting onto nitrocellulose using tris-glycine buffers. Thanks for your time Karen Spink From owner-proteins@net.bio.net Sun Aug 02 23:00:00 1998 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!news.bbnplanet.com!news1.best.com!newsfeed.nacamar.de!news-kar1.dfn.de!news-fra1.dfn.de!news-ber1.dfn.de!news-ham1.dfn.de!news.mu-luebeck.de!not-for-mail From: "Lars Komorowski" Newsgroups: bionet.molbio.proteins Subject: pEt-Expression-Mystery Date: Mon, 3 Aug 1998 10:29:49 +0100 Organization: Med. Universitaet zu Luebeck Lines: 12 Message-ID: <6q3sl4$dmt$1@gwsun.medinf.mu-luebeck.de> NNTP-Posting-Host: 141.83.167.168 X-Newsreader: Microsoft Outlook Express 4.71.1712.3 X-MimeOLE: Produced By Microsoft MimeOLE V4.71.1712.3 I am trying to overexpress a 17kDa-Protein in E.coli BL21 with Novagens pET24a Vector. I inocculate my LB medium from an overnight culture, let the culture grow until the OD reaches 0.6 and induce with 1 mM IPTG. After one night there is a band at 25 kDa in SDS-PAGE made with total cell protein. When I try to separate cytosolic proteins from the rest and make another SDS-PAGE with both fractions there is no band there. I have two questions: 1. If the protein band is not the desired protein what can it be ? 2. If the band represents the protein, is it possible that it is proteolysed within minutes ? From owner-proteins@net.bio.net Sun Aug 02 23:00:00 1998 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!news.bbnplanet.com!news1.best.com!newsxfer3.itd.umich.edu!nntp.news.xara.net!xara.net!server5.netnews.ja.net!server3.netnews.ja.net!server4.netnews.ja.net!server2.netnews.ja.net!leicester!usenet From: "Dr E. Buxbaum" Newsgroups: alt.bio.technology,bionet.cellbiol,bionet.immunology,bionet.molbio.proteins Subject: Re: cross reactivty on Western immunoblotting Date: Mon, 03 Aug 1998 15:01:34 -0700 Organization: University of Leicester (PCFS User) Lines: 8 Message-ID: <35C6333E.1D10@le.ac.uk> References: <6pqku3$7gj$1@heliodor.xara.net> NNTP-Posting-Host: pc222.tulip5.le.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Win16; I) Xref: biosci bionet.cellbiol:9912 bionet.immunology:14441 bionet.molbio.proteins:13108 david pryce wrote: > Can anyone tell me if there are any "cheap" alternatives to using milk > powder as a blocking agent A former boss of mine swore by fish skin gelatine, a liquid of the colour and consitency of used motor oil, but freely soluble in PBS. The stuff is available from Sigma, but be carefull: their bottle cap is ill designed and you won't be able to open it a second time. BSA or common gelatine are also possible. From owner-proteins@net.bio.net Sun Aug 02 23:00:00 1998 From: Cornelius Krasel Subject: Re: pEt-Expression-Mystery Newsgroups: bionet.molbio.proteins References: <6q3sl4$dmt$1@gwsun.medinf.mu-luebeck.de> User-Agent: tin/pre-1.4-980514 (UNIX) (Linux/2.0.34 (i486)) Date: Mon, 3 Aug 1998 15:31:49 +0200 Message-ID: <54e4q6.tc.ln@wpxx02.toxi.uni-wuerzburg.de> NNTP-Posting-Host: wpxx02.toxi.uni-wuerzburg.de Lines: 15 Path: biosci!agate!newsfeed.berkeley.edu!howland.erols.net!newsfeed.nacamar.de!frankfurt.de.uu.net!news.uni-stuttgart.de!uni-erlangen.de!uni-wuerzburg.de!not-for-mail Lars Komorowski wrote: > I am trying to overexpress a 17kDa-Protein in E.coli BL21 with Novagens > pET24a Vector. I inoculate my LB medium from an overnight culture, let the > culture grow until the OD reaches 0.6 and induce with 1 mM IPTG. After one > night there is a band at 25 kDa in SDS-PAGE made with total cell protein. If your protein is unstable, it will not survive overnight induction. Can you detect your protein by western blotting? --Cornelius. -- /* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */ /* D-97078 Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de SP4 */ /* "Science is the game we play with God to find out what His rules are." */ From owner-proteins@net.bio.net Sun Aug 02 23:00:00 1998 Path: biosci!SLIP.NET!grizzly From: grizzly@SLIP.NET (Michael Sherrell) Newsgroups: bionet.molbio.proteins Subject: Sequencers/synthesizers Date: 3 Aug 1998 16:07:19 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 55 Sender: daemon@net.bio.net Distribution: world Message-ID: <01BDBEF8.7256CD20@oak-pm1-50-178.dialup.slip.net> NNTP-Posting-Host: net.bio.net I have a number of peptide and oligo synthesizers and sequencers for = sale: Licor 4200S-2 two-laser Sequencer, 4 mos. old Licor 4000L sequencer, ~$30K PerSeptive 9050, ~$6K PerSeptive Expedite 8909, <=3D $14K ABI 394, rebuilt, warranteed, Oligonet-ready, ~$12K ABI 394, rebuilt, warranteed, non-Oligonet, ~$11K ABI 391, rebuilt, warranteed, $7K ABI 430, rebuilt, warranteed, ~$12K ABI 431, ~$14K ABI 432 Synergy ABI 433, rebuilt by ABI, warranteed, < $40K ABI 373 classic, $10,500 ABI 373 stretch, 5-filter, 36-lane, $29K ABI 477, <=3D $10K ABI Catalyst 800 ABI 120, 130, $2.5K [We rebuild valve blocks for ABI 430, 431 and 39x @ $65/port, 90-day = warranty] also: LC-MS: Finnigan TSQ 7000, ~$200K Fisons VG 2000, <$100K Finnigan MAT 900, <$50K Finnigan MAT 90, ~$45K =09 MALDI-TOF: HP, masses to 500 KDa, lightly used, <=3D$60K Finnigan Laser MAT 2000, <$40K Other expensive hi-tech items: Bio-Dot sub-microliter 8-channel aspirate/dispense system (typically = 96-well microplate source, glass slide, microwell plate or membrane = target), < 1 year old Molecular Devices 445SI-MAC Phosphorimager, ~ 3 years old, currently = operating and under maintenance contact I also have available a few other synthesizers and sequencers and a = wide selection of HPLCs, mass specs, robots and other lab instruments. Please contact me to discuss any of these items, or if you have any = items you might like to sell. Michael Sherrell Grizzly Analytical (USA) 707 887 2919/fax 707 887 9834 www.grizzlyanalytical.com From owner-proteins@net.bio.net Sun Aug 02 23:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!howland.erols.net!newsfeed.nacamar.de!nntp.news.xara.net!xara.net!server5.netnews.ja.net!server3.netnews.ja.net!server4.netnews.ja.net!server2.netnews.ja.net!leicester!usenet From: "Dr E. Buxbaum" Newsgroups: bionet.molbio.proteins Subject: Re: Blotting onto PVDF Date: Mon, 03 Aug 1998 15:09:56 -0700 Organization: University of Leicester (PCFS User) Lines: 14 Message-ID: <35C63534.137A@le.ac.uk> References: NNTP-Posting-Host: pc222.tulip5.le.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Win16; I) Karen spink wrote: > > Has anyone had any problems Western blotting onto PVDF in CAPS buffer? I am > trying to get a sequence on my protein and it appears that the efficiency > of transfer is letting me down. Could it be the buffer, (10mM CAPS, 10% > methanol, pH 11) ? I have had no problems blotting onto nitrocellulose > using tris-glycine buffers. It may be the high detergent concentration which is causing problems. If you want to avoid the glycine, why not use Dunn's buffer (published in Anal. Biochem. a couple of years back, NaHCO3/Na2CO3, which can be supplemented with methanol or SDS as needed). I use it routinely (because I found the transfer efficiency better than with tris-gly, and also because it is a lot cheaper). From owner-proteins@net.bio.net Sun Aug 02 23:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!howland.erols.net!newsfeed.internetmci.com!139.130.250.2!intgwpad.nntp.telstra.net!nsw.nntp.telstra.net!news.syd.connect.com.au!news.mel.connect.com.au!munnari.OZ.AU!news.unimelb.edu.au!ludwignt-4 From: murphy_r@licre.ludwig.edu.au (Roger Murphy) Newsgroups: bionet.molbio.proteins Subject: T-cell epitope prediction Date: Tue, 04 Aug 98 01:29:09 GMT Organization: Ludwig Institute for Cancer Research Lines: 20 Message-ID: <6q5rf8$gch$1@izvestia.its.unimelb.edu.au> NNTP-Posting-Host: ludwignt-4.austin.unimelb.edu.au X-Newsreader: News Xpress 2.0 Beta #0 Can anyone point me towards software that can predict T-cell epitopes, either web-based, shareware, or commercial? Thanks in advance! (Please also reply to my email) Roger Murphy Roger Murphy, Ph.D. Biological Production Facility Ludwig Institute for Cancer Research Austin & Repatriation Medical Centre Studley Road, Heidelberg, Vic. 3084 Australia. Tel 61-3-94965463 Fax 61-3-94965436 Email murphy_r@licre.ludwig.edu.au From owner-proteins@net.bio.net Mon Aug 03 23:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!howland.erols.net!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news-fra.maz.net!news-fra1.dfn.de!news-ber1.dfn.de!news-ham1.dfn.de!news.mu-luebeck.de!not-for-mail From: "Lars Komorowski" Newsgroups: bionet.molbio.proteins Subject: Re: pEt-Expression-Mystery Date: Tue, 4 Aug 1998 12:29:44 +0100 Organization: Med. Universitaet zu Luebeck Lines: 10 Message-ID: <6q6o1v$o8e$1@gwsun.medinf.mu-luebeck.de> References: <6q3sl4$dmt$1@gwsun.medinf.mu-luebeck.de> <54e4q6.tc.ln@wpxx02.toxi.uni-wuerzburg.de> NNTP-Posting-Host: 141.83.167.168 X-Newsreader: Microsoft Outlook Express 4.71.1712.3 X-MimeOLE: Produced By Microsoft MimeOLE V4.71.1712.3 >If your protein is unstable, it will not survive overnight induction. >Can you detect your protein by western blotting? > >--Cornelius. No, I can not. But I find a very huge band at 25kDa and it is IPTG-dependant. From owner-proteins@net.bio.net Mon Aug 03 23:00:00 1998 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!london-news-feed1.bbnplanet.com!news.bbnplanet.com!diablo.theplanet.net!btnet-feed2!btnet-peer!btnet!nntp.news.xara.net!xara.net!interpath.net!news-relay.ncren.net!news.wfu.edu!news From: "Julianne M. Braun" Newsgroups: alt.bio.technology,bionet.molbio.proteins Subject: Fish Skin Gelatin Date: Tue, 04 Aug 1998 14:00:45 -0400 Organization: Dept of Chemistry, Wake Forest University Lines: 16 Message-ID: <35C74C4D.D8022736@wfu.edu> References: <6pqku3$7gj$1@heliodor.xara.net> <35C6333E.1D10@le.ac.uk> NNTP-Posting-Host: braun.computer.wfu.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.05 [en] (Win95; I) To: "Dr E. Buxbaum" , EB15@le.ac.uk I just purchased some fish skin gelatin from Sigma. Any suggestions on how to store the stuff so that it can be opened more than once? Is the problem with the bottle related to the gelatin acting as a glue to stick the cap to the bottle? TIA. Julianne M. Braun Dr E. Buxbaum wrote: > A former boss of mine swore by fish skin gelatine, a liquid of the > colour and consitency of used motor oil, but freely soluble in PBS. The > stuff is available from Sigma, but be carefull: their bottle cap is ill > designed and you won't be able to open it a second time. BSA or common > gelatine are also possible. From owner-proteins@net.bio.net Mon Aug 03 23:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!island.idirect.com!feed.nntp.acc.ca!newsfeed.nyu.edu!newsfeed.sgi.net!pitt.edu!not-for-mail From: pxpst2@SPAM.SUXS.unixs.cis.pitt.edu (Peter) Newsgroups: bionet.molbio.proteins Subject: Re: T-cell epitope prediction Date: Tue, 04 Aug 1998 11:26:57 -0500 Organization: University of Pittsburgh Lines: 28 Message-ID: References: <6q5rf8$gch$1@izvestia.its.unimelb.edu.au> NNTP-Posting-Host: pelli.pathology.pitt.edu Mail-Copies-To: pxpst2@SPAM.SUXS.unixs.cis.pitt.edu X-Newsreader: MT-NewsWatcher 2.4.4 In article , bpmurray*STUFFER*@socrates.ucsf.edu (Bernard P. Murray, PhD) wrote: > In article <6q5rf8$gch$1@izvestia.its.unimelb.edu.au>, > murphy_r@licre.ludwig.edu.au (Roger Murphy) wrote: > > > Can anyone point me towards software that can predict T-cell epitopes, either > > web-based, shareware, or commercial? > > Thanks in advance! (Please also reply to my email) > > Roger Murphy > > You don't say what platform on which you intend to use it > but if DOS will do I have used a program called EPIPLOT that > I grabbed from the EMBL fileserver > eg. ftp://ftp.ebi.ac.uk/pub/software/dos/epiplot$.exe Just out of curiousity, How good is that program at such a prediction? Does it come close to accurate predictions? Peter -- "Don't you eat that yellow snow watch out where the Huskies go" FZ --------------------------------------------------------------------- From owner-proteins@net.bio.net Mon Aug 03 23:00:00 1998 From: Cornelius Krasel Subject: Re: pEt-Expression-Mystery Newsgroups: bionet.molbio.proteins References: <6q3sl4$dmt$1@gwsun.medinf.mu-luebeck.de> <54e4q6.tc.ln@wpxx02.toxi.uni-wuerzburg.de> <6q6o1v$o8e$1@gwsun.medinf.mu-luebeck.de> User-Agent: tin/pre-1.4-980514 (UNIX) (Linux/2.0.34 (i486)) Date: Tue, 4 Aug 1998 16:06:50 +0200 Message-ID: NNTP-Posting-Host: wpxx02.toxi.uni-wuerzburg.de Lines: 18 Path: biosci!news.stanford.edu!newsfeed.concentric.net!feed2.news.erols.com!erols!newsfeed.internetmci.com!194.162.162.196!newsfeed.nacamar.de!uni-erlangen.de!uni-wuerzburg.de!not-for-mail Lars Komorowski wrote: >>If your protein is unstable, it will not survive overnight induction. >>Can you detect your protein by western blotting? > > No, I can not. But I find a very huge band at 25kDa and it is > IPTG-dependant. Have you looked for this band in cells which were transformed with empty pET vector? This is usually the best control since induced beta-galactosidase, T4 RNA polymerase etc. will show up there as well. --Cornelius. -- /* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */ /* D-97078 Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de SP4 */ /* "Science is the game we play with God to find out what His rules are." */ From owner-proteins@net.bio.net Mon Aug 03 23:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!howland.erols.net!news-peer.sprintlink.net!news-backup-east.sprintlink.net!news.sprintlink.net!128.218.95.22!itssrv1.ucsf.edu!macmac-2.ucsf.edu!user From: bpmurray*STUFFER*@socrates.ucsf.edu (Bernard P. Murray, PhD) Newsgroups: bionet.molbio.proteins Subject: Re: T-cell epitope prediction Date: Tue, 04 Aug 1998 02:00:14 -0700 Organization: University of California, San Francisco Lines: 24 Message-ID: References: <6q5rf8$gch$1@izvestia.its.unimelb.edu.au> NNTP-Posting-Host: macmac-2.ucsf.edu In article <6q5rf8$gch$1@izvestia.its.unimelb.edu.au>, murphy_r@licre.ludwig.edu.au (Roger Murphy) wrote: > Can anyone point me towards software that can predict T-cell epitopes, either > web-based, shareware, or commercial? > Thanks in advance! (Please also reply to my email) > Roger Murphy You don't say what platform on which you intend to use it but if DOS will do I have used a program called EPIPLOT that I grabbed from the EMBL fileserver eg. ftp://ftp.ebi.ac.uk/pub/software/dos/epiplot$.exe If this isn't exactly what you want there may be something else for you on that server. It may also be worth a look at ExPasy to see if their web server has this facility (http://expasy.hcuge.ch) If you don't receive any useful replies here you may wish to try bionet.software as another newsgroup. I hope that this helps, Bernard -- Bernard P. Murray, PhD Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA From owner-proteins@net.bio.net Mon Aug 03 23:00:00 1998 Newsgroups: bionet.molbio.proteins Path: biosci!agate!newsfeed.berkeley.edu!howland.erols.net!newsfeed.internetmci.com!194.162.162.196!newsfeed.nacamar.de!nntp.news.xara.net!xara.net!server6.netnews.ja.net!leeds.ac.uk!news From: bmbjmm@bmb.leeds.ac.uk (Jez Murray) Subject: dialysis X-Accept-Language: en Message-ID: <35C74387.8CD35692@bmb.leeds.ac.uk> NNTP-Posting-Host: bmbsgi18.leeds.ac.uk X-Mailer: Mozilla 4.5b1 [en] (X11; I; IRIX 6.3 IP32) Content-Type: text/plain; charset=us-ascii Organization: University of Leeds MIME-Version: 1.0 Date: Tue, 4 Aug 1998 18:26:02 +0100 (BST) Lines: 17 Content-Transfer-Encoding: 7bit check out D. Doonan protocols in protein purification part of the humana press spiral bound series ...loads of practical tips that work -- . .