From owner-proteins@net.bio.net Sun Feb 01 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!ais.net!uunet!in2.uu.net!vega.hkstar.com!hkstar2!news@hkstar.com From: "Ronnie Cheng" Newsgroups: bionet.molbio.proteins Subject: Help - chemical testing Date: 1 Feb 1998 15:44:48 GMT Organization: Hong Kong Star Internet Ltd. Lines: 11 Message-ID: <01bd2f28$a4f29300$863252ca@joshua> NNTP-Posting-Host: ip-50-134.dialup.hkstar.com X-Newsreader: Microsoft Internet News 4.70.1161 Can anyone tell me in detail ways to test the presence and quantity of the following substances? - ethoxyresorufin-o-dealkylase (EROD) - glutathione - metallotheonin If description in detail is difficult in email, please tell me papers or books in which I can find the information. Thanks. Ronnie From owner-proteins@net.bio.net Sun Feb 01 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!news-peer.gip.net!news.gsl.net!gip.net!newsfeed.internetmci.com!164.67.42.145!awabi.library.ucla.edu!134.139.1.31!csulb.edu!drivel.ics.uci.edu!news.service.uci.edu!design.eng.uci.edu!egak From: Evgeny Gak Newsgroups: bionet.molbio.proteins Subject: polyaspartic and polyglutamic acid Date: Mon, 2 Feb 1998 16:25:02 -0800 Organization: University of California, Irvine Lines: 11 Message-ID: NNTP-Posting-Host: design.eng.uci.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII I need polyASP(10-20) and polyGLU(10-20) where can I get it? ------------ Dr. Evgueni Gak 952 Engineering Tower Dept. Chemical and Biochemical Engineering UCI Irvine, CA 92697-2575 Tel:714-824-8389 Fax: 714-824-2541 E-mail:egak@design.eng.uci.edu From owner-proteins@net.bio.net Sun Feb 01 22:00:00 1998 From: "Lars Komorowski" Newsgroups: bionet.molbio.proteins Subject: HPLC-Manager-Software Date: Mon, 2 Feb 1998 20:49:24 +0100 Organization: Med. Universitaet zu Luebeck Lines: 6 Message-ID: <6b587t$9db$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 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!fu-berlin.de!news.CS.Uni-Magdeburg.De!RRZ.Uni-Koeln.DE!news-koe1.dfn.de!news-han1.dfn.de!news-ham1.dfn.de!news.mu-luebeck.de!not-for-mail Who can help me ? I lost data from HPLCManager 3.0 for DOS from Pharmacia LKB. And I don't have the disks anymore (they were from 1991). Who can send me the installation files or maybe newer ones if they work ? Lars From owner-proteins@net.bio.net Sun Feb 01 22:00:00 1998 Newsgroups: bionet.molbio.proteins Path: biosci!agate!ihnp4.ucsd.edu!sdd.hp.com!usc!howland.erols.net!news.maxwell.syr.edu!rill.news.pipex.net!pipex!server1.netnews.ja.net!yama.mcc.ac.uk!liv!news From: adrian@liv.ac.uk (Adrian Milton) Subject: Apologies.... Message-ID: Sender: news@liverpool.ac.uk (News System) NNTP-Posting-Host: pc011008.envbio.liv.ac.uk Organization: The University of Liverpool X-Newsreader: Forte Free Agent v0.55 Date: Mon, 2 Feb 1998 11:43:55 GMT Lines: 23 Dear Newsgroup, This may be the wrong group to send this request for information to, and if so I apologise and would welcome advice on the correct newsgroup. My problem is quite simple I am intending to investigate the effects of protein restriction on the effects of a virus on small mammals. To achieve this I want to produce (or have produced) diets with varying protein levels. Unfortunately, I am having difficulty finding the minimal dietary levels of crude protein required for bank voles and wood (field) mice, any information, references, detaisl of supplies (pref. UK) would be gratefully accepted. TIA. Adrian Milton University of Liverpool UK adrian@liv.ac.uk From owner-proteins@net.bio.net Sun Feb 01 22:00:00 1998 Newsgroups: bionet.molbio.proteins Path: biosci!agate!logbridge.uoregon.edu!newsfeed.direct.ca!torn!utnut!utzoo!pbowser From: pbowser@zoo.utoronto.ca (Paul Bowser) Subject: Iodo-beads iodination of peptides Organization: U of Toronto Zoology Message-ID: Date: Mon, 2 Feb 1998 17:21:14 GMT Lines: 17 Hi, I'm looking for protocols for iodinating small (<2000 daltons) peptides using the iodo-beads product from Pierce. The protocol that comes with the beads is pretty vague and I don't want to spend forever optimizing the procedure. While I'm at it I might as well ask if anyone has any tips or tricks for iodinating peptides with the Chloramine T method. Thanks Paul -- -- Paul Bowser "The Tobe Lab" RWZL 534 978-3522 pbowser(at)zoo(dot)utoronto(dot)ca From owner-proteins@net.bio.net Sun Feb 01 22:00:00 1998 Path: biosci!NETVIGATOR.COM!sentosa