-. .-. .-. .-. .-. .-. .-. .-. .-. .-. .-. .-. .-. .- |\ /|||X|||\ /|||X|||\ /|||X|||\ /|||X|||\ /|||X|||\ /|||X|||\ /|||X|| ||X|||/ \|||X|||/ \|||X|||/ \|||X|||/ \|||X|||/ \|||X|||/ \|||X|||/ \| -' `-' `-' `-' `-' `-' `-' `-' `-' `-' `-' `-' `-' `-' ` Jeremy Murray MAIL: bmbjmm@bmb.leeds.ac.uk Biochemistry & Molecular Biology TEL: +44 113 233 2591 Leeds University, LEEDS LS2 9JT FAX: +44 113 233 3167 http://www.smb.leeds.ac.uk/wwwsb/jez.html . .-. .-. .-. .-. .-. .-. .-. .-. .-. .-. .-. .-. .-. .- |\ /|||X|||\ /|||X|||\ /|||X|||\ /|||X|||\ /|||X|||\ /|||X|||\ /|||X|| ||X|||/ \|||X|||/ \|||X|||/ \|||X|||/ \|||X|||/ \|||X|||/ \|||X|||/ \| -' `-' `-' `-' `-' `-' `-' `-' `-' `-' `-' `-' `-' `-' ` From owner-proteins@net.bio.net Mon Aug 03 23:00:00 1998 Newsgroups: bionet.molbio.proteins Path: biosci!agate!newsfeed.berkeley.edu!howland.erols.net!news-peer.sprintlink.net!news.sprintlink.net!vixen.cso.uiuc.edu!uchinews!keith.bsd.uchicago.edu!user From: keith@cummings.uchicago.edu (Brian Keith) Subject: src homology domains X-Nntp-Posting-Host: keith.bsd.uchicago.edu Message-ID: Followup-To: srao1@midway.uchicago.edu Sender: news@midway.uchicago.edu (News Administrator) Organization: University of Chicago Date: Tue, 4 Aug 1998 16:45:00 GMT Lines: 5 I was wondering if anyone know of a databse where protein/DNA sequences could be submitted and analyzed for the presence of SH1, SH2, SH3, SH4, proline-rich domains, etc.. Thanks, Sid From owner-proteins@net.bio.net Tue Aug 04 23:00:00 1998 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.internetmci.com!193.190.198.39!news.belnet.be!inf6serv.rug.ac.be!not-for-mail From: srikrishnan Newsgroups: bionet.molbio.proteins Subject: Re: Blotting onto PVDF Date: Wed, 05 Aug 1998 10:12:31 +0200 Organization: lab of genetics / univ gent /belgium Lines: 23 Message-ID: <35C813EF.3226@gengenp.rug.ac.be> References: <35C63534.137A@le.ac.uk> NNTP-Posting-Host: altion.rug.ac.be Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01Gold (Win95; I) Dr E. Buxbaum wrote: > > Karen spink wrote: > > > > Has anyone had any problems Western blotting onto PVDF in CAPS buffer? I am > > trying to get a sequence on my protein and it appears that the efficiency > > of transfer is letting me down. Could it be the buffer, (10mM CAPS, 10% > > methanol, pH 11) ? I have had no problems blotting onto nitrocellulose > > using tris-glycine buffers. > > It may be the high detergent concentration which is causing problems. If > you want to avoid the glycine, why not use Dunn's buffer (published in > Anal. Biochem. a couple of years back, NaHCO3/Na2CO3, which can be > supplemented with methanol or SDS as needed). I use it routinely > (because I found the transfer efficiency better than with tris-gly, and > also because it is a lot cheaper). hi karen, i read about your problems with caps buffer, we too here in the lab used this buffer , but i also tried to blot on pvdf with tris glycine buffer and it works good too. try it and let me know. krish From owner-proteins@net.bio.net Tue Aug 04 23:00:00 1998 Path: biosci!news.stanford.edu!logbridge.uoregon.edu!news-peer.gip.net!news-dc.gip.net!news.gsl.net!gip.net!duke.telepac.pt!news.telepac.pt!not-for-mail From: "Ricardo" Newsgroups: bionet.molbio.proteins Subject: Protein overload Date: 5 Aug 1998 11:32:54 GMT Lines: 9 Message-ID: <01bdbf83$6d27fc80$fda941c2@default> NNTP-Posting-Host: 194.65.169.254 X-Newsreader: Microsoft Internet News 4.70.1161 Dear reader, I'm a Medicine student in Portugal, and I would like to know if anyone can tell me something about protein overload effects, after protein ingestion, in the liver. Thanks in advance, Retrieve message to: rjtribeiro@mail.telepac.pt From owner-proteins@net.bio.net Tue Aug 04 23:00:00 1998 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsxfer3.itd.umich.edu!news.eecs.umich.edu!news.bu.edu!ppp1 From: mlamkin@bu.edu (Mark Lamkin) Newsgroups: bionet.molbio.proteins Subject: Re: Vaccuum dialysis? Date: Wed, 05 Aug 98 15:14:34 GMT Organization: Boston University Lines: 24 Message-ID: <6q9t2s$78b$1@news1.bu.edu> References: NNTP-Posting-Host: ppp-75-8.bu.edu X-Newsreader: News Xpress 2.0 In article , i.mcfarlane@icrf.icnet.uk (Ian McFarlane) wrote: >Once upon a time somebody mentioned that there was some trick with >dialysis tubing and vaccuum that could be performed to concentrate >proteins (flash dialysis?). Does anyone know how to do this or is my >memory failing me? > >Thanks, > >Ian Mc It's been a while since I did it but as I recall my vacuum dialysis set was as follows: Use an empty vacuum flask and rubber stopper with two large holes. Put one P1000 pipette tip (with end melted) in one hole in the stopper as a plug. You will want to use this to release the vacuum when you are finished. Fill the dialysis tubing with your sample leaving one end open. Slip it up through the second hole in the stopper. Now use the other pipette tip (P1000) with its tapered end cut off and slip it in open end of the tubing and press it tight in the stopper hole. The dialysis tubing should be in the flask and now you apply vacuum using a water aspirator. Buffer will be drawn out of the membrane and into the flask. You can continue to add more sample to the dialysis membrane through the pipette tip. Mark Lamkin From owner-proteins@net.bio.net Tue Aug 04 23:00:00 1998 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.wli.net!howland.erols.net!news-peer.gip.net!news-raspail.gip.net!news.gsl.net!gip.net!news.belnet.be!inf6serv.rug.ac.be!not-for-mail From: srikrishnan Newsgroups: bionet.molbio.proteins Subject: enzyme activity with fad as cofactor Date: Wed, 05 Aug 1998 10:21:00 +0200 Organization: lab of genetics / univ gent /belgium Lines: 6 Message-ID: <35C815EC.63AF@gengenp.rug.ac.be> NNTP-Posting-Host: altion.rug.ac.be Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01Gold (Win95; I) hi readers, i would like to know if anyone has used fad as cofactor to do enzyme activity from crude extracts or has anyone tried in gel staining with native proteins. i would really apprecite if someone can help me in this regard. krish From owner-proteins@net.bio.net Wed Aug 05 23:00:00 1998 Path: biosci!agate!not-for-mail From: lhom@OCF.Berkeley.EDU (Louis Hom) Newsgroups: bionet.molbio.proteins Subject: Re: Electron microscopy of proteins Date: 6 Aug 1998 21:06:02 GMT Organization: Univ. of California Berkeley Open Computing Facility Lines: 12 Message-ID: <6qd5rq$h6a$1@agate.berkeley.edu> References: <35C98E51.7F7C@uni-koeln.de> <6qced1$3f4_002@doit.wisc.edu> NNTP-Posting-Host: ocf.berkeley.edu In article <6qced1$3f4_002@doit.wisc.edu>, Dima Klenchin wrote: > >As far as I know, the density of all proteins is ~ the same. Hence, if you and I *think* the density of typical proteins is about 1.3 g/mL. maybe Creighton or Lehninger has a more precise figure. -- ______________________________________________________________________________ Lou Hom >K'93 lhom@ocf.berkeley.edu http://www.ocf.berkeley.edu/~lhom/ From owner-proteins@net.bio.net Wed Aug 05 23:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!newsxfer3.itd.umich.edu!news.internetMCI.com!newsfeed.internetmci.com!194.72.7.126!btnet-peer!btnet!rill.news.pipex.net!pipex!main.de.uu.net!news-reader.dortmund.de.uu.net!not-for-mail From: Kay Hofmann Newsgroups: bionet.molbio.proteins Subject: Re: src homology domains Date: Thu, 06 Aug 1998 16:41:31 +0000 Organization: MEMOREC Lines: 17 Message-ID: <35C9DCBB.5D22CF56@isrec-sun1.unil.ch> References: NNTP-Posting-Host: 194.175.75.210 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: goof.de.uu.net 902421475 28816 194.175.75.210 (6 Aug 1998 16:37:55 GMT) X-Complaints-To: abuse@de.uu.net NNTP-Posting-Date: 6 Aug 1998 16:37:55 GMT X-Mailer: Mozilla 4.05 [en] (X11; I; Linux 2.0.32 i686) Brian Keith wrote: > > I was wondering if anyone know of a databse where protein/DNA sequences > could be submitted and analyzed for the presence of SH1, SH2, SH3, SH4, > proline-rich domains, etc.. Yes, you can find all of this at http://www.isrec.isb-sib.ch/software/PFSCAN_form.html =========================================================== Kay Hofmann, PhD Tel: +49 (221) 950 4814 Bioinformatics Group FAX: +49 (221) 950 4848 MEMOREC Stoffel GmbH Stoeckheimer Weg 1 D50829 Koeln/Germany E-mail: Kay.Hofmann@memorec.com From owner-proteins@net.bio.net Wed Aug 05 23:00:00 1998 Path: biosci!news.stanford.edu!logbridge.uoregon.edu!news-peer.gip.net!news-lond.gip.net!news.gsl.net!gip.net!nntp.news.xara.net!xara.net!server6.netnews.ja.net!server4.netnews.ja.net!server2.netnews.ja.net!news.reading.ac.uk!suma3!sar96mb From: Mayank Bhalla Newsgroups: bionet.molbio.proteins Subject: His tagged protein expression Date: Thu, 6 Aug 1998 15:27:22 +0100 Organization: University of Reading Lines: 17 Message-ID: References: <01BDBEF8.7256CD20@oak-pm1-50-178.dialup.slip.net> Reply-To: Mayank Bhalla NNTP-Posting-Host: suma3.rdg.ac.uk Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII In-Reply-To: <01BDBEF8.7256CD20@oak-pm1-50-178.dialup.slip.net> Hi, I am trying to express a 15 kDa protozoan protein as a His tag fusion in Qiagen's pQE30 vector. My problem is that the protein is being expressed as a trimer (possibly!?) as the over expressed protein appears to be about 45 kDa on the SDS-PAGE. This high molecular weight protein gets purified on the Ni-NTA resin supplied by Qiagen. Can any one offer ideas so as to why it may be happening and what can I do to get the protein of the right size? I have confirmed the construct by sequencing the whole of the insert and the junctions. Any help will be greatly appreciated. Many thanks Mayank From owner-proteins@net.bio.net Wed Aug 05 23:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!newsfeed.nyu.edu!btnet-peer!btnet!server2.netnews.ja.net!susx.ac.uk!sunx1!baps9 From: Matthew Hicks Newsgroups: bionet.molbio.proteins Subject: Re: His tagged protein expression Date: Fri, 7 Aug 1998 00:23:58 +0100 Organization: University of Sussex Lines: 22 Message-ID: References: <01BDBEF8.7256CD20@oak-pm1-50-178.dialup.slip.net> NNTP-Posting-Host: sunx1.central.susx.ac.uk Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII X-Sender: baps9@sunx1 In-Reply-To: If the protein is non-covalently linked as a trimer the SDS will (almost certainly!) denature it - ie the protein will run as monomer on the gel. However, if the polypeptide chains are disulphide linked then you will have to reduce these (covalent) bonds to observe the monomer molecular weight on your gel: To reduce the disulphide bonds the best plan is to use a mercaptan (eg. mercaptoethanol: HSCH2CH2OH - smells like farts/eggs though! ) in your gel. These are usually used in "denaturing" gels - check your recipie. If there is a disulphide-reducing agent in your gel then some weird stuff is going on (have you checked to see if the nucleotide sequence has been inserted more than once, ie the protein is being expressed as three repeats of the sequence, - sorry I am not a molecular biologist so I may be talking out of my ****/patronising you - but cannot think what else may be happening!). Let me know how you get on and good luck. Matt Hicks University of Sussex Falmer East Sussex UK From owner-proteins@net.bio.net Wed Aug 05 23:00:00 1998 Path: biosci!bloom-beacon.mit.edu!senator-bedfellow.mit.edu!usenet From: David Green Newsgroups: bionet.molbio.proteins Subject: Re: Electron microscopy of proteins Date: Thu, 06 Aug 1998 09:21:22 -0400 Organization: Massachusetts Institute of Technology Lines: 34 Message-ID: <35C9ADD2.6EBDBDB2@lms.mit.edu> References: <35C98E51.7F7C@uni-koeln.de> NNTP-Posting-Host: born.lms.mit.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.05 [en] (X11; U; Linux 2.0.30 i686) Dr. Christian Kupfer wrote: > I have some EM-Pictures of protein-komplexes bound to DNA. > I can determine the size of the proteins in angstroms, because > I know the size of the DNA. > > Is there anybody who can tell me how to determine the molecular > mass just by knowing the radius in angstroms? Or is there any good > review/ book out there where I can find answers? > > thanks in advance > > christian You could get a rough estimate by assuming close packing in the protein to calculate the expected number of heavy atoms and then plug in an average heavy atom mass (14 is probably a good number). Note that this will only give you only an estimate of the mass (maybe to an order of magnitude?). -- ************************************************ David F. Green Department of Chemistry Massachusetts Institute of Technology 77 Massachusetts Ave., Rm. 6-133 Cambridge, MA 02139 E-mail: dfgreen@lms.mit.edu Phone: (617) 258-6229 ************************************************ From owner-proteins@net.bio.net Wed Aug 05 23:00:00 1998 Message-ID: <35CA04DC.DC218016@f-t.de> Date: Thu, 06 Aug 1998 21:32:44 +0200 From: COA X-Mailer: Mozilla 4.05 [en] (WinNT; I) MIME-Version: 1.0 Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Far Western Blotting Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit NNTP-Posting-Host: p146.ftb.zet.net Lines: 9 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.corridex.com!newspump.monmouth.com!newspeer.monmouth.com!newsfeed.ecrc.net!newsfeed2.ecrc.net!news.zeitung-online.net!p146.ftb.zet.net Xref: biosci bionet.molbio.methds-reagnts:69661 bionet.molbio.proteins:13127 Hi everybody, who can tell me something about the method Far Western Blotting ? Thanks Chris From owner-proteins@net.bio.net Wed Aug 05 23:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!news.maxwell.syr.edu!newsfeed1.swip.net!swipnet!masternews.telia.net!fu-berlin.de!RRZ.Uni-Koeln.DE!usenet From: "Dr. Christian Kupfer" Newsgroups: bionet.molbio.proteins Subject: Electron microscopy of proteins Date: Thu, 06 Aug 1998 13:06:57 +0200 Organization: Regional Computing Center, University of Cologne Lines: 11 Message-ID: <35C98E51.7F7C@uni-koeln.de> NNTP-Posting-Host: kemp3.genetik.uni-koeln.de Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.04Gold (Win95; I) I have some EM-Pictures of protein-komplexes bound to DNA. I can determine the size of the proteins in angstroms, because I know the size of the DNA. Is there anybody who can tell me how to determine the molecular mass just by knowing the radius in angstroms? Or is there any good review/ book out there where I can find answers? thanks in advance christian From owner-proteins@net.bio.net Wed Aug 05 23:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!newsfeed.wli.net!newsfeed.sgi.net!nntp.itis.com!news.doit.wisc.edu!Home From: klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins Subject: Re: Electron microscopy of proteins Date: Thu, 06 Aug 1998 14:25:37 GMT Organization: UW-Madison Lines: 13 Message-ID: <6qced1$3f4_002@doit.wisc.edu> References: <35C98E51.7F7C@uni-koeln.de> NNTP-Posting-Host: t-22-181-189.dialup.wisc.edu X-Newsreader: News Xpress 2.01 X-No-Archive: Yes In article <35C98E51.7F7C@uni-koeln.de>, "Dr. Christian Kupfer" wrote: >I have some EM-Pictures of protein-komplexes bound to DNA. >I can determine the size of the proteins in angstroms, because >I know the size of the DNA. > >Is there anybody who can tell me how to determine the molecular >mass just by knowing the radius in angstroms? Or is there any good >review/ book out there where I can find answers? As far as I know, the density of all proteins is ~ the same. Hence, if you know dimensions (is it _really_ sphere?), you know mass. Dima From owner-proteins@net.bio.net Thu Aug 06 23:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!news.maxwell.syr.edu!news-was.dfn.de!news-fra1.dfn.de!news-koe1.dfn.de!news-han1.dfn.de!news.tu-bs.de!rzlimes.gbf-braunschweig.de!not-for-mail From: Frank Bartels Newsgroups: bionet.molbio.proteins Subject: Re: pEt-Expression-Mystery Date: Fri, 07 Aug 1998 23:22:44 +0200 Organization: Gesellschaft fuer Biotechnologische Forschung mbH Lines: 27 Message-ID: <35CB7024.536@gbf.de> References: <6q3sl4$dmt$1@gwsun.medinf.mu-luebeck.de> NNTP-Posting-Host: mikdeg.gbf-braunschweig.de Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01Gold (WinNT; I) Lars Komorowski wrote: > > I am trying to overexpress a 17kDa-Protein in E.coli BL21 with Novagens > pET24a Vector. I inocculate my LB medium from an overnight culture, let the > culture grow until the OD reaches 0.6 and induce with 1 mM IPTG. After one > night there is a band at 25 kDa in SDS-PAGE made with total cell protein. > When I try to separate cytosolic proteins from the rest and make another > SDS-PAGE with both fractions there is no band there. > I have two questions: > 1. If the protein band is not the desired protein what can it be ? > 2. If the band represents the protein, is it possible that it is proteolysed > within minutes ? Hi Lars, It is certainly possible that your 17 kD protein appears as a 25 kD on a SDS gel since there are many reports about that (the reason can be that your 17 kD protein is basic or acidic). I dont know what your protocol is to separate the cytosolic proteins from the rest but can it be that your expressed protein is insoluble (mybe forming inclusion bodies)? If your prepare a crude cell extract and centrifuge afterwards the protein may precipitate. Load the supernatant and the pellet on the gel. The insoluble protein should appear in the pellet. Regards From owner-proteins@net.bio.net Thu Aug 06 23:00:00 1998 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.internetmci.com!165.87.194.242!newsm2.ibm.net!newsfeed.de.ibm.net!ibm.net!newsfeed.Austria.EU.net!fleetstreet.Austria.EU.net!not-for-mail From: Gerald Loeffler Newsgroups: bionet.molbio.proteins Subject: Re: src homology domains Date: Fri, 07 Aug 1998 17:28:05 +0200 Organization: Boehringer Ingelheim R&D Vienna Lines: 42 Message-ID: <35CB1D05.44DDD265@vienna.at> References: NNTP-Posting-Host: apollon.bender.co.at Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: fleetstreet.Austria.EU.net 902503718 13752 193.154.107.33 (7 Aug 1998 15:28:38 GMT) X-Complaints-To: abuse@Austria.EU.net NNTP-Posting-Date: 7 Aug 1998 15:28:38 GMT X-Mailer: Mozilla 4.04 [en] (WinNT; I) Hi! These are some important protein domain servers that should contain the Shn domains as well as many others: http://www.sanger.ac.uk/Software/Pfam/ and http://www.isrec.isb-sib.ch/software/PFSCAN_form.html and http://www.bork.embl-heidelberg.de/Modules/sinput.shtml and http://www.biochem.ucl.ac.uk/bsm/dbbrowser/PRINTS/PRINTS.html and http://www.blocks.fhcrc.org/blocks_search.html hope this helps, best wishes, gerald Brian Keith wrote: > > I was wondering if anyone know of a databse where protein/DNA sequences > could be submitted and analyzed for the presence of SH1, SH2, SH3, SH4, > proline-rich domains, etc.. > > Thanks, Sid -- Gerald Loeffler - Bioinformatics Scientist Boehringer Ingelheim R&D Vienna, Molecular Biology Department Email: Gerald.Loeffler@vienna.at Phone: +43 676 3289588 (and +43 1 80105 634) Fax: +43 1 80105 683 Smail: Bender+Co, Dr. Boehringer-Gasse 5-11, A-1121 Vienna, Austria From owner-proteins@net.bio.net Thu Aug 06 23:00:00 1998 Path: biosci!tcd.ie!Brackena From: Brackena@tcd.ie (Adrian Bracken) Newsgroups: bionet.molbio.proteins Subject: S-35 Methionine labelling yeast cells? Date: 7 Aug 1998 08:55:35 -0700 Organization: Trinity College Lines: 12 Sender: daemon@net.bio.net Distribution: world Message-ID: <35CB2450.608@tcd.ie> NNTP-Posting-Host: net.bio.net Hi, I am wondering if it is possible to S-35 Methionine label yeast cells? I want to label cells, prepare extracts, immunoprecipitate a ribonuclear-protein complex and run the protein content out on an SDS gel. Because the protein content will not be detectable by silver-staining, I hope the Methinine labelled proteins will be visable after exposure to film. Do you know if this approach has been used before? If so, have you any recent publications on it? Please, if you could mail me at brackena@tcd.ie it would be great, Thanks a million, Adrian Bracken From owner-proteins@net.bio.net Thu Aug 06 23:00:00 1998 From: "Titus R.Wapato" Subject: Medical Sales Rep (FLEX-LA) Date: Fri, 7 Aug 1998 15:11:32 -0700 Lines: 29 X-Newsreader: Microsoft Outlook Express 4.71.1712.3 X-MimeOLE: Produced By Microsoft MimeOLE V4.71.1712.3 Message-ID: Newsgroups: bionet.molbio.proteins NNTP-Posting-Host: 1Cust87.tnt1.santa-monica2.ca.da.uu.net [208.250.88.87] Path: biosci!agate!newsfeed.berkeley.edu!howland.erols.net!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.internetmci.com!207.68.152.12!upnetnews01!upnetnews05 Job Posting re : LA Area Our client, an innovative clinical diagnostic company with exclusive proprietary technology, is seeking a “Flex” Sales Representative to call on physicians in the LA area. You would join a national sales force of 80 representatives who market and sell diagnostic products to physicians within commuting distance of their homes. These positions are “flex”, that is, the sales rep typically works 25-30 hours per week based on a schedule that they set for themselves. The position is an excellent opportunity for working parents with small children, or can provide a substantial income for second wage earner in a family. This is also a good position for an individual who is semi-retired who like to still be active . The ideal candidate should have prior sales experience selling medical products or have worked in a technical capacity in a laboratory setting. The company offers an compensation program which includes base salary, commission, sales incentives, paid expenses, stock options and 401(k) savings program. For more information please call Dish Taylor at 310-394-7858 or else E-mail at Ty_n_Dish@msn.com Fax resumes to (310) 394 7061 From owner-proteins@net.bio.net Thu Aug 06 23:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!news.maxwell.syr.edu!newscore.univie.ac.at!Cabal.CESspool!bofh.vszbr.cz!pegasus.csx.cam.ac.uk!hgmp.mrc.ac.uk!not-for-mail From: Rolf Apweiler Newsgroups: bionet.molbio.proteins Subject: Release 7 of TrEMBL, the protein sequence database supplementing SWISS-PROT Date: Fri, 07 Aug 1998 17:28:51 +0100 Organization: MRC Human Genome Mapping Project Resource Centre Lines: 96 Message-ID: <35CB2B43.F374B699@ebi.ac.uk> NNTP-Posting-Host: dhcp4.ebi.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.04 [en] (Win95; I) INTRODUCTION ============ TrEMBL is the protein sequence database supplementing the SWISS-PROT Protein Sequence Data Bank. TrEMBL contains the translations of all coding sequences (CDS) present in the EMBL Nucleotide Sequence Database not yet integrated in SWISS-PROT. TrEMBL can be considered as a preliminary section of SWISS-PROT. For all TrEMBL entries which should finally be upgraded to the standard SWISS-PROT quality, SWISS-PROT accession numbers have been assigned. RELEASE 7.0 OF TREMBL ===================== This TrEMBL release is created from the EMBL Nucleotide Sequence Database release 55 and contains 193'860 sequence entries, comprising 53'601'062 amino acids. TrEMBL is split in two main sections; SP-TrEMBL and REM-TrEMBL: SP-TrEMBL (SWISS-PROT TrEMBL) contains the entries (165'420), which should be eventually incorporated into SWISS-PROT. SWISS-PROT accession numbers have been assigned for all SP-TrEMBL entries. SP-TrEMBL is organized in subsections: arc.dat (Archea): 7434 entries fun.dat (Fungi): 5261 entries hum.dat (Human): 6976 entries inv.dat (Invertebrates): 21991 entries mam.dat (Other Mammals): 2684 entries mhc.dat (MHC proteins): 3601 entries org.dat (Organelles): 12699 entries phg.dat (Bacteriophages): 1604 entries pln.dat (Plants): 12668 entries pro.dat (Prokaryotes): 35857 entries rod.dat (Rodents): 6346 entries unc.dat (Unclassified): 88 entries vrl.dat (Viruses): 44561 entries vrt.dat (Other Vertebrates): 3650 entries REM-TrEMBL (REMaining TrEMBL) contains the entries (28'440) that we do not want to include in SWISS-PROT. WEEKLY UPDATES OF TREMBL AND NON-REDUNDANT DATA SETS ==================================================== Weekly cumulative updates of TrEMBL are available by anonymous FTP and from the EBI SRS server. We also produce every week a complete non-redundant protein sequence collection by providing three compressed files (these are in the directory /pub/databases/sp_tr_nrdb on the EBI FTP server): sprot.dat.Z, trembl.dat.Z and trembl_new.dat.Z. ACCESS/DATA DISTRIBUTION ======================== FTP server: ftp.ebi.ac.uk/pub/databases/trembl SRS server: http://srs.ebi.ac.uk/ TrEMBL is also available on the SWISS-PROT CD-ROM. SWISS-PROT + TrEMBL is searchable on the following servers at the EBI: FASTA3 (http://www2.ebi.ac.uk/fasta3/) BLAST2 (http://www2.ebi.ac.uk/blast2/) Bic_sw (http://www2.ebi.ac.uk/bic_sw/) Scanps (http://www2.ebi.ac.uk/scanps/) SSearch (http://www2.ebi.ac.uk/ssearch3/) TREMBL HAS BEEN PREPARED BY: ============================ Rolf Apweiler, Sergio Contrino, Wolfgang Fleischmann, Henning Hermjakob, Vivien Junker, Stephanie Kappus, Fiona Lang, Michele Magrane, Maria Jesus Martin, Steffen Moeller, Nicoletta Mitaritonna and Claire O'Donovan at the EMBL Outstation - European Bioinformatics Institute (EBI) in Hinxton, UK; Amos Bairoch and Alain Gateau at the Swiss Institute of Bioinformatics in Geneva, Switzerland. ======================================================================= Rolf Apweiler | SWISS-PROT Coordinator EMBL Outstation | Email:apweiler@ebi.ac.uk European Bioinformatics Institute (EBI) | URL: http://www.ebi.ac.uk Wellcome Trust Genome Campus, Hinxton | Tel: +44 (1223) 494435 Cambridge CB10 1SD, UK | Fax: +44 (1223) 494968 ======================================================================== From owner-proteins@net.bio.net Thu Aug 06 23:00:00 1998 Path: biosci!news.stanford.edu!logbridge.uoregon.edu!news.maxwell.syr.edu!ptdnetP!newsgate.ptd.net!news.cc.ukans.edu!not-for-mail From: PGegen@UKans.edu (Dr. Peter Gegenheimer) Newsgroups: bionet.molbio.proteins Subject: Re: Electron microscopy of proteins Date: 7 Aug 1998 23:38:24 GMT Organization: Univ. Kansas (Biochemistry) Lines: 53 Message-ID: <7opiGDf98QgB-pn2-75d0dhnrQzsW@rnaworld.bio.ukans.edu> References: <35C98E51.7F7C@uni-koeln.de> <6qced1$3f4_002@doit.wisc.edu> Reply-To: PGegen@UKans.edu NNTP-Posting-Host: rnaworld.bio.ukans.edu Mime-Version: 1.0 Content-Type: text/plain; charset="us-ascii" Content-Transfer-Encoding: 8bit X-Newsreader: ProNews/2 Version 1.00 On Thu, 6 Aug 1998 14:25:37, klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) wrote: > In article <35C98E51.7F7C@uni-koeln.de>, "Dr. Christian Kupfer" wrote: --- snip --- > >Is there anybody who can tell me how to determine the molecular > >mass just by knowing the radius in angstroms? Or is there any good > >review/ book out there where I can find answers? > > As far as I know, the density of all proteins is ~ the same. Hence, if you > know dimensions (is it _really_ sphere?), you know mass. Another response gave protein density as ~1.3 g/ml. This refers to the *buoyant* density (theta) of protein (e.g., in solutions of CsCl, etc.). This is not the same as the physical density (rho), which is the reciprocal of the partial specific volume (nu). I looked around for a value of nu and couldn't find one--it should be in any biological physical chemistry text. I did have at hand ME Hamilton, Meth Enz. 20:512-521 (1971). This gives a value for anhydrous ribosomal protein of nu=0.74, said to have been calculated from the amino acid composition. The corresponding density=1.35 g/ml. I have no idea how to calculate this, but the partial specific volumes of amino acids are not secret. (For the same protein in Cs salt solutions, an extrapolated value of nu=~0.8, so density=1.25. My understanding is that a protein's apparent density will decrease as it is solvated by H2O, and increase as it binds various salts.) Density of 1.30 corresponds to nu=0.77, 1.32 to nu=0.76, and 1.33 to nu=0.75. My bottom line would be that using a density of 1.33 g/ml (nu=0.75) would be a reasonable estimate. o------------------------------------------------------------------- --o | Dr. Peter Gegenheimer | Vox: 785-864-3939 FAX: 785-864-5321 | | Departments of Biochemistry | PGegen@UKans.edu | | and of Botany | http://RNAworld.Bio.UKans.edu/ | | | | | University of Kansas | | | 2045 Haworth Hall | "The sleep of reason produces | | Lawrence KS 66045-2106 | monsters." Goya | o_____________________________|_____________________________________ __o From owner-proteins@net.bio.net Thu Aug 06 23:00:00 1998 Path: biosci!news.stanford.edu!logbridge.uoregon.edu!howland.erols.net!newshub.northeast.verio.net!ptdnetP!newsgate.ptd.net!news.cc.ukans.edu!not-for-mail From: PGegen@UKans.edu (Dr. Peter Gegenheimer) Newsgroups: bionet.molbio.proteins Subject: Re: pEt-Expression-Mystery Date: 7 Aug 1998 23:57:32 GMT Organization: Univ. Kansas (Biochemistry) Lines: 54 Message-ID: <7opiGDf98QgB-pn2-IBbv8MwbIcDl@rnaworld.bio.ukans.edu> References: <6q3sl4$dmt$1@gwsun.medinf.mu-luebeck.de> Reply-To: PGegen@UKans.edu NNTP-Posting-Host: rnaworld.bio.ukans.edu Mime-Version: 1.0 Content-Type: text/plain; charset="us-ascii" Content-Transfer-Encoding: 8bit X-Newsreader: ProNews/2 Version 1.00 On Mon, 3 Aug 1998 09:29:49, "Lars Komorowski" wrote: > I am trying to overexpress a 17kDa-Protein in E.coli BL21 with Novagens > pET24a Vector. I inocculate my LB medium from an overnight culture, let the > culture grow until the OD reaches 0.6 and induce with 1 mM IPTG. After one > night there is a band at 25 kDa in SDS-PAGE made with total cell protein. > When I try to separate cytosolic proteins from the rest and make another > SDS-PAGE with both fractions there is no band there. An another poster answered, it is common that overexpressed proteins will aggregate into insoluble "inclusion bodies". If you were really just examining the soluble cell fraction by SDS-PAGE, examine the cell debris. (If the protein is there, the cell debris ought to be a big white pellet.) The lesson from this is that when you think you've lost something, make sure you've accounted for 100% of the places where it could be--no matter how unlikely. If the protein is in the pellet, you can get a reasonable purification by washing the pellet to remove trapped cytoplasmic protein. Homogenize the pellet (in