From: sentosa@NETVIGATOR.COM ("Erik A.J. Miljan") Newsgroups: bionet.molbio.proteins Subject: Sequencing of Phospho-amino acids? Date: 2 Feb 1998 05:24:21 -0800 Organization: The University of Hong Kong Lines: 68 Sender: daemon@net.bio.net Distribution: world Message-ID: <2D247FE1.7B891D68@netvigator.com> NNTP-Posting-Host: net.bio.net --------------61D3B841393E0186C2EABBA0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit I would like to determine the position of the phosphorylated amino acid within the tryptic peptide I have isolated from a protein kinase. The Problem- I am using a Hewlett-Packard G1000A protein sequencer and it uses a non-standard sample cartridge. The peptide is applied to and remains bound to a small HIC (hydrophobic interaction chromatography) column. A SAX (ion exchange) column is placed directly on the HIC half - presumably to clean up cleavage by products??- and the entire cartridge is loaded into the sequencer The question- what happens to the phospho-amino acid and the 32P in this automated amino acid sequencer? Can I follow the 32P in order to determine the position of the phosphorylated amino acid? I suspect the 32P and the peptide are retained on the HIC until the phospho amino acid is hit. The 32P is then hydrolyzed from the aa and is washed onto the SAX portion. The aa is difficult to detect. To identify the specific phosphorylated amino acid I am using 1D TLC (what a contrast of technology!). Can anybody help me? If there is any references available I would be grateful. Cheers ERiK Miljan The University of Hong Kong Dept. of Biochemistry --------------61D3B841393E0186C2EABBA0 Content-Type: text/html; charset=us-ascii Content-Transfer-Encoding: 7bit I would like to determine the position of the phosphorylated amino acid within the tryptic peptide I have isolated from a protein kinase.

The Problem- I am using a Hewlett-Packard G1000A protein sequencer and it uses a non-standard sample cartridge.  The peptide is applied to and remains bound to a small HIC (hydrophobic interaction chromatography) column.  A  SAX (ion exchange) column is placed directly on the HIC half - presumably to clean up cleavage by products??- and the entire cartridge is loaded into the sequencer

The question- what happens to the phospho-amino acid and the 32P in this automated amino acid sequencer?  Can I follow the 32P in order to determine the position of the phosphorylated amino acid?

I suspect the 32P and the peptide are retained on the HIC until the phospho amino acid is hit.  The 32P is then hydrolyzed from the aa and is washed onto the SAX portion.  The aa is difficult to detect. To identify the specific phosphorylated  amino acid I am using 1D TLC (what a contrast of technology!).

Can anybody help me?  If there is any references available I would be grateful.

Cheers
ERiK Miljan
The University of Hong Kong
Dept. of Biochemistry --------------61D3B841393E0186C2EABBA0-- From owner-proteins@net.bio.net Sun Feb 01 22:00:00 1998 Message-ID: <34D5BC2B.41C6@sgiclu.chemie.uni-konstanz.de> Date: Mon, 02 Feb 1998 13:29:31 +0100 From: Marcus Macht X-Mailer: Mozilla 3.04 (X11; I; IRIX 6.2 IP22) MIME-Version: 1.0 Newsgroups: bionet.molbio.proteins Subject: Re: affinity chromatography References: <34C46368.B42D79AD@kfunigraz.ac.at> Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit NNTP-Posting-Host: sg17.chemie.uni-konstanz.de Organization: University of Constance, Germany Lines: 28 Path: biosci!agate!logbridge.uoregon.edu!sunqbc.risq.qc.ca!newsfeed.nacamar.de!fu-berlin.de!news.belwue.de!news.uni-konstanz.de!sg17.chemie.uni-konstanz.de Markus Zettl wrote: > > Does anyone know some ways besides classical affinity chromatography to > detect protein-protein interactions? Hello! What do you think about: - yeast two hybrid systems, - analytical ultracentrifugation, - surface plasmon resonance (BIAcore), - electrospray mass spectrometry, - gel permeation chromatography, - native gel electrophoresis ? I'm quite sure that this is not a complete list of methods for the analysis of non-covalent interactions. I hope I could help you with this. Yours sincerely, Marcus -- * Marcus Macht, AG Przybylski, Faculty Chemistry, University of Konstanz * Box M 732, 78457 Konstanz, Germany * Tel:++49-07531-883436/ Fax:++49-07531-883097 * Email: macht@sgiclu.chemie.uni-konstanz.de * URL: http://www.ag-przybylski.chemie.uni-konstanz.de/~macht From owner-proteins@net.bio.net Sun Feb 01 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!news-peer.gip.net!news.gsl.net!gip.net!news-xfer.netaxs.com!nntp.texas.net!news.io.com!news.tamu.edu!not-for-mail From: Paul Fitzpatrick Newsgroups: bionet.molbio.proteins Subject: Trial Date: Mon, 02 Feb 1998 12:29:09 -0600 Organization: TAMU Lines: 11 Message-ID: <34D61074.63C2A098@bioch.tamu.edu> Reply-To: gmoran@bioch.tamu.edu NNTP-Posting-Host: claudius.tamu.