tris/edta/NaCl or whatever you're using for cell lysis) and spin it back down; repeat 1-2X.] Addition of Triton or sarkosyl would help remove membrane proteins. Inclusion proteins are formed when nascent polypeptides don't fold completely, and the exposed "interior" regions, which are usually hydrophobic, of only polypeptide associate with adjacent polypeptide chains rather than with their "own" chain. To avoid inclusion body formation, two methods which have been well-documented in the literature to work _sometimes_ are: 1) Induce your target gene to a very low-level; use 10-100 micromolar IPTG and let the cells incubate (plenty of aeration!) for a day or more. 2) Grow the cells below room temperature--15 to 19C--just prior to and during induction. o------------------------------------------------------------------- --o | Dr. Peter Gegenheimer | Vox: 785-864-3939 FAX: 785-864-5321 | | Departments of Biochemistry | PGegen@UKans.edu | | and of Botany | http://RNAworld.Bio.UKans.edu/ | | | | | University of Kansas | | | 2045 Haworth Hall | "The sleep of reason produces | | Lawrence KS 66045-2106 | monsters." Goya | o_____________________________|_____________________________________ __o From owner-proteins@net.bio.net Fri Aug 07 23:00:00 1998 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.wli.net!feed2.news.erols.com!erols!newsfeed.internetmci.com!206.172.150.11!news1.bellglobal.com!torn!testinfo.uoguelph.ca!ccshst01!skrishna From: skrishna@uoguelph.ca (Sankaran Krishnaraj) Newsgroups: bionet.molbio.proteins Subject: Post doctoral position Date: 7 Aug 1998 20:01:52 GMT Organization: University of Guelph Lines: 20 Message-ID: <6qfmfg$hrj$2@testinfo.uoguelph.ca> NNTP-Posting-Host: 131.104.96.14 X-Newsreader: TIN [version 1.2 PL2] POST DOCTORAL POSITION AVAILABLE The Plant Cell Technology Laboratory in the Department of Plant Agriculture at the University of Guelph invites applications for a post doctoral position. Candidates must have a Ph.D. in plant sciences with demonstrated research productivity and experience in classical/ molecular genetics, or related field. Experience in plant tissue culture and genetic transformation techniques will be an asset. The successful candidate will be involved in a variety of research programs currently in progress in the PCT laboratory. Further details about the various program can be obtained at: http://www.uoguelph.ca/hortsci/cellculture/. Salary is competitive and commensurate with experience and qualifications. Screening of candidates will continue until the position is filled. Interested candidates should submit a letter of application, and an complete curriculum vitae, representative reprints and names, addresses of 3 referees, by 30 August 1998, to: Dr. Praveen K. Saxena, Department of Plant Agriculture, Plant Biotechnology Division, University of Guelph, Guelph, Ontario, Canada, N1G 2W1; E-mail: psaxena@ uoguelph.ca; FAX: 519-767-0755. From owner-proteins@net.bio.net Fri Aug 07 23:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!ihnp4.ucsd.edu!news1.ucsd.edu!not-for-mail From: "Antonin Tutter" Newsgroups: bionet.molbio.proteins Subject: Re: pEt-Expression-Mystery Date: Sat, 08 Aug 1998 00:47:56 -0700 Organization: University of California, San Deigo Lines: 38 Message-ID: <6qgvpn$gia$1@news1.ucsd.edu> References: <6q3sl4$dmt$1@gwsun.medinf.mu-luebeck.de> NNTP-Posting-Host: atutter.extern.ucsd.edu Mime-Version: 1.0 Content-Type: text/plain; charset="US-ASCII" Content-Transfer-Encoding: 7bit X-Newsreader: Microsoft Outlook Express for Macintosh - 4.01 (297) In article <6q3sl4$dmt$1@gwsun.medinf.mu-luebeck.de> , "Lars Komorowski" wrote: >I am trying to overexpress a 17kDa-Protein in E.coli BL21 with Novagens >pET24a Vector. I inocculate my LB medium from an overnight culture, let the >culture grow until the OD reaches 0.6 and induce with 1 mM IPTG. After one >night there is a band at 25 kDa in SDS-PAGE made with total cell protein. >When I try to separate cytosolic proteins from the rest and make another >SDS-PAGE with both fractions there is no band there. >I have two questions: >1. If the protein band is not the desired protein what can it be ? >2. If the band represents the protein, is it possible that it is proteolysed >within minutes ? > > 1) You probably should experiment with induction times and induction temperatures. Even when I express very stable proteins using the pET system in BL-21 cells, I only induce for 3 hours maximum. You may even try 1.5 hours at 30 degrees. 2) Some proteins migrate differently than expected. Ususally highly cationic peptides will migrate slower in the gel, appearing as higher molecular weight. This variance in apparent MW can be as much as 50%. 3) Has this protein been successfully expressed before? Have you verified the sequence? _______________________________________ Antonin Tutter Salk Institute for Biological Studies RBIO-J 10010 N. Torrey Pines Rd. La Jolla, CA 92037 email: atutter@aim.salk.edu web: http://www-biology.ucsd.edu/~atutter/ From owner-proteins@net.bio.net Fri Aug 07 23:00:00 1998 Path: biosci!news.stanford.edu!logbridge.uoregon.edu!fu-berlin.de!cpeter.dialup.fu-berlin.DE!not-for-mail From: deleteme_cpeter@zedat.fu-berlin.de (Christoph Peter) Newsgroups: bionet.molbio.proteins Subject: On-line dialysis for chromatography? Date: Fri, 07 Aug 1998 08:03:22 GMT Organization: Freie Universitaet Berlin Lines: 19 Message-ID: <35cab000.1477207@news.fu-berlin.de> Reply-To: deleteme_cpeter@zedat.fu-berlin.de NNTP-Posting-Host: cpeter.dialup.fu-berlin.de (130.133.221.185) Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Access: 16 17 18 X-Trace: fu-berlin.de 902573804 29803 (none) 130.133.221.185 X-Newsreader: Forte Free Agent 1.11/16.235 Hi All! I am looking for a gadget to change the buffer-composition in the effluent of a chromatographic column - on-line! Who knows names, web-pages etc.? Somebody told me he used something like that for FIA - any idea? TIA Christoph -- Christoph Peter PhD Student (Biotechnology) cpeter at zedat dot fu-berlin dot de >Alles wird gut!<(Beate) From owner-proteins@net.bio.net Fri Aug 07 23:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!newsfeed.nyu.edu!btnet-peer!btnet!dispose.news.demon.net!demon!newsfeed.nacamar.de!fu-berlin.de!cpeter.dialup.fu-berlin.DE!not-for-mail From: deleteme_cpeter@zedat.fu-berlin.de (Christoph Peter) Newsgroups: bionet.molbio.proteins Subject: BCA-Protein-Quantification: Time Drive? Date: Fri, 07 Aug 1998 08:03:24 GMT Organization: Freie Universitaet Berlin Message-ID: <35cab1d3.1944564@news.fu-berlin.de> Reply-To: deleteme_cpeter@zedat.fu-berlin.de NNTP-Posting-Host: cpeter.dialup.fu-berlin.de (130.133.221.185) Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Access: 16 17 18 X-Trace: fu-berlin.de 902573806 29803 (none) 130.133.221.185 X-Newsreader: Forte Free Agent 1.11/16.235 Lines: 27 Hi all! I use the bicinchoninic-acid-assay in a 96well format to determine/estimate protein concentrations. I always have a standard-dilution-row on every plate. When I tried to determine the concentration of a simple yeast-extract, I noted the following: - the slope of the calibration-line (mg as a function of A550) decreases with time (15min to 3hrs) - the concentration of my extract also decreases with time!!!! (calculated with corresponding slope!) As the assay is designed to be read after 30min and 30degrees C - when do I get the _correct_ concentration????????????????? MTIA Christoph -- Christoph Peter PhD Student (Biotechnology) cpeter at zedat dot fu-berlin dot de >Alles wird gut!<(Beate) From owner-proteins@net.bio.net Fri Aug 07 23:00:00 1998 Path: biosci!agate!not-for-mail From: lhom@OCF.Berkeley.EDU (Louis Hom) Newsgroups: bionet.molbio.proteins Subject: Caesar Salad Date: 9 Aug 1998 00:54:42 GMT Organization: Univ. of California Berkeley Open Computing Facility Lines: 13 Message-ID: <6qis0i$irp$1@agate.berkeley.edu> NNTP-Posting-Host: ocf.berkeley.edu Here in the States, Salmonella contamination of eggs is a significant problem -- significant enough that in some places it was against the law to serve raw egg products (such as Caesar salad dressing) in restaurants. It is often suggested, however, that you can acidify the beaten egg (with, e.g., lemon juice or vinegar), then heat it to a temperature that will kill the bugs, but the egg won't get cooked. My question: is increased thermostability following acidification common among proteins? Any ideas on how it would stabilize ovalbumin? -- ______________________________________________________________________________ Lou Hom >K'93 lhom@ocf.berkeley.edu http://www.ocf.berkeley.edu/~lhom/ From owner-proteins@net.bio.net Sat Aug 08 23:00:00 1998 Path: biosci!STUDENT2.MAHIDOL.AC.TH!g3937506 From: g3937506@STUDENT2.MAHIDOL.AC.TH (Jongrak Kittiworakarn) Newsgroups: bionet.molbio.proteins Subject: Re: How to get rid of DNA from sample of western blot? Date: 9 Aug 1998 20:21:06 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 10 Sender: daemon@net.bio.net Distribution: world Message-ID: <35CE658A.411C03EA@student.mahidol.ac.th> NNTP-Posting-Host: net.bio.net Hi Platini: If you sure that sticky thing is DNA, not carbohydrate, adding MgCl2 to a final concantration of 100mM into your sample together with SDS-PAGE sample buffer might be helpful. I found this method in Analytical Biochemistry a long time ago and so far it work well for me. Good luck, Jongrak From owner-proteins@net.bio.net Sat Aug 08 23:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!newsfeed.nyu.edu!newsfeed.sgi.net!pitt.edu!not-for-mail From: pxpst2@SPAM.SUXS.unixs.cis.pitt.edu (Peter) Newsgroups: bionet.molbio.proteins Subject: Re: BCA-Protein-Quantification: Time Drive? Date: Sun, 09 Aug 1998 14:12:58 -0500 Organization: University of Pittsburgh Lines: 35 Message-ID: References: <35cab1d3.1944564@news.fu-berlin.de> NNTP-Posting-Host: pelli.pathology.pitt.edu Mail-Copies-To: pxpst2@SPAM.SUXS.unixs.cis.pitt.edu X-Newsreader: MT-NewsWatcher 2.4.4 In article <35cab1d3.1944564@news.fu-berlin.de>, deleteme_cpeter@zedat.fu-berlin.de wrote: > Hi all! > > I use the bicinchoninic-acid-assay in a 96well format to > determine/estimate protein concentrations. I always have a > standard-dilution-row on every plate. > > When I tried to determine the concentration of a simple yeast-extract, > I noted the following: > > - the slope of the calibration-line (mg as a function of A550) > decreases with time (15min to 3hrs) > > - the concentration of my extract also decreases with time!!!! > (calculated with corresponding slope!) > > As the assay is designed to be read after 30min and 30degrees C - when > do I get the _correct_ concentration????????????????? > As long as all decreases occur to all samples , It does not matter. Incubation of the reaction at 37 C for 30 minutes and then read should be fine. The key is that all are read at the same time( samples and calibration curve) variability will take care of itself. Peter -- "Don't you eat that yellow snow watch out where the Huskies go" FZ --------------------------------------------------------------------- From owner-proteins@net.bio.net Sat Aug 08 23:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!news.maxwell.syr.edu!ix.netcom.com!news From: Mark Atlas Newsgroups: bionet.molbio.proteins Subject: Lab Equipment auction liquidation sale online with Going, Going...Sold! Date: Sun, 09 Aug 1998 15:21:22 GMT Organization: ICGNetcom Lines: 36 Message-ID: <6qkem6$l7s@dfw-ixnews6.ix.netcom.com> NNTP-Posting-Host: sji-ca4-79.ix.netcom.com X-NETCOM-Date: Sun Aug 09 10:19:34 AM CDT 1998 X-Newsreader: NETCOMplete/4.0 Lab Equipment auction liquidation sale online. Microscopes, and much more Visit http://www.going-going-sold.com for its upcoming liquidation auction. You may register (free) to see all of the details ahead of time before bidding. Aug 24th features a liquidation of a small chemistry lab. Featured items include Waters Alliance System opens 12,000. PDA based 2 years old. This is a $45,000 system FTIR ( Nicolet 410 and software) opens $7000. Fume hood Glassware Lab furniture Refrigerator ( explosion proof) General labware Also available is a large selection of mlabe equipment and instruments including GC, HPLC, Microscopes and much more. All equipment includes an in lab evaluation prior to relaesing funds to the seller. This insures that you recieve the equipment as it was defined on the Web site. Unlike other online auctions. All equipment must sell if reserve prices are met. Dealers and sellers do not have the ability to pull back on sale if bid is unfavorable please visit http://www.going-going-sold.com Going, Going...Sold! is a bonded auctioneer int the state of California and is owned and operated by Internet Auctioneers Intl (IAI) bond # SUR2721119. We are technically experienced with now over 25 years of sales background in lab instrumentation. This knowledge base is not available from ANY Dealer. IAI acts as an intermediary on your behalf. Please visit us today at http://www,going-going-sold.com Leasing and custom equipment search services for any item are available. From owner-proteins@net.bio.net Sat Aug 08 23:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!newsgate.cuhk.edu.hk!news.glink.net.hk!news From: Platini Kwok Newsgroups: bionet.molbio.proteins Subject: How to get rid of DNA from sample of western blot? Date: Mon, 10 Aug 1998 08:10:40 +0800 Organization: Global Link Information Services Ltd. Lines: 16 Message-ID: <35CE3A80.A546EB18@glink.net.hk> NNTP-Posting-Host: dialup97.glink.net.hk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.01 [tw] (Win95; I) X-Priority: ¤@¯ë) > Hi, > > Recently, I was involved in doing western blot on retina of SD rat and > I > found that there was a lot sticky, jelly like structure after boiling > with TBS with SDS, triton x-100 buffer although I centrifuged it can > sonicate the supernatant, it still here. How should I do so that I can > > get rid of it? These sticky material gave vertically line rather than > horizontal band in SDS page or NC membrane. > > Thank you! From owner-proteins@net.bio.net Sat Aug 08 23:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!newsfeed.nyu.edu!newsfeed.sgi.net!nntp.itis.com!news.doit.wisc.edu!Home From: klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins Subject: Re: BCA-Protein-Quantification: Time Drive? Date: Mon, 10 Aug 1998 00:05:35 GMT Organization: UW-Madison Lines: 30 Message-ID: <6qldgf$108_002@doit.wisc.edu> References: <35cab1d3.1944564@news.fu-berlin.de> NNTP-Posting-Host: t-43-183-142.dialup.wisc.edu X-Newsreader: News Xpress 2.01 X-No-Archive: Yes In article <35cab1d3.1944564@news.fu-berlin.de>, deleteme_cpeter@zedat.fu-berlin.de wrote: >Hi all! > >I use the bicinchoninic-acid-assay in a 96well format to >determine/estimate protein concentrations. I always have a >standard-dilution-row on every plate. > >When I tried to determine the concentration of a simple yeast-extract, >I noted the following: > >- the slope of the calibration-line (mg as a function of A550) >decreases with time (15min to 3hrs) > >- the concentration of my extract also decreases with time!!!! >(calculated with corresponding slope!) > >As the assay is designed to be read after 30min and 30degrees C - when >do I get the _correct_ concentration????????????????? Anywhere it is convenient for you! That's what calibration curve is for. I, for example, to achive higher sensitivity, do the following: 1) put twice recommended copper (Pierce's BCA kit), 2) leave samples for 3 hours at 50C. Works like a charm. Quite frankly, I have difficult time understanding how and where the color that forms in BCA reaction can disappear with time. - Dima From owner-proteins@net.bio.net Sun Aug 09 23:00:00 1998 Path: biosci!news.stanford.edu!Cabal.CESspool!bofh.vszbr.cz!howland.erols.net!woodstock.news.demon.net!demon!dispose.news.demon.net!demon!peer.news.zetnet.net!peer.news.bb.u-net.net!u-net!yama.mcc.ac.uk!hydraulix.bangor.ac.uk!manager From: "S.Kammerer" Newsgroups: bionet.molbio.proteins Subject: enzyme assay Date: Mon, 10 Aug 1998 10:16:32 +0100 Organization: University of Wales, Bangor. Message-ID: <35CEBA70.6773@bangor.ac.uk> Reply-To: bsx404@bangor.ac.uk NNTP-Posting-Host: bramlab1.bangor.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (Win95; I) Lines: 6 I want to do an elastase enzyme assay with a protease. I tried to stop the reaction with hydrogen chloride but the colour of the solution changed and therefore I can not quantify the reaction. I would like to know how to stop my reaction at regular time to process a kinetics study. From owner-proteins@net.bio.net Sun Aug 09 23:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!howland.erols.net!newsfeed.nacamar.de!rill.news.pipex.net!pipex!server1.netnews.ja.net!leicester!usenet From: Dr Engelbert Buxbaum Newsgroups: bionet.molbio.proteins Subject: Re: S-35 Methionine labelling yeast cells? Date: Mon, 10 Aug 1998 14:33:44 -0700 Organization: University of Leicester (PCFS User) Lines: 16 Message-ID: <35CF6738.1AE4@le.ac.uk> References: <35CB2450.608@tcd.ie> Reply-To: eb15@le.ac.uk NNTP-Posting-Host: pc220.tulip5.le.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Win16; I) Adrian Bracken wrote: > > Hi, > I am wondering if it is possible to S-35 Methionine label yeast cells? You can label proteins in any actively metabolizing organism by incubatin in a medium without "cold" Met (or Cys) by adding the labeled amino acid (there are mixtures of 35-S Met and Cys available which are cheaper and offer better labeling than either aa alone). Incubate the cells for an appropriate amount of time (try a time series with a small amount first, say 10 min to 2-3 h). The correct time depends on the sort of protein (synthesis and turnover rates, posttranslational processing ect). Avoid complex mixtures of aa like pepton, yeast extract, serum ect in the medium as they would dilute the radioactivity. From owner-proteins@net.bio.net Sun Aug 09 23:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!news.maxwell.syr.edu!news-was.dfn.de!news-fra1.dfn.de!news-ber1.dfn.de!news-ham1.dfn.de!news.mu-luebeck.de!not-for-mail From: "Lars Komorowski" Newsgroups: bionet.molbio.proteins Subject: pET-Expression Mystery - The Sequel Date: Mon, 10 Aug 1998 09:10:07 +0100 Organization: Med. Universitaet zu Luebeck Lines: 9 Message-ID: <6qm6jm$h88$1@gwsun.medinf.mu-luebeck.de> NNTP-Posting-Host: 141.83.167.168 X-Newsreader: Microsoft Outlook Express 4.71.1712.3 X-MimeOLE: Produced By Microsoft MimeOLE V4.71.1712.3 Indeed the protein which I am trying to express is very acidic - pI is 4-4.5 - and I am certain that there are no inclusion bodies during my expression because the pellet doesn't look like those from other expressions which form inclusion bodies (white an huge). So one could guess that the protein is disintegrated during purification which seems to be very fast since the purifaction takes only 2 hours. Who has experienced similar things ? From owner-proteins@net.bio.net Sun Aug 09 23:00:00 1998 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news-peer.sprintlink.net!news-backup-east.sprintlink.net!news.sprintlink.net!128.218.95.22!itssrv1.ucsf.edu!itsa!thorn From: thorn@itsa.ucsf.edu (Kurt Thorn) Newsgroups: bionet.molbio.proteins,bionet.xtallography Subject: 6xHis vs. 8xHis in Dictyostelium Date: 10 Aug 1998 20:37:18 GMT Organization: ITS, University of California, San Francisco Lines: 19 Message-ID: <6qnllu$1cni@itssrv1.ucsf.edu> NNTP-Posting-Host: itsa.ucsf.edu Xref: biosci bionet.molbio.proteins:13152 bionet.xtallography:4345 Hello. I am trying to purify a 380 kDa recombinant protein from Dictyostelium for crystallography. I have been using a 6xHis tagged protein and have been having a lot of trouble with poor binding capacities of the metal resin and lots of contamination of the eluted product. Lately I have heard that 8x or 10x His tags work better than 6x His tags for organisms with lots of his-rich proteins (like Dicty). Can anybody give me references or personal experiences of working with these larger tags? Any other tips on difficult his-tag purifications are welcome. Thanks, Kurt ------------------------------------------------------------------------------- Kurt Thorn - thorn@itsa.ucsf.edu - http://motorhead.ucsf.edu/~thorn ------------------------------------------------------------------------------- From owner-proteins@net.bio.net Sun Aug 09 23:00:00 1998 From: Platini Kwok Newsgroups: bionet.molbio.proteins Subject: What is the major factor to affect the immunostaining of cytochrome C on embryo of ICR Date: Mon, 10 Aug 1998 23:31:16 +0800 Organization: Global Link Information Services Ltd. Lines: 23 Message-ID: <35CF1243.C39397CE@glink.net.hk> NNTP-Posting-Host: dialup183.glink.net.hk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.01 [tw] (Win95; I) X-Priority: ¤@¯ë) Path: biosci!news.stanford.edu!logbridge.uoregon.edu!newsfeed.direct.ca!newsfeed.corridex.com!ameritech.ais.net!jamie!ais.net!ameritech.net!uunet!in4.uu.net!news-hk.telia.net!d2o501.telia.com!newsgate.cuhk.edu.hk!news.glink.net.hk!news Hi, Recently, I was involved in the immunostaining of cytochrome c in embryo tissue. The tissue was fixed in 4% paraformaldehyde and dehydrated in graded alcohol, xylene and finally it was embared in wax block. It is found that only few positive signals were found in heart and bone tissue of 14.5 dpc of ICR embryo. My question are listed as follows 1. Will the embaring process destroy the antigen (cytochrome c)? 2. The antibody is rightly selected for "native protein" rather than denaturing protein which is usually done by SDS. Am I right? 3. there is negative signal in liver tissue which was expected to find a great deal of signal because live is a detoxification organ and it was expected very metabolic active. thank you for your advice! Platini (Anatomy, CUHK, HK) From owner-proteins@net.bio.net Tue Aug 11 23:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!news.maxwell.syr.edu!news.msfc.nasa.gov!bcm.tmc.edu!news From: Alex Hutagalung Newsgroups: bionet.molbio.proteins Subject: Transient protein interactions Date: Wed, 12 Aug 1998 12:34:13 -0500 Organization: Baylor College of Medicine Lines: 4 Message-ID: <35D1D215.CC2EB5C9@bcm.tmc.edu> Reply-To: ah692010@bcm.tmc.edu NNTP-Posting-Host: bcm02042.neuro.bcm.tmc.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.04 [en] (Win95; U) Hi! Could anyone please suggest the best way to show an interaction between proteins that transiently interact? Any suggestions would be terrific. Thank you. From owner-proteins@net.bio.net Wed Aug 12 23:00:00 1998 Message-ID: <35D2EDF7.5BC96026@bota.unine.ch> Date: Thu, 13 Aug 1998 15:45:51 +0200 From: David Humair Reply-To: David.Humair@bota.unine.ch Organization: Labo de biochimie =?iso-8859-1?Q?Universit=E9?= de =?iso-8859-1?Q?Neuch=E2tel?= X-Mailer: Mozilla 4.05 (Macintosh; I; PPC) MIME-Version: 1.0 Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins To: COA Subject: Re: Far Western Blotting References: <35CA04DC.DC218016@f-t.de> Content-Type: text/plain; charset=us-ascii; x-mac-type="54455854"; x-mac-creator="4D4F5353" Content-Transfer-Encoding: 7bit NNTP-Posting-Host: mac20-24.unine.ch Lines: 29 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.wli.net!198.138.0.5!newshub.northeast.verio.net!europa.clark.net!152.158.16.50!newsfeed.uk.ibm.net!ibm.net!news-ge.switch.ch!news-zh.switch.ch!sitelnet.unine.ch!mac20-24.unine.ch Xref: biosci bionet.molbio.methds-reagnts:69873 bionet.molbio.proteins:13155 Dear Christian, The idea of far western blot is like a western blot , but your probe is not an antibody but a protein which is supposed to have an interaction with your protein. This method allows you to check interactions between proteins. I can have a protocol Good luck David -- ==================================== David Humair Laboratoire de biochimie Emile-Argand 9 2007 Neuchatel Switzerland Tel: +41(0)327182226 Fax: +41(0)327182201 Email: David.Humair@bota.unine.ch http://www.unine.ch/biol/bioch/biochimie.html Silent soul leaves 308. holes. No brain, no headaches ! ==================================== From owner-proteins@net.bio.net Wed Aug 12 23:00:00 1998 Path: biosci!internet!biosci!not-for-mail From: biohelp (BIOSCI Administrator) Newsgroups: bionet.molbio.proteins Subject: BIOSCI/bionet miniFAQ & Fundraiser Date: 13 Aug 1998 02:00:08 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 233 Sender: daemon@net.bio.net Distribution: world Message-ID: <199808130900.CAA15962@net.bio.net> NNTP-Posting-Host: net.bio.net (LAST REVISION: 30-JUL-95) This BIOSCI "miniFAQ" is designed to answer the questions that come up the *most frequently*. The main BIOSCI FAQ (Frequently Asked Questions) is accessible on the World Wide Web at URL http://www.bio.net/. If you can not find an answer to your question in this or other documentation, the BIOSCI technical support staff answers e-mail queries sent to biosci-help@net.bio.net We can only answer questions about the use of the newsgroups and mailing lists. We unfortunately do not have the staff to do Internet information searches or answer scientific questions. Please post those to the appropriate BIOSCI/bionet newsgroups. Contents: -------- 0) BIOSCI NEEDS YOUR SUPPORT!! 1) Using the WWW to access the BIOSCI/bionet newsgroups. 2) What to do about "spams," i.e., junk mail, ads, etc. 3) Examples of subscribing and unsubscribing to the mailing lists. 4) The BIOSCI user address and research interest directory. 0) BIOSCI NEEDS YOUR SUPPORT!! ------------------------------ BIOSCI's government funding has been expended, and we are now operating solely from advertising revenue that we have raised from our Web site at http://www.bio.net/. We need just a few minutes of your time to help us serve you. You can do two important things which will take very little time for you individually and will immensely help us continue to help you. First, please use our WWW system at http://www.bio.net/ to access the archives. You can post or reply to messages via your Web browser as described in item #1 below. Your usage helps attract sponsors. If you contact any of our sponsors, please be sure to thank them for supporting BIOSCI. It is critical for them to get this feedback if they are to continue their sponsorship for the long term. Second, if you work for a company or organization that provides products or services of interest to the biology community, please pass this message on to your marketing or marketing communications department or other appropriate group. Please ask them to help support BIOSCI by sponsoring our Web site and explain the uses and benefits of the system to the biology community. If they are interested, they can then contact us for further information at our tech support address, biosci-help@net.bio.net. 1) Using the WWW to access the BIOSCI/bionet newsgroups. -------------------------------------------------------- As of 10 December 1995, all BIOSCI/bionet full newsgroups are accessible through the World Wide Web (WWW) at URL http://www.bio.net. One can read and reply publicly or privately to both recent postings and archived messages through one's Web browser if it is configured properly to send e-mail. Each newsgroup is equipped with its own WAIS index. The main BIOSCI home page also has access to the BIO-JOURNALS Table of Contents database WAIS index and the BIOSCI user address database described in another item further below. 2) What to do about "spams," i.e., junk mail, ads, etc. ------------------------------------------------------- BIOSCI is a set of parallel USENET newsgroups (the "bionet" groups), mailing lists, and a hypermail archive at URL http://www.bio.net/. The same postings are distributed on all media (except for a small number of mailing-list-only groups at net.bio.net). Unfortunately it is becoming a despicable practice on the Internet (by a few people out to make a fast buck) to do automated mass postings to thousands of newsgroups and mailing lists. These attempts to grab free advertising are refered to as "spams" in the usual, somewhat boneheaded, net terminology. USENET is more susceptible to this practice, and many spams originate on the USENET groups and then are passed on to the mailing lists. However, spammers also get lists of mailing addresses and hit these too, so neither medium is immune. What should you do personally if you get junk mail? --------------------------------------------------- Just delete it and move on without reading it further. Filing a protest is becoming increasingly useless because spammers are often disguising the addresses where the messages are sent from. Unless you really understand Internet mail systems, your attempt at protest by sending replies to the message will often end up being sent to the address of an innocent person that the spammer is victimizing. What can BIOSCI/bionet do to protect its newsgroups? ---------------------------------------------------- The only solution currently available is to moderate the newsgroup. If this newsgroup is already moderated, then you are in good shape. Moderation protects the USENET distribution from about 95% of the spams that are being sent to date and protects the mailing lists completely. Moderation means, however, that someone has to take the time to review each message before it goes out. We have set up software here that simply allows the moderator to forward to an address at net.bio.net messages that (s)he wishes to have distributed. This takes no more time than that needed to read the message and pass it on, say about 1 min. per message. Most newsgroups currently have a discussion leader who is responsible for their newsgroup. The discussions leaders and their e-mail addresses are listed in the BIOSCI Information Sheet which is available on the Web at http://www.bio.net/. If a newsgroup is being hit with too many junk postings, please contact the discussion leader for that group and see if there is interest in moderating the group. Please do not assume that by simply posting a complaint to the newsgroup itself, anyone on the BIOSCI staff will act on your complaint. With close to 100 newsgroups to run, the BIOSCI staff has to rely on the discussion leaders of each newsgroup to report problems directly to us at biosci-help@net.bio.net. We will moderate any of our newsgroups if the discussion leader tells us that the readership of the group wishes to do so and if a moderator is willing to do the work. For most BIOSCI/bionet groups, this entails only a few minutes of work each day. Moderating a newsgroup will resolve probably 95% of the junk postings on the USENET distribution. Unfortunately there are easy ways for determined spammers to override the moderation mechanism on USENET, but we can protect our e-mail subscribers from unwanted postings if the newsgroup is moderated. You can also access our newsgroups over the WWW at URL http://www.bio.net. While this Web interface will not stop spammers from trying to post to the groups, this will give you yet another way, besides using USENET news, to keep the junk out of your personal mail files. For those of you with local USENET news systems, the Web interface will also give you faster access to new newsgroups and recent postings. 