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii; x-mac-type="54455854"; x-mac-creator="4D4F5353" Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.01 (Macintosh; I; PPC) X-Priority: 3 (Normal) 1) Does anybody know if aminopeptidase M is able to cut the N-terminus of a formylated peptidase? 2) Is aminopeptidase M working in the presence of detergents, i.e., on membrane proteins which need to be in the presence of detergents? The help of anybody who can give me refs concerning these questions is appreciated. Goga From owner-proteins@net.bio.net Mon Feb 02 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!news-peer.gip.net!news.gsl.net!gip.net!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!portc02.blue.aol.com!pitt.edu!newsfeed.pitt.edu!pxpst2 From: pxpst2@vms.cis.pitt.spam.stinks.edu (Peter Pediaditakis) Newsgroups: bionet.molbio.proteins Subject: Re: Iodo-beads iodination of peptides Date: Tue, 03 Feb 1998 09:37:38 -0500 Organization: University of Pittsburgh Lines: 45 Message-ID: References: NNTP-Posting-Host: pelli.pathology.pitt.edu X-Newsreader: MT-NewsWatcher 2.3.5 In article , pbowser@zoo.utoronto.ca (Paul Bowser) wrote: > Hi, > > I'm looking for protocols for iodinating small (<2000 daltons) > peptides using the iodo-beads product from Pierce. The protocol that > comes with the beads is pretty vague and I don't want to spend forever > optimizing the procedure. > > While I'm at it I might as well ask if anyone has any tips or tricks > for iodinating peptides with the Chloramine T method. I have used this kit many times with great success. As long as the bead is in contact with the Iodine and protein then the reaction proceeds. When the bead is removed the reaction stops, thus no quencing is needed. The problem with the standard Clor-T method is theat it is a 3D reaction and proceeds extremely fast. often what happens is that the protein will get cross linked to itself. The beads are a 2D reaction and proceed much slower. As for optimizing, forget it. It is not needed. If you nknow the sequence then you can count the tyr residues and multiply by ~1.5 and that is approx how many Iodines will be added. For a small protein, 6 min should be adequate and 10m will work as well. Regards Peter Pediaditakis -- "Don't you eat that yellow snow WAtch out where the Huskies go" --------------------------------------------------------------------- Let us not forget those marketing boys from SPAM CENTRAL bestrealtor@marketingmaster.com info@herbchew.com clickthru@timefreedom.com invite@onlinenow.net zippydj@nevwest.com Offer@shire.com empower@empowerlabs.com dynamarket@vaprnet.com root@mail.icongrp.com cashrewards@hotmail.com From owner-proteins@net.bio.net Mon Feb 02 22:00:00 1998 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!4.1.16.34!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!baron.netcom.net.uk!netcom.net.uk!nntp.news.xara.net!xara.net!rill.news.pipex.net!pipex!server1.netnews.ja.net!bham!bcs113.bham.ac.uk!user From: email@domain.com (LC475 (r121)) Newsgroups: bionet.molbio.proteins Subject: mdm2 Date: Tue, 03 Feb 1998 14:10:06 +0000 Organization: The University of Birmingham, UK. Lines: 10 Message-ID: NNTP-Posting-Host: bcs113.bham.ac.uk Has anyone encountered any difference between the induction of MDM2 mRNA after treatment with Ionizing Radiation compared to the induction of the MDM2 protein? Also has anyone found a 130kDa protein that cross reacts with the 2A10 anti-MDM2 monoclonal when doing a Western Blot on whole cell lysate? Grant Email: gss643@sun1.bham.ac.uk From owner-proteins@net.bio.net Tue Feb 03 22:00:00 1998 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!199.0.154.56!ais.net!uunet!in3.uu.net!ozemail!news.mel.aone.net.au!newsfeed-in.aone.net.au!inferno.mpx.com.au!news.unimelb.edu.au!ludwignt-4 From: murphy_r@licre.ludwig.edu.au (Roger Murphy) Newsgroups: bionet.molbio.proteins Subject: Re: 6His protein Date: Thu, 05 Feb 98 01:11:58 GMT Organization: Ludwig Institute