3) Examples of subscribing and unsubscribing to the mailing lists. ------------------------------------------------------------------ PLEASE NOTE: The BIOSCI management does NOT act on subscription/unsubscription requests that are posted improperly to the newsgroups and mailing lists. People who do this only bother everyone on the lists to no avail. Please be sure to follow the proper procedures below. Gory details are in the BIOSCI Information sheets on the Web at http://www.bio.net. Below we give an example utilizing the METHODS-AND-REAGENTS list at both of our two BIOSCI sites: Users in the Americas and Pacific Rim countries who use the BIOSCI ------------------------------------------------------------------ node at computer net.bio.net: ---------------------------- A) Determine the "listname" which is the <=8 character mail address ^^^^^^^^^^^^^ for the group. These can be found in the BIOSCI Info. Sheet. For the METHODS-AND-REAGENTS group the mailing address is methods@net.bio.net. The listname is the portion of the address to the left of the @ sign, i.e., "methods". The listname is used with the "subscribe" and "unsubscribe" commands illustrated below. B) Mail all commands in the body of a mail message addressed to biosci-server@net.bio.net. Do NOT send commands to the newsgroup posting addresses! Leave the Subject: line blank, any text on it will be ignored. C) In the body of your message put one or more of the following commands with an "end" command on the last line, e.g., subscribe methods unsubscribe methods end Do NOT put your e-mail address or other text on these lines. The server only allows you to cancel your subscription if the address on your mail header matches the address on our mailing list. Please ask for help at biosci-help@net.bio.net if your address has changed, e.g., if you know you are on the list but the server tells you that you are not a member. Users in Europe, Africa, and Central Asia who use the BIOSCI node at -------------------------------------------------------------------- computer daresbury.ac.uk (also known as dl.ac.uk): ------------------------------------------------- To subscribe and unsubscribe to/from the BIOSCI lists, you need to specify the full USENET newsgroup name with "bionet-news." prepended. The USENET newsgroup names are listed in the BIOSCI Information sheet on the Web at http://www.bio.net/. For the METHODS-AND-REAGENTS list the USENET newsgroup name is bionet.molbio.methds-reagnts, thus the appropriate commands are sub bionet-news.bionet.molbio.methds-reagnts unsub bionet-news.bionet.molbio.methds-reagnts These commands are included in a message addressed to mxt@dl.ac.uk, NOT to the newsgroup mailing addresses. As usual, include the text in the body of the message as text on the Subject: line is ignored. To unsubscribe from all the lists at the UK node, use unsub bionet-news Please note that if the address in the list is different than the one in your mail message header, you will not be able to unsubscribe by this method. If you have problems, please mail biosci@daresbury.ac.uk. 4) The BIOSCI user address and research interest directory. ----------------------------------------------------------- Please take this opportunity to add your name, address, and research interest information to the BIOSCI User Address Database if you have not already done so. You can fill out the address form directly through our Web page at URL http://www.bio.net/adrform.html. The address database is reindexed nightly for WWW access (the URL is http://www.bio.net/). If you are not directly on the Internet but can reach it by e-mail, please use our waismail server to access the user directory. waismail use is described above. You can also request a user address form by e-mail from biosci-help@net.bio.net. Please check your database entry from time-to-time to see if your address information is still up-to-date. Because of our limited personnel resources, we ask that you resubmit a *complete* form to revise your entry; we only replace complete entries and do not have resources to edit old forms. From owner-proteins@net.bio.net Wed Aug 12 23:00:00 1998 Path: biosci!METHYLGENE.COM!liz From: liz@METHYLGENE.COM (liz) Newsgroups: bionet.molbio.proteins Subject: help! binding to nickle column Date: 13 Aug 1998 13:24:33 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 4 Sender: daemon@net.bio.net Distribution: world Message-ID: <35D34A59.62C0@methylgene.com> NNTP-Posting-Host: net.bio.net I am trying to bind a 180 KDa protein with a His tag to a nickle column (invitrogen) but most of the protein comes out in the flow through. I have tried both native and denaturing conditions. Can anybody offer any suggestions? From owner-proteins@net.bio.net Thu Aug 13 23:00:00 1998 Path: biosci!YAHOO.COM!rjdudley From: rjdudley@YAHOO.COM ("Richard J. Dudley") Newsgroups: bionet.molbio.proteins Subject: Re: help! binding to nickle column Date: 14 Aug 1998 05:45:15 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 27 Sender: daemon@net.bio.net Distribution: world Message-ID: <19980814124147.29797.rocketmail@send101.yahoomail.com> Reply-To: rjdudley@yahoo.com NNTP-Posting-Host: net.bio.net ---liz wrote: > > I am trying to bind a 180 KDa protein with a His tag to a nickle column > (invitrogen) but most of the protein comes out in the flow through. I > have tried both native and denaturing conditions. Can anybody offer any > suggestions? Make sure there is no EDTA nor ionic detergents in your protein suspension. Also, search these archives at www.bio.net; this problem comes up a lot. rich == --- --- --- -- -- -- --- --- --- Richard J. Dudley (rdudley@iname.com) Research Specialist V Dept. of Cell Biology and Physiology The University of Pittsburgh ->biosci archives can be searched at www.bio.net <- _________________________________________________________ DO YOU YAHOO!? Get your free @yahoo.com address at http://mail.yahoo.com From owner-proteins@net.bio.net Thu Aug 13 23:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!cyclone.news.idirect.com!newshub.northeast.verio.net!btnet-peer!btnet!nntp.news.xara.net!xara.net!server6.netnews.ja.net!server4.netnews.ja.net!server2.netnews.ja.net!leicester!usenet From: Dr Engelbert Buxbaum Newsgroups: bionet.molbio.proteins Subject: Re: Transient protein interactions Date: Fri, 14 Aug 1998 11:02:33 -0700 Organization: University of Leicester (PCFS User) Lines: 18 Message-ID: <35D47BB9.3BF2@le.ac.uk> References: <35D1D215.CC2EB5C9@bcm.tmc.edu> Reply-To: eb15@le.ac.uk NNTP-Posting-Host: pc222.tulip5.le.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Win16; I) Alex Hutagalung wrote: > Could anyone please suggest the best way to show an interaction between > proteins that transiently interact? Any suggestions would be terrific. > Thank you. Crosslinking with bifunctional reagent (check Pierce catalogue for a good selection). Emanescent wave detectors (like a Pharmacia BiaCore). Analytical ultracentrifugation or gelchromatography. A genetic approach would be the yeast two hybrid system. Low angle laser light scattering or neutron scattering. Radiation target size analysis (not my prefered option, though). You may try to grow crystals of the associated proteins (if that actually works, it would be the ideal solution). CD spectrum of the protein complex may differe from what is expected from a mixture of the two proteins, indication conformational changes upon binding. Well, at least that's what I can think of at the top of my head. Get a good book on physical biochemistry, and you may find further options. From owner-proteins@net.bio.net Thu Aug 13 23:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!howland.erols.net!newsfeed.nacamar.de!nntp.news.xara.net!xara.net!server6.netnews.ja.net!server4.netnews.ja.net!server2.netnews.ja.net!leicester!usenet From: Dr Engelbert Buxbaum Newsgroups: bionet.molbio.proteins Subject: Re: What is the major factor to affect the immunostaining of cytochrome C on embryo of ICR Date: Fri, 14 Aug 1998 10:49:53 -0700 Organization: University of Leicester (PCFS User) Lines: 25 Message-ID: <35D478C1.7C2E@le.ac.uk> References: <35CF1243.C39397CE@glink.net.hk> Reply-To: eb15@le.ac.uk NNTP-Posting-Host: pc222.tulip5.le.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Win16; I) Platini Kwok wrote: > Recently, I was involved in the immunostaining of cytochrome c in embryo > tissue. The tissue was fixed in 4% paraformaldehyde and dehydrated in > graded alcohol, xylene and finally it was embared in wax block. It is > found that only few positive signals were found in heart and bone tissue The harsh treatment during tissue processing may or may not prevent proper staining of an antigen. The risk is obviously greatest with monoclonal antibodies, which have only a single binding epitope. Polyclonal antibodies, with their multiple binding sites, are less susceptible. There are several ways to control for this effect. One is to use frozen sections from unfixed tissues instead of parafine embedding. This avoids a lot of the denaturing processes. The second experiment you can perform is to carefully trypsinize the sections before antibody exposure to recover blocked antigenic sites. Works sometimes and sometimes not. Briefly microwaving sections in citrate buffer has also been suggested for this purpose. If you want to know only wether a particular antigen is present in a particular tissue, rather than its subcellular localisation, you may try immunoprecipitation of tissue homogenisates instead of immunocytochemistry. Also, it may be worthwhile to do your experiments with several different antibodies (there should be enough available for a popular protein like cytochrome c). From owner-proteins@net.bio.net Thu Aug 13 23:00:00 1998 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.internetmci.com!164.67.42.145!awabi.library.ucla.edu!132.239.1.220!ihnp4.ucsd.edu!news1.ucsd.edu!not-for-mail From: "Antonin Tutter" Newsgroups: bionet.molbio.proteins Subject: Re: Transient protein interactions Date: Fri, 14 Aug 1998 00:57:55 -0700 Organization: University of California, San Deigo Lines: 23 Message-ID: <6r0qju$gke$1@news1.ucsd.edu> References: <35D1D215.CC2EB5C9@bcm.tmc.edu> NNTP-Posting-Host: atutter.extern.ucsd.edu Mime-Version: 1.0 Content-Type: text/plain; charset="US-ASCII" Content-Transfer-Encoding: 7bit X-Newsreader: Microsoft Outlook Express for Macintosh - 4.01 (297) >Hi! >Could anyone please suggest the best way to show an interaction between >proteins that transiently interact? Any suggestions would be terrific. >Thank you. That's tough. Often transient interactions are not detectable by Far western or other more traditional assays. How about UV crosslinking? You haven't mentioned whether this is between two recombinant proteins, or one recombinant protein and an extract, or what... I couldn't suggest a protocol, but I'm sure there's a lot of literature on UV crosslinking of proteins. _______________________________________ Antonin Tutter Salk Institute for Biological Studies RBIO-J 10010 N. Torrey Pines Rd. La Jolla, CA 92037 email: atutter@aim.salk.edu web: http://www-biology.ucsd.edu/~atutter/ From owner-proteins@net.bio.net Thu Aug 13 23:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!ihnp4.ucsd.edu!news1.ucsd.edu!not-for-mail From: "Antonin Tutter" Newsgroups: bionet.molbio.proteins Subject: Re: help! binding to nickle column Date: Fri, 14 Aug 1998 00:54:00 -0700 Organization: University of California, San Deigo Lines: 22 Distribution: world Message-ID: <6r0qck$gk9$1@news1.ucsd.edu> References: <35D34A59.62C0@methylgene.com> NNTP-Posting-Host: atutter.extern.ucsd.edu Mime-Version: 1.0 Content-Type: text/plain; charset="US-ASCII" Content-Transfer-Encoding: 7bit X-Newsreader: Microsoft Outlook Express for Macintosh - 4.01 (297) >I am trying to bind a 180 KDa protein with a His tag to a nickle column >(invitrogen) but most of the protein comes out in the flow through. I >have tried both native and denaturing conditions. Can anybody offer any >suggestions? Have you checked your buffering conditions during binding to make sure they are not reducing the Ni++? Make sure the pH is >7, and make sure you have little reducing agent (<1mM DTT, <20mMM BME). _______________________________________ Antonin Tutter Salk Institute for Biological Studies RBIO-J 10010 N. Torrey Pines Rd. La Jolla, CA 92037 email: atutter@aim.salk.edu web: http://www-biology.ucsd.edu/~atutter/ From owner-proteins@net.bio.net Thu Aug 13 23:00:00 1998 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!howland.erols.net!vixen.cso.uiuc.edu!jestone From: jestone@staff.uiuc.edu (stone john e) Newsgroups: bionet.molbio.proteins Subject: VMD "Visual Molecular Dynamics" 1.2 Released Date: 15 Aug 1998 03:26:25 GMT Organization: University of Illinois at Urbana-Champaign Lines: 136 Message-ID: <6r2v51$8mh$1@vixen.cso.uiuc.edu> NNTP-Posting-Host: staff1.cso.uiuc.edu VMD "Visual Molecular Dynamics" 1.2 Release Announcement -------------------------------------------------------- The Theoretical Biophysics group at the University of Illinois and the Beckman Institute For Advanced Science and Technology is happy to announce the availability of version 1.2 of the program VMD, a package for the vizualization and analysis of biomolecular systems. This software is being made available to the structural biology research community free of charge, and includes the source code for VMD, documentation, and precompiled binaries for IBM, HP, Linux, Sun, and SGI Unix systems. The documentation includes an installation guide, a users guide, and a programmers guide for interested researchers. VMD also provides on-line help through the use of an external HTML viewer. A full description of VMD is available via the VMD WWW home page: http://www.ks.uiuc.edu/Research/vmd/ Changes to VMD 1.2 since VMD 1.1: --------------------------------- o Many updates to VMD documentation o VMD now supports OSF1/Digital Unix 4.x on DEC Alpha with OpenGL or Mesa o Dihedral angles are now reported in the proper range o Various small bug fixes, and portability enhancements -- 1.2 beta 4 o Improved display of 3-D axis labels "X","Y","Z". o Fixed POV, POV3, Art, Raster3D, and Rayshade renderer export codes. o Fixed the "write" function found in the "edit animation" dialog. o VMD runs in the Cave simulator now. -- 1.2 beta 3 o VMD now supports AIX 4.x with the native AIX OpenGL implementation o OpenGL rendering speed has been improved by 2-5x on several platforms o OpenGL version now correctly supports stereo-in-a-window o OpenGL/GL versions are now able to interoperate with VMD scripts created in by one or the other. o Upgraded to use newer XForms libraries, which fix some GUI/Mouse bugs o VMD upgraded to compile with the 2.6c release of the CAVE libraries o VMD code now builds with much pickier C++ compilers (i.e. HP aCC) o New Linux binary distributions have working file browser now. o New Linux RPM style binary distribution o New HPUX10-OpenGL binaries for Visualize-FX hardware accelerators o New AIX4-OpenGL binaries for IBM OpenGL hardware accelerators o Many bug fixes -- 1.2 beta 2 o VMD has been ported to Sun Solaris 2. o Upgraded to support Linux RedHat 5.0 o OpenGL support works with native HP-UX and Sun OpenGL implementations o Added Tk support and upraded to Tcl/Tk 8.0 o Upgraded to Babel 1.6 o Added support for MSMS, a program for calculating molecular surfaces o Added support for Grasp file format o Typing in a command without arguments will print a help message o Added more commands to Tcl scripting interface o Many bug fixes -- 1.2 beta 1 o This biggest improvement in version 1.2b1 support for platforms other than GL-based SGIs. In addition to the full source and SGI binary distributions, VMD is now available for HP-UX (tested under 9 and 10) and Linux. o Greatly enhanced Tcl scripting commands for performing molecular analysis, writing scripts, developing tutorials, etc. o New rendering styles, a fast (and cheap) solvent accessible surface and C-alpha and P trace method, and improvements to the existing styles. o New output renderer formats: Postscript, VRML and STL (a stereo-lithography format) o Support for Amber structure and animation file formats ============= Basic information about VMD ================= Features -------- VMD is designed for the visualization and analysis of biological systems such as proteins, nucleic acids, lipid bilayer assemblies, etc. It may be used to view more general molecules, as VMD can read standard Protein Data Bank (PDB) files and display the contained structure. VMD provides a wide variety of methods for rendering and coloring a molecule: simple points and lines, CPK spheres and cylinders, licorice bonds, backbone tubes and ribbons, cartoon drawings, and others. VMD can be used to animate and analyze the trajectory of a molecular dynamics (MD) simulation. In particular, VMD can act as a graphical front end for an external MD program by displaying and animating a molecule undergoing simulation on a remote computer. The program has many features, which include: o No limits on the number of molecules, atoms, residues or number of animation frames, excepting available memory. o Many molecular rendering and coloring methods. o Stereo display capability. o Extensive atom selection syntax for choosing subsets of atoms for display (includes boolean operators, regular expressions, and more). o Integration with the program 'Babel' which allows VMD to read many molecular data file formats. Even without the use of Babel, VMD can read PDB files, as well as CHARMM- and X-PLOR compatible binary DCD files and X-PLOR compatible PSF files. o Ability to write the current image to a file which may be processed by a number of popular raytracing and image rendering packages, including POV-Ray, Rayshade, Raster3D, and Radiance. o Extensive graphical and text-based user interfaces, which use the Tcl package to provide full scripting capabilities. o Extensions to the Tcl language which enable researchers to write their own routines for molecular analysis o Modular, extensible source code using an object-oriented design in C++, with a programmer's guide describing the source code o Integration with the program NAMD, a fast, parallel, and scalable molecular dynamics program developed in conjunction with VMD in the Theoretical Biophysics Group at the University of Illinois. See the NAMD WWW home page for more info: http://www.ks.uiuc.edu/Research/namd/ VMD can be used to set up and concurrently display an MD simulation using NAMD. The two programs, along with the intermediary communcations package (called MDComm) constitute the 'MDScope' interactive environment. Availability ------------ The software is available for downloading from http://www.ks.uiuc.edu/Research/vmd/ Please email any questions to vmd@ks.uiuc.edu. VMD, NAMD, and the entire MDScope environment are part of an ongoing project within the Theoretical Biophysics group to help provide free, effective tools for molecular dynamics studies in structural biology. For more information, see http://www.ks.uiuc.edu/Research/MDScope/ This project is funded by the National Institutes of Health. (grant number PHS 5 P41 RR05969) John Stone vmd@ks.uiuc.edu August 1, 1998 From owner-proteins@net.bio.net Thu Aug 13 23:00:00 1998 Path: biosci!news.stanford.edu!logbridge.uoregon.edu!newsfeed.nyu.edu!newsfeed.sgi.net!nntp.itis.com!news.doit.wisc.edu!default From: klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins Subject: Re: Transient protein interactions Date: Fri, 14 Aug 1998 16:23:45 GMT Organization: UW-Madison Lines: 27 Message-ID: <6r1oa1$eq4$1@news.doit.wisc.edu> References: <35D1D215.CC2EB5C9@bcm.tmc.edu> NNTP-Posting-Host: martinlab.biochem.wisc.edu Mime-Version: 1.0 Content-Type: text/plain; charset=KOI8-R Content-Transfer-Encoding: 8bit X-Newsreader: News Xpress 2.01 X-No-Archive: Yes In article <35D1D215.CC2EB5C9@bcm.tmc.edu>, ah692010@bcm.tmc.edu wrote: :Hi! :Could anyone please suggest the best way to show an interaction between :proteins that transiently interact? Any suggestions would be terrific. :Thank you. What exactly do you mean? In every solution, every molecule transiently interacts with another molecule (they hit each other and then have higher or lower chances of staying together for some time). Do you mean that the association constant is so low so that half-time of the complex is very short? Or you mean that there is some high affinity interaction which is disturbed by a third party in a timely fashion? The approaches, I think, would be very different. Also, it is important to know what system you have - in vitro or in vivo, whether you are dealing with pure proteins available in large quantities or not, etc. There are so amny ways to study interactions between molecules that the answer to your question becomes impossible without knowing what's given. - Dima From owner-proteins@net.bio.net Sun Aug 16 23:00:00 1998 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news1.bellglobal.com!torn!resunix.sickkids.on.ca!michael.didonato From: michael.didonato@utoronto.ca (Michael DiDonato) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Thrombin Cleavage of GST Fusions Date: Mon, 17 Aug 1998 14:09:41 -0400 Organization: Hospital for Sick Children Lines: 15 Message-ID: NNTP-Posting-Host: 142.20.54.12 X-Newsreader: MT-NewsWatcher 2.4.4 Xref: biosci bionet.molbio.methds-reagnts:69943 bionet.molbio.proteins:13166 Does anyone know of a way to optimize thrombin cleavage of GST fusion such that only the five base site is cleaved and not the three base sites. I am getting the majority of cleavage at the five base site but also some annoying cleavage at the three base sites present in my protein. I have heard of using heparin in the cleavage reaction, does anyone know of this? Any help would be appreciated. Mike -- Michael DiDonato Structural Biology and Biochemistry The Hospital for Sick Children Toronto, Ontario, Canada From owner-proteins@net.bio.net Sun Aug 16 23:00:00 1998 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!news.bbnplanet.com!news1.best.com!newsfeed.slip.net!news.slip.net!not-for-mail From: "Aaron Lewis" Newsgroups: bionet.molbio.proteins Subject: Tracking proteins? Date: Mon, 17 Aug 1998 14:35:37 -0700 Organization: Slip.Net (http://www.slip.net) Lines: 11 Message-ID: <6ra7ic$6al$1@owl.slip.net> NNTP-Posting-Host: 207.171.204.96 X-Newsreader: Microsoft Outlook Express 4.72.3110.5 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3110.3 Does anyone have any experience (or suggestions) on how to track proteins and pepidoglycans as they move through a food chain? E.g., Biological transport and fate of nutrients from speciefic sources. We want to know which critters in a mixed culture are actually using the proteins (or energy from the proteins) from the specific nutrient source as opposed to metabolizing other organic material in the microcosm. The only technology we can think of is C-14 labeling. From owner-proteins@net.bio.net Sun Aug 16 23:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!howland.erols.net!news-nyc.telia.net!uunet!uunet!in1.uu.net!news1-gui.server.ntli.net!news-feed.ntli.net!news5-gui.server.ntli.net!news-feed.ntli.net!server4.netnews.ja.net!server2.netnews.ja.net!pegasus.csx.cam.ac.uk!macg6-1.mrc-lmb.cam.ac.uk!user From: jyl@mrc-lmb.cam.ac.uk (Jan Lowe) Newsgroups: bionet.molbio.proteins Subject: Re: Stereo viewers Date: Mon, 17 Aug 1998 19:52:45 +0100 Organization: MRC-LMB, Cambridge (UK) Lines: 29 Message-ID: References: <35D87131.CA733F5C@acpub.duke.edu> NNTP-Posting-Host: macg6-1.mrc-lmb.cam.ac.uk Mike, have a look at: http://www.hamptonresearch.com/Misc.html#anchor100008 Jan ______________________________________________________________________________ Jan Lowe E-mail: jyl@mrc-lmb.cam.ac.uk Laboratory of Molecular Biology phone : +uk (0)1223 402402 Medical Research Council fax : +uk (0)1223 213556 Hills Road Cambridge CB2 2QH UK ______________________________________________________________________________ In article <35D87131.CA733F5C@acpub.duke.edu>, Michael J Morales wrote: > I'm one of those who just can't see those stereo images of > proteins published in journals in 3D. Does anyone > know where I can purchase the glasses for viewing these > images? > > Thanks, > > Mike Morales From owner-proteins@net.bio.net Sun Aug 16 23:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!newsgate.duke.edu!usenet From: Michael J Morales Newsgroups: bionet.molbio.proteins Subject: Stereo viewers Date: Mon, 17 Aug 1998 14:06:41 -0400 Organization: Duke University, Durham, NC, USA Lines: 8 Message-ID: <35D87131.CA733F5C@acpub.duke.edu> NNTP-Posting-Host: async249-157.async.duke.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.04 [en] (Win95; U) X-Corel-MessageType: EMail I'm one of those who just can't see those stereo images of proteins published in journals in 3D. Does anyone know where I can purchase the glasses for viewing these images? Thanks, Mike Morales From owner-proteins@net.bio.net Mon Aug 17 23:00:00 1998 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news1.bellglobal.com!torn!garnet.nbnet.nb.ca!news.unb.ca!coranto.ucs.mun.ca!cyanolab.bioc.mun.ca!user From: tbelbin@morgan.ucs.mun.ca (Tom Belbin) Newsgroups: bionet.molbio.proteins Subject: Re: help! binding to nickle column Date: 18 Aug 1998 12:24:29 GMT Organization: Memorial University of Newfoundland Lines: 18 Distribution: world Message-ID: References: <35D34A59.62C0@methylgene.com> NNTP-Posting-Host: cyanolab.bioc.mun.ca X-Newsreader: Yet Another NewsWatcher 2.1.8 In article <35D34A59.62C0@methylgene.com>, liz@METHYLGENE.COM (liz) wrote: > I am trying to bind a 180 KDa protein with a His tag to a nickle column > (invitrogen) but most of the protein comes out in the flow through. I > have tried both native and denaturing conditions. Can anybody offer any > suggestions? Is the tag at the N-term end? If so, are you sure it is actually present on the protein being expressed? Beware of an internal start codons. You may be getting translation starting downstream so that the tag is not translated at all. I speak from experience on this one. Tom Belbin Biochemistry Department Memorial University of Newfoundland From owner-proteins@net.bio.net Mon Aug 17 23:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!news.maxwell.syr.edu!newsfeed.nacamar.de!nntp.news.xara.net!xara.net!server5.netnews.ja.net!server3.netnews.ja.net!server4.netnews.ja.net!server2.netnews.ja.net!leicester!usenet From: Dr Engelbert Buxbaum Newsgroups: bionet.molbio.proteins Subject: Re: Tracking proteins? Date: Tue, 18 Aug 1998 16:33:52 -0700 Organization: University of Leicester (PCFS User) Lines: 9 Message-ID: <35DA0F5F.160D@le.ac.uk> References: <6ra7ic$6al$1@owl.slip.net> Reply-To: eb15@le.ac.uk NNTP-Posting-Host: pc221.tulip5.le.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Win16; I) Aaron Lewis wrote: > > Does anyone have any experience (or suggestions) on how to track proteins > and pepidoglycans as they move through a food chain? > The only technology we can think of is C-14 labeling. There are people who use stable isotope labeling (like C-13, O-18) for such purposes. Obviously a lot safer than radioisotopes, but you need access to a mass spectrometer. From owner-proteins@net.bio.net Tue Aug 18 23:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!newsfeed.nyu.edu!news.maxwell.syr.edu!firehose.mindspring.net!firehose.mindspring.com!user-38h1tpk.dialup.mindspring.com!user From: petter@mindspring.com (Petter) Newsgroups: bionet.molbio.proteins Subject: Re: Protein dip stick Date: Wed, 19 Aug 1998 19:30:14 -0500 Organization: MindSpring Enterprises Lines: 15 Distribution: world Message-ID: References: <35DAF3D0.D5664525@ornl.gov> NNTP-Posting-Host: user-38h1tpk.dialup.mindspring.com X-Server-Date: 20 Aug 1998 00:24:57 GMT In article <35DAF3D0.D5664525@ornl.gov>, isolan@ornl.gov wrote: >This is a multi-part message in MIME format. >--------------EC8DC33FAEF588173FE685DC >Content-Type: text/plain; charset=us-ascii >Content-Transfer-Encoding: 7bit > >A friend of mine has developed a protein dip stick for rapid >quantitation of proteins. Is there any interest in such a product. > >Narayana R.Isola depends on the accurracy i suppose wasn't there one of these for DNA in the market at one point? From owner-proteins@net.bio.net Tue Aug 18 23:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!sunqbc.risq.qc.ca!newsfeed.nacamar.de!oleane!rain.fr!wanadoo.fr!not-for-mail From: bruno collinet Newsgroups: bionet.molbio.proteins Subject: Re: Transient protein interactions Date: Wed, 19 Aug 1998 21:54:05 +0200 Organization: Wanadoo - (Client of French Internet Provider) Lines: 61 Message-ID: <35DB2D5D.1A60B529@wanadoo.fr> References: <35D1D215.CC2EB5C9@bcm.tmc.edu> NNTP-Posting-Host: bjn10-28.abo.wanadoo.fr Mime-Version: 1.0 Content-Type: multipart/mixed; boundary="------------940C47600C8D282DE9CA3E35" X-Mailer: Mozilla 4.05 [en] (Win95; I) This is a multi-part message in MIME format. --------------940C47600C8D282DE9CA3E35 Content-Type: multipart/alternative; boundary="------------385F3896D107A81817A7A8BD" --------------385F3896D107A81817A7A8BD Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit Alex Hutagalung wrote: > Hi! > Could anyone please suggest the best way to show an interaction between > proteins that transiently interact? Any suggestions would be terrific. > Thank you. Hi, in our lab we've detected transient multimeric species during the refolding of yeast PGK using a crosslinking method with glutaraldehyde. ref: Pecorari etal (1996) JBC 271, 5270-5276 --------------385F3896D107A81817A7A8BD Content-Type: text/html; charset=us-ascii Content-Transfer-Encoding: 7bit  

Alex Hutagalung wrote:

Hi!