for Cancer Research Lines: 30 Message-ID: <6bavmj$pau$1@izvestia.its.unimelb.edu.au> References: <01bd2c0c$ad136100$2d187889@henri> NNTP-Posting-Host: ludwignt-4.austin.unimelb.edu.au X-Newsreader: News Xpress 2.0 Beta #0 In article <01bd2c0c$ad136100$2d187889@henri>, "Henri M.H. Spronk" wrote: >After expression (in E.coli) of a recombinant human protein as part of a >6His-DHFR-fusion protein and purification using a Ni-NTA-column, the >protein is insolluble. The protein is soluble in a solution of pH 12, but >not at 7.5. Does anybody have experience with protein solubillization? > >H.M.H. Spronk >Department of Biochemistry >University Maastricht >The Netherlands > >henri.spronk@bioch.unimaas.nl Have you tried various concentrations of urea or guanadinium hydrochloride in your protein solution? Or some of the non-ionic surfactants? 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 Tue Feb 03 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!sunqbc.risq.qc.ca!news-peer-east.sprintlink.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!worldnet.att.net!news.u.washington.edu!not-for-mail From: "Gerd Kleemann" Newsgroups: bionet.molbio.proteins Subject: Re: 6His protein Date: Wed, 4 Feb 1998 23:04:30 -0800 Organization: University of Washington Lines: 35 Message-ID: <6bboq9$44p$1@nntp3.u.washington.edu> References: <01bd2c0c$ad136100$2d187889@henri> NNTP-Posting-Host: cs112-12.u.washington.edu X-Trace: nntp3.u.washington.edu 886662793 4249 (None) 140.142.64.4 X-Complaints-To: help@cac.washington.edu X-Newsreader: Microsoft Outlook Express 4.72.2106.4 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.2106.4 I think that DHFR is the problem. DHFR itself is not very soluble and stable and therefore a terrible fusion partner. Even point mutations can have dramatic effects on solubility. Solubilizing the DHFR inclusion bodies in GdmCl and refolding via Dialysis brings poor yields of folded functional protein also. I would suggest to avoid DHFR as fusion construct. Maltose Binding Protein (MBP)is very soluble also as fusion protein with any other fusion partner (Biolabs). MBP itself you can purify via affinity chromatography. MBP-fusions are expressed with excellent yields. Another good fusion construct would be with GST, which you can purify also via affinity chromatography (Boehringer Mannheim). Good Luck ! Gerd Kleemann University of Washington Dept. of Biotechnology Seattle kleemann@u.washington.edu Henri M.H. Spronk schrieb in Nachricht <01bd2c0c$ad136100$2d187889@henri>... >After expression (in E.coli) of a recombinant human protein as part of a >6His-DHFR-fusion protein and purification using a Ni-NTA-column, the >protein is insolluble. The protein is soluble in a solution of pH 12, but >not at 7.5. Does anybody have experience with protein solubillization? > >H.M.H. Spronk >Department of Biochemistry >University Maastricht >The Netherlands > >henri.spronk@bioch.unimaas.nl From owner-proteins@net.bio.net Tue Feb 03 22:00:00 1998 From: murphy_r@licre.ludwig.edu.au (Roger Murphy) Newsgroups: bionet.molbio.proteins Subject: Re: HPLC-Manager-Software Date: Thu, 05 Feb 98 01:15:11 GMT Organization: Ludwig Institute for Cancer Research Lines: 22 Message-ID: <6bavsl$pau$2@izvestia.its.unimelb.edu.au> References: <6b587t$9db$1@gwsun.medinf.mu-luebeck.de> NNTP-Posting-Host: ludwignt-4.austin.unimelb.edu.au X-Newsreader: News Xpress 2.0 Beta #0 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!139.130.250.2!intgwpad.nntp.telstra.net!news.telstra.net!sa.news.telstra.net!news.chariot.net.au!news.box.net.au!news.camtech.net.au!duster.adelaide.on.net!news.ade.connect.com.au!news.mel.connect.com.au!news.mel.aone.net.au!newsfeed-in.aone.net.au!inferno.mpx.com.au!news.unimelb.edu.au!ludwignt-4 In article <6b587t$9db$1@gwsun.medinf.mu-luebeck.de>, "Lars Komorowski" wrote: >Who can help me ? I lost data from HPLCManager 3.0 for DOS from Pharmacia >LKB. And I don't have the disks anymore (they were from 1991). Who can send >me the installation files or maybe newer ones if they work ? >Lars > > You should contact Pharmacia, I'm sure they'd have a copy somewhere! 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 Wed Feb 04 22:00:00 1998 Path: biosci!agate!howland.erols.net!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news.mindspring.com!user-37kbbdl.dialup.mindspring.com!user From: petter@mindspring.com (Petter) Newsgroups: bionet.molbio.proteins Subject: Re: HPLC-Manager-Software Date: Thu, 05 Feb 1998 05:10:30 -0600 Organization: MindSpring Enterprises Lines: 33 Message-ID: References: <6b587t$9db$1@gwsun.medinf.mu-luebeck.de> <6bavsl$pau$2@izvestia.its.unimelb.edu.au> NNTP-Posting-Host: user-37kbbdl.dialup.mindspring.com X-Server-Date: 5 Feb 1998 11:07:00 GMT In article <6bavsl$pau$2@izvestia.its.unimelb.edu.au>, murphy_r@licre.ludwig.edu.au (Roger Murphy) wrote: >In article <6b587t$9db$1@gwsun.medinf.mu-luebeck.de>, "Lars Komorowski" wrote: >>Who can help me ? I lost data from HPLCManager 3.0 for DOS from Pharmacia >>LKB. And I don't have the disks anymore (they were from 1991). Who can send >>me the installation files or maybe newer ones if they work ? >>Lars >> >> >You should contact Pharmacia, I'm sure they'd have a copy somewhere! > > > >Roger Murphy, Ph.D. >Biological Production Facility >Ludwig Institute for Cancer Research >Austin & Repatriation Medical Centre >Studley Road, >Heidelberg, Vic. 3084 >Australia. Not true! Surprisingly, we are in a similar situation, got a hand me down instrument, dead computer, no disks, and pharmacia no longer has the software....pretty amazing... Peter > >Tel 61-3-94965463 >Fax 61-3-94965436 >Email murphy_r@licre.ludwig.edu.au From owner-proteins@net.bio.net Wed Feb 04 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!europa.clark.net!4.1.16.34!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!rill.news.pipex.net!pipex!server1.netnews.ja.net!news.nott.ac.uk!pdxkgs From: pdxkgs@pdn1.gene.nottingham.ac.uk (Karen Spink) Newsgroups: bionet.molbio.proteins Subject: Band-shift assays Date: Thu, 05 Feb 1998 12:54:04 +0000 Organization: University of Nottingham Lines: 24 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 I am very confused! Recently, I have been trying to purify a DNA-binding protein from yeast and have constructed a specific DNA affinity column, attaching the binding site for my protein to CnBr-activated Sepharose. I bound some of my partially purified protein extract to this column and was successfull in retrieving a band-shift with one of the fraction. SDS-PAGE informed me that I now had a dozen or so bands in this fraction, so I re-applied this fraction to the column and eluted with a narrower range of salt concentrations, but my protein has now DISAPPEARED! It is neither in the flow through nor any of the fractions. I have read in a couple of publications that loss of activity in a band-shift can occur with a highly pure DNA binding protein and that activity can be regained by the addition of 1mg/ml casein to the binding reactions. I am hoping someone can enlighten me on this phenomena. 1) Does this really work ? 2) Why does this work, (BSA supposedly does not have this effect on activity) 3) Is it better to use casein or B-casein I do not really understand how the addition of casein, rather than BSA, can affect activity in this way and am hoping not to have to do the whole purification thing again Thanks very much for your time, Karen Spink From owner-proteins@net.bio.net Wed Feb 04 22:00:00 1998 Path: biosci!agate!ihnp4.ucsd.edu!sdd.hp.com!usc!news1.chicago.iagnet.net!qual.net!iagnet.net!howland.erols.net!news-peer.sprintlink.net!news-backup-east.sprintlink.net!news-in-east.sprintlink.net!news.sprintlink.net!Sprint!130.199.128.185!nntp.bnl.gov!not-for-mail From: Dawei Lin Newsgroups: bionet.molbio.proteins Subject: To hold coodinates, or not to hold coordinates, that's poll Date: Thu, 05 Feb 1998 11:22:36 -0500 Organization: Protein Data Bank, Brookhaven National Lab Lines: 48 Message-ID: <34D9E74C.167E@pdb.pdb.bnl.gov> NNTP-Posting-Host: latimeria.pdb.bnl.gov Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (X11; I; IRIX 6.2 IP22) Dear Macromolecular Structure funs, Please see the note from Guy Riddihough. You can also find some discussions are going on in the PDB listserver. --------------------------------------------------------- In light of the various ongoing discussions of the one year hold for structure coordinates, we are hosting a vote on the matter on the Nature Structural Biology web site, based on a petition to the IUCr proposed by Alex Wlodawer. I know that the journals Science and Nature, the IUCr, the PDB and the NIH are interested in the results we will obtain so potentially, the data may be of some importance in guiding discussions of future policy. For the vote to best represent the feelings in and