Could anyone please suggest the best way to show an interaction between
proteins that transiently interact?  Any suggestions would be terrific.
Thank you.
  Hi,
in our lab we've detected transient multimeric species during the refolding of yeast PGK using a crosslinking method with glutaraldehyde.
ref: Pecorari etal (1996) JBC 271, 5270-5276 --------------385F3896D107A81817A7A8BD-- --------------940C47600C8D282DE9CA3E35 Content-Type: text/x-vcard; charset=us-ascii; name="vcard.vcf" Content-Transfer-Encoding: 7bit Content-Description: Card for bruno Collinet Content-Disposition: attachment; filename="vcard.vcf" begin: vcard fn: bruno Collinet n: Collinet;bruno email;internet: brunoc1@wanadoo.fr x-mozilla-cpt: ;0 x-mozilla-html: FALSE version: 2.1 end: vcard --------------940C47600C8D282DE9CA3E35-- From owner-proteins@net.bio.net Tue Aug 18 23:00:00 1998 Path: biosci!ORNL.GOV!isolan From: isolan@ORNL.GOV ("Dr.Narayana R.Isola") Newsgroups: bionet.molbio.proteins Subject: Protein dip stick Date: 19 Aug 1998 08:59:56 -0700 Organization: Oak Ridge National Laboratory Lines: 30 Sender: daemon@net.bio.net Distribution: world Message-ID: <35DAF3D0.D5664525@ornl.gov> Reply-To: isolan@ornl.gov NNTP-Posting-Host: net.bio.net This is a multi-part message in MIME format. --------------EC8DC33FAEF588173FE685DC Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit A friend of mine has developed a protein dip stick for rapid quantitation of proteins. Is there any interest in such a product. Narayana R.Isola isolan@ornl.gov --------------EC8DC33FAEF588173FE685DC Content-Type: text/x-vcard; charset=us-ascii; name="vcard.vcf" Content-Transfer-Encoding: 7bit Content-Description: Card for isolan@ornl.gov Content-Disposition: attachment; filename="vcard.vcf" begin: vcard fn: isolan@ornl.gov n: ;isolan@ornl.gov email;internet: isolan@ornl.gov x-mozilla-cpt: ;0 x-mozilla-html: FALSE version: 2.1 end: vcard --------------EC8DC33FAEF588173FE685DC-- From owner-proteins@net.bio.net Tue Aug 18 23:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!newsgate.cuhk.edu.hk!news.glink.net.hk!news From: Platini Kwok Newsgroups: bionet.molbio.proteins Subject: VEGF-b of 23 Kd and 46 Kd Date: Wed, 19 Aug 1998 23:23:59 +0800 Organization: Global Link Information Services Ltd. Lines: 18 Message-ID: <35DAEE0F.9E8882A3@glink.net.hk> NNTP-Posting-Host: dialup-17-67.glink.net.hk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.01 [tw] (Win95; I) X-Priority: ¤@¯ë) Hi, Why it is difficult to detect the 46 Kd by vegf-b antibody? Although I already put the sample into unreducing loading buffer (no 2-mercaethanol), run the SDS page and then electroblotting onto NC membrane by wet method. After incubating with primary vegf-b polychonal antibody (Santa Cruze) and goat ABC (Vector stain) and development of DAB, it is found that the intensity of the band of 46 Kd is very week. Instead, the intensity of 23 Kd is very strong in reducing condition. Why is that? My sample is rat retina. Thank you for your opinion! Platini From owner-proteins@net.bio.net Tue Aug 18 23:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!newsfeed.axxsys.net!uunet!uunet!in2.uu.net!news1-gui.server.ntli.net!news-feed.ntli.net!news5-gui.server.ntli.net!news-feed.ntli.net!server4.netnews.ja.net!server2.netnews.ja.net!pegasus.csx.cam.ac.uk!hgmp.mrc.ac.uk!gmorley From: gmorley@hgmp.mrc.ac.uk (Mr. G. Morley) Newsgroups: bionet.molbio.proteins Subject: Bcl2 and Bcl xl Date: 19 Aug 1998 12:13:15 GMT Organization: MRC Human Genome Mapping Project Resource Centre Lines: 14 Message-ID: <6refgr$om8$1@niobium.hgmp.mrc.ac.uk> NNTP-Posting-Host: tin.hgmp.mrc.ac.uk Dear all, I was wondering if anyone has Bcl2 and Bclxl in a plasmid or (preferably) a mammalian retroviral vector that they could let me have. If anyone could take the time to send me some I w ould be deeply grateful! Thanks in advance, Gary Morley Department of haematology University College London 98 Chennies Mews London WCE1 6HX. g.morley@ucl.ac.uk From owner-proteins@net.bio.net Tue Aug 18 23:00:00 1998 Path: biosci!PILOT.MSU.EDU!delgadoi From: delgadoi@PILOT.MSU.EDU (Ivan J Delgado Orlic) Newsgroups: bionet.molbio.proteins Subject: Help with SDS-PAGE of Arabidopsis samples Date: 19 Aug 1998 22:39:12 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 27 Sender: daemon@net.bio.net Distribution: world Message-ID: <35DBB5F7.EA85C605@pilot.msu.edu> NNTP-Posting-Host: net.bio.net Dear All, I am having problems with 1-D SDS-PAGE. After a lot of troubleshooting, I have concluded that there may be something wrong with my Arabidopsis protein samples (since other protein samples run fine into the same gel, including older Arabidopsis protein samples, but every single sample, old and new, tends to show protein bands only larger than about 25 kD). A copy of a Ponceau stained gel showing the "smiling" effect I see starting at around 29 kD (the greater the protein concentration, the more pronounced the smiling is) and the lack of polypeptides smaller than 25 kD can be seen here: http://www.msu.edu/~delgadoi/ponceau.html The extracts are from Arabidopsis seedlings ground in a mortar and pestle, spun at 2,000g twice (each time the supernatant was transferred to a clean tube) and then desalted using a Centricon (5 kD cutoff) filter before resuspending in PBS (plus 0.1% Triton X-100). The buffers I have use for extraction include Laemmli buffer and a Tris+sucrose based buffer. Since I have never seen this before, any help would be greatly appreciated. I am running out of things to try (the acrylamide, Running buffer and the other reagents are nearly all from lab stocks that show consistent good results -they work!- so I believe the problem is with the protein sample, but what?). Thank you for your time, Ivan Ivan J Delgado Orlic Lab: 517-353-3519 MSU-DOE Plant Research Laboratory delgadoi@pilot.msu.edu East Lansing, MI 48824-1312 http://www.msu.edu/user/delgadoi From owner-proteins@net.bio.net Wed Aug 19 23:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!sunqbc.risq.qc.ca!newsfeed.nacamar.de!fu-berlin.de!news.uni-stuttgart.de!news.urz.uni-heidelberg.de!usenet From: James Fethiere Newsgroups: bionet.molbio.proteins Subject: protein precipitation Date: Thu, 20 Aug 1998 19:27:35 +0200 Organization: MPI-Med Forschung Lines: 15 Message-ID: <35DC5C87.1CFB@mpimf-heidelberg.mpg.de> NNTP-Posting-Host: phe.mpimf-heidelberg.mpg.de Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01SGoldC-SGI (X11; I; IRIX 6.3 IP32) Hello, I was just preparing to purify a soluble kinase from the cytoplasm of sf9 cells but ran into a little problem. After polytron, and high speed centrifugation, the published method indicates that the supernatant (soluble fraction) should be completed with a HEPES buffer pH 7.2 containing EDTA, and 0.02% Triton X-100 before going to an S-sepharose column. Well, the prepared buffer looks very clear, but as soon as I dilute the protein fraction with this buffer, everything becomes cloudy, and a light precipitate forms. I don't see any reason for this. Any ideas???? Thank you James From owner-proteins@net.bio.net Wed Aug 19 23:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!howland.erols.net!dca1-hub1.news.digex.net!digex!lynx.unm.edu!news.NMSU.Edu!NMSU.Edu!dkim From: D. KIM Newsgroups: bionet.molbio.proteins Subject: Re: Protein dip stick Date: 20 Aug 1998 19:02:21 GMT Organization: New Mexico State University Lines: 25 Distribution: world Message-ID: <6rhrrt$bqh$1@bubba.NMSU.Edu> References: <35DAF3D0.D5664525@ornl.gov> NNTP-Posting-Host: hector.nmsu.edu X-Trace: bubba.NMSU.Edu 903639741 12113 128.123.34.9 (20 Aug 1998 19:02:21 GMT) X-Complaints-To: usenet@bubba.NMSU.Edu NNTP-Posting-Date: 20 Aug 1998 19:02:21 GMT X-Newsreader: TIN [UNIX 1.3 unoff BETA 970324; AIX 4.2] There was the DNA Dipstick from Invitrogen. I think they made an RNA version and maybe a protein dipstick, but I am not sure. There is a company that makes the DotMetric protein quantitation method, which is kind of a dipstick method. I forget the company name. Daniel Kim Petter wrote: : In article <35DAF3D0.D5664525@ornl.gov>, isolan@ornl.gov wrote: : >This is a multi-part message in MIME format. : >--------------EC8DC33FAEF588173FE685DC : >Content-Type: text/plain; charset=us-ascii : >Content-Transfer-Encoding: 7bit : > : >A friend of mine has developed a protein dip stick for rapid : >quantitation of proteins. Is there any interest in such a product. : > : >Narayana R.Isola : depends on the accurracy i suppose : wasn't there one of these for DNA in the market at one point? From owner-proteins@net.bio.net Thu Aug 20 23:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!arclight.uoregon.edu!news.cc.ukans.edu!not-for-mail From: PGegen@UKans.nolospamare.edu (Dr. Peter Gegenheimer) Newsgroups: bionet.molbio.proteins Subject: Re: Help with SDS-PAGE of Arabidopsis samples Date: 21 Aug 1998 23:33:33 GMT Organization: Univ. Kansas (BCMB) Lines: 58 Message-ID: <7opiGDf98QgB-pn2-GGxdWcpJ0viB@rnaworld.bio.ukans.edu> References: <35DBB5F7.EA85C605@pilot.msu.edu> Reply-To: PGegen@UKans.nolospamare.edu NNTP-Posting-Host: rnaworld.bio.ukans.edu Mime-Version: 1.0 Content-Type: text/plain; charset="iso-8859-1" Content-Transfer-Encoding: quoted-printable X-Newsreader: ProNews/2 Version 1.00 On Thu, 20 Aug 1998 05:39:12, delgadoi@PILOT.MSU.EDU (Ivan J Delgado Orlic) wrote: > Dear All, > I am having problems with 1-D SDS-PAGE. After a lot of > troubleshooting, I > have concluded that there may be something wrong with my Arabidopsis protein > samples (since other protein samples run fine into the same gel, including > older Arabidopsis protein samples, but every single sample, old and new, tends > to show protein bands only larger than about 25 kD). A copy of a Ponceau > stained gel showing the "smiling" effect I see starting at around 29 kD (the > greater the protein concentration, the more pronounced the smiling is) and the > lack of polypeptides smaller than 25 kD can be seen here: http://www.msu.edu/~delgadoi/ponceau.html > The extracts are from Arabidopsis seedlings ground in a mortar and pestle, > spun at 2,000g twice (each time the supernatant was transferred to a clean > tube) and then desalted using a Centricon (5 kD cutoff) filter before > resuspending in PBS (plus 0.1% Triton X-100). The buffers I have use for > extraction include Laemmli buffer and a Tris+sucrose based buffer. > Since I have never seen this before, any help would be greatly > appreciated. I am running out of things to try (the acrylamide, Running buffer > and the other reagents are nearly all from lab stocks that show consistent > good results -they work!- so I believe the problem is with the protein sample, > but what?). > Thank you for your time, > Ivan > > > Ivan J Delgado Orlic Lab: 517-353-3519 > MSU-DOE Plant Research Laboratory delgadoi@pilot.msu.edu > East Lansing, MI 48824-1312 http://www.msu.edu/user/delgadoi Thanks for the photo -- great idea! Looks to me like your samples have a ton of salt in them. That would account for the smearing, smiling, and retardation of low-mol wt polypeptides while the larger species aren't so much affected. The salt front can be spread out a lot, and would interfere with lower-mol wt species or make them run together. I think you'd want at most 50 mM monovalent salt, and preferably <5 mM divalent cation. Can you desalt by dialysis? On a micro-scale, use a 3500-MWCO membrane stretched over a 'decapped' eppitube & held on with a ~2 mm ring of rubber tubing or a rubber band, then floated upside down on buffer. Sucrose in your buffer might well interfere with the centrifugal dialysis your trying. I'd also recommend a more conventional extraction buffer unless, as I infer, you're running a non-denaturing gel. Have a look at the Methods in Plant Molecular Biology series book or other protein methods guide. For denaturing gels, an extraction buffer with Tris, SDS, DTT,