beyond the structure community it is vital that as many people vote as possible. Thus I am e-mailing you to ask if you, and your colleagues, would be interested in contributing your opinions on the matter. The URL for the site of the vote is: http://us.nature.com/survey/nsb_poll.nclk I would not object if you wished to forward this message on to other colleagues, either structural biologists or scientists with an interest in the availability of structural data, whom you think may also want to express their opinion on the matter. If you have any questions regarding this or any other matter, please don't hesitate to contact me at this address or tel: 212 726 9313, fax: 212 679 0735. Sincerely Guy Riddihough Editor, Nature Structural Biology -- --------< *** Dr. Dawei LIN *** >--------------- Protein Data Bank Biology Department Brookhaven National Laboratory Upton, NY 11973 U.S.A Telepone: 516-344-6359 (office) Fax: 516-344-5751 Email: lin@bnl.gov ------------------------------------------------ From owner-proteins@net.bio.net Wed Feb 04 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!howland.erols.net!wnfeed!204.127.130.5!worldnet.att.net!news.u.washington.edu!not-for-mail From: "Gerd Kleemann" Newsgroups: bionet.molbio.proteins Subject: Re: 6His protein Date: Thu, 5 Feb 1998 10:41:12 -0800 Organization: University of Washington Lines: 43 Message-ID: <6bd150$5bc$1@nntp3.u.washington.edu> References: <01bd2c0c$ad136100$2d187889@henri> <6bboq9$44p$1@nntp3.u.washington.edu> NNTP-Posting-Host: chinook.mbt.washington.edu X-Trace: nntp3.u.washington.edu 886704096 5484 (None) 140.142.64.4 X-Complaints-To: help@cac.washington.edu X-Newsreader: Microsoft Outlook Express 4.72.2106.4 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.2106.4 Sorry the GST fusion vectors are availble at Pharmacia and not at Boehringer Mannheim !!! Gerd Kleemann wrote in message <6bboq9$44p$1@nntp3.u.washington.edu>... >I think that DHFR is the problem. DHFR itself is not very soluble and stable >and therefore a terrible fusion partner. Even point mutations can have >dramatic effects on solubility. Solubilizing the DHFR inclusion bodies in >GdmCl and refolding via Dialysis brings poor yields of folded functional >protein also. I would suggest to avoid DHFR as fusion construct. Maltose >Binding Protein (MBP)is very soluble also as fusion protein with any other >fusion partner (Biolabs). MBP itself you can purify via affinity >chromatography. MBP-fusions are expressed with excellent yields. Another >good fusion construct would be with GST, which you can purify also via >affinity chromatography (Boehringer Mannheim). > >Good Luck ! > >Gerd Kleemann >University of Washington >Dept. of Biotechnology >Seattle >kleemann@u.washington.edu > > > >Henri M.H. Spronk schrieb in Nachricht <01bd2c0c$ad136100$2d187889@henri>... >>After expression (in E.coli) of a recombinant human protein as part of a >>6His-DHFR-fusion protein and purification using a Ni-NTA-column, the >>protein is insolluble. The protein is soluble in a solution of pH 12, but >>not at 7.5. Does anybody have experience with protein solubillization? >> >>H.M.H. Spronk >>Department of Biochemistry >>University Maastricht >>The Netherlands >> >>henri.spronk@bioch.unimaas.nl > > From owner-proteins@net.bio.net Wed Feb 04 22:00:00 1998 Path: biosci!agate!howland.erols.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!newsfeed.internetmci.com!193.174.75.126!news-was.dfn.de!news-fra1.dfn.de!news-koe1.dfn.de!news.rhrz.uni-bonn.de!news.uni-stuttgart.de!news.urz.uni-heidelberg.de!not-for-mail From: January Weiner Newsgroups: bionet.molbio.proteins Subject: Methods for labelling 3' Date: 5 Feb 1998 16:26:15 GMT Organization: University of Heidelberg, Germany Lines: 29 Message-ID: <6bcp77$dgc@sun0.urz.uni-heidelberg.de> Reply-To: nospam_jweiner1@ix.urz.uni-heidelberg.de NNTP-Posting-Host: ix.urz.uni-heidelberg.de X-Newsreader: TIN [UNIX 1.3 unoff BETA 970820; AIX 4.2] Xcanpos: shelf.01/199802192101!0011141836 Anyone has experience with 3' radioactive end labelling of PCR products? I have been using T4 Polymerase [1] but it seems that I get sometimes into trouble and that the method is not very reliable nor effective. Any suggestions? TIA! j. [1] ca. 1 ug DNA 3 U T4 2 ul T4 10x Puffer 30 uCi alfa-32P dATP 2 ul 1 mM d(C,G,T)TP H20 ad 20 ul --> 2-5' 37 deg C + 1 ul 1 mM dATP --> 5-10' 37 deg C + 100 ul STOP mix (phenol, EDTA etc.) | +----> fractioning on 50 Sephadex It sometimes works perfectly (or almost), sometimes I get nothing with the same PCR product. -- ----)-\//-///-----------------------------------January-Weiner-3------- Two things are infinite: the universe and human stupidity; and I'm not sure about the the universe. [Albert Einstein] From owner-proteins@net.bio.net Wed Feb 04 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!news.maxwell.syr.edu!howland.erols.net!vixen.cso.uiuc.edu!staff1.cso.uiuc.edu!sdavidso From: Sean Davidson Newsgroups: bionet.molbio.proteins Subject: sequence search question Date: Thu, 5 Feb 1998 17:11:40 -0600 Organization: University of Illinois at Urbana-Champaign Lines: 18 Message-ID: NNTP-Posting-Host: staff1.cso.uiuc.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII X-Sender: sdavidso@staff1.cso.uiuc.edu Hi, I am trying to figure out a way to search the structural databases (i.e. crystal structures) for proteins that have only one Tryptophan in the sequence. Most search programs only allow you to use names or sequences for the search. Anyone have any ideas? Thanks. -Sean _______________________________________________________________________________ Dr. Sean Davidson, Ph.D. e-mail: sdavidso@uiuc.edu Department of Biochemistry Office: (217) 333-7294 University of Illinois Home: (217) 355-0526 Urbana, IL 61821 www.staff.uiuc.edu/~sdavidso/index.html _______________________________________________________________________________ From owner-proteins@net.bio.net Wed Feb 04 22:00:00 1998 Newsgroups: bionet.molbio.proteins Path: biosci!agate!logbridge.uoregon.edu!news.maxwell.syr.edu!newsfeed.internetmci.com!199.117.161.1!csn!nntp-xfer-1.csn.net!news.acsu.buffalo.edu!srv1.drenet.dnd.ca!crc-news.doc.ca!nott!utnut!utinfo!nntp From: Darryl Yu Subject: SASP's X-Nntp-Posting-Host: rbm06.phm.utoronto.ca Content-Type: text/plain; charset=us-ascii Message-ID: <34DB7CDF.47AB0B9F@utoronto.ca> Sender: nntp@utcc.utoronto.ca (News) Content-Transfer-Encoding: 7bit Organization: Faculty of Pharmacy, University of Toronto Mime-Version: 1.0 Date: Fri, 6 Feb 1998 21:13:04 GMT X-Mailer: Mozilla 4.04 [en] (Win95; U) Lines: 6 I am interested whether anyone has created a small molecule probe or an antibody probe to the Small Acid Solube Proteins in Bacillus Subtilus spores. I am interested in using such a probe to co-localize with the SASP's and view them under an electron microscope. If anyone has heard of this please send me the literature source. From owner-proteins@net.bio.net Wed Feb 04 22:00:00 1998 Path: biosci!rutgers!uwm.edu!vixen.cso.uiuc.edu!howland.erols.net!news.idt.net!nntp2.cerf.net!moon.pepperdine.edu!usenet From: zhuqi Newsgroups: bionet.molbio.proteins Subject: Re: Iodo-beads iodination of peptides Date: Fri, 06 Feb 1998 10:49:30 +0800 Organization: Pepperdine University Lines: 13 Message-ID: <34DA7A3A.B512DBF7@hunnu.edu.cn> References: NNTP-Posting-Host: 202.197.120.110 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.03 [en] (Win95; I) To: Peter Pediaditakis ear Dr.Peter: I am a Grad-student in China.I need iodinate a toxin (33 amino acids)which have no Tyr but His.I want label it with 125I. I used mothod of Chloramine T.But the product is so little.So I think the the reason is this peptide have no Tyr.Do you have some useful method to improve the product of Iodination?Would you mind give me some advice? Thank you in advance. Sincerely Yours : Qi Zhu From owner-proteins@net.bio.net Wed Feb 04 22:00:00 1998 Path: biosci!webtv.net!uunet!in5.uu.net!news.new-york.net!news.columbia.edu!news-not-for-mail From: Baohe Shen Newsgroups: bionet.molbio.proteins Subject: Phosphoinositol 3,4,5-P3 Date: Tue, 27 Jan 1998 13:53:11 -0500 Organization: Columbia University Lines: 3 Message-ID: <34CE2D17.6F6B@columbia.edu> NNTP-Posting-Host: manleylab-pc.bio.columbia.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01Gold (Win95; I) Hi. Does anyone know any commercial Phosphoinositol 3,4,5-P3 which is the product of PI3 kinase? I want to study whether it has effects on the activity of my enzyme. Thanks. From owner-proteins@net.bio.net Thu Feb 05 22:00:00 1998 Path: biosci!bloom-beacon.mit.edu!news.kodak.com!news-pen-16.sprintlink.net!newsfeed.nysernet.net!news.nysernet.net!207.41.200.14!news-pen-14.sprintlink.net!206.229.87.26!news-east.sprintlink.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!news-peer.gip.net!news-raspail.gip.net!news.gsl.net!gip.net!newsfeed.eerie.fr!jussieu.fr!saphir.jouy.inra.fr!NewsWatcher!user From: herrbach@colmar.inra.fr (Etienne Herrbach) Newsgroups: bionet.molbio.proteins Subject: Postdoc position: VIRUS RECEPTORS IN INSECTS Date: 6 Feb 1998 14:58:23 GMT Organization: INRA Lines: 25 Message-ID: NNTP-Posting-Host: 192.93.67.13 Postdoc position available: VIRUS RECEPTORS IN INSECTS A postdoctoral position opens at INRA (Institut National de la Recherche Agronomique), Colmar, France, to study plant virus receptors in aphid vectors. The objectives include the identification and isolation of epithelial receptors. Experience required in protein biochemistry, and appreciated in molecular biology (development of a cDNA library). The position is funded for one year, renewable after scientific evaluation, and will start July 1988. Net salary is about 8,500 Francs monthly, or about 6,500 as a complement to current salary. Applicants should not be French citizen. Please send applications, with CV, list of publications, relevant reprints, and references, to: Dr Etienne Herrbach Laboratoire de Zoologie INRA, BP 507 68021 Colmar, France phone +33 (0) 3 89 22 49 42 fax +33 (0) 3 89 22 49 33 e-mail: herrbach@colmar.inra.fr From owner-proteins@net.bio.net Thu Feb 05 22:00:00 1998 Path: biosci!agate!howland.erols.net!news-peer.gip.net!news-raspail.gip.net!news.gsl.net!gip.net!newscore.univie.ac.at!newsfeed.Austria.EU.net!Austria.EU.net!not-for-mail From: Gerald Loeffler Newsgroups: bionet.molbio.proteins Subject: Re: sequence search question Date: Fri, 06 Feb 1998 12:12:23 +0100 Organization: Boehringer Ingelheim Vienna (Bender Wien) Lines: 51 Message-ID: <34DAF017.201D@bender.co.at> References: Reply-To: loeffler@bender.co.at NNTP-Posting-Host: apollon.bender.co.at Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.02 (WinNT; I) Hi! tricky question. i tried this, which in principle works: using a Smith-Waterman alignment algorithm (like http://www.dna.affrc.go.jp/htdocs/MPsrch/index_PP.html) search in PDB (or nrl3d) for a sequence containing only TRPs using the identity matrix for scoring. Then you should (and do) get a ranking of 3 if a sequence contains 3 TRPs, a ranking of 2 if the sequence contains 2 TRPs and finally of 1 if the sequence contains just 1 TRP. However!!: the above site returns a max of 200 hits, which consist only of ranks 3 and 2, i.e. the hits with rank 1 are not returned to you )-: Any help with that? gerald Gerald Loeffler - Bioinformaticist Boehringer Ingelheim Vienna (aka: Bender Wien) Email: loeffler@bender.co.at Phone: +43 1 80105 634 and +43 676 3289588 Fax: +43 1 8041540 Smail: Dr. Boehringer-Gasse 5-11, A-1121 Vienna, Austria Sean Davidson wrote: > > Hi, > > I am trying to figure out a way to search the structural databases (i.e. > crystal structures) for proteins that have only one Tryptophan in the > sequence. Most search programs only allow you to use names or sequences > for the search. Anyone have any ideas? Thanks. > > -Sean > > _______________________________________________________________________________ > > Dr. Sean Davidson, Ph.D. e-mail: sdavidso@uiuc.edu > Department of Biochemistry Office: (217) 333-7294 > University of Illinois Home: (217) 355-0526 > Urbana, IL 61821 www.staff.uiuc.edu/~sdavidso/index.html > _______________________________________________________________________________ -- Gerald Loeffler - Bioinformaticist Boehringer Ingelheim Vienna (aka: Bender Wien) Email: loeffler@imp.univie.ac.at Phone: +43 1 80105 634 and +43 676 3289588 Fax: +43 1 8041540 Smail: Dr. Boehringer-Gasse 5-11, A-1121 Vienna, Austria From owner-proteins@net.bio.net Thu Feb 05 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!news-peer.gip.net!news.gsl.net!gip.net!nntp.abs.net!fu-berlin.de!newscore.univie.ac.at!03-newsfeed.univie.ac.at!news.univie.ac.at!not-for-mail From: a8803349.nospam@unet.univie.ac.at (Martin Offterdinger) Newsgroups: bionet.molbio.proteins Subject: sensitive protein assay Date: Fri, 06 Feb 1998 09:13:43 GMT Organization: AKH Lines: 13 Message-ID: <34dad398.4953740@news.univie.ac.at> NNTP-Posting-Host: grunt.imed1.akh-wien.ac.at X-Newsreader: Forte Free Agent 1.1/16.230 Hi ! Is there anyone who tried the nano orange protein assay from molecular probes? I am currently using Bradford, but it is not sensitive enough as I have only got 5-8ul of a very dilute sample and would like to get an exact protein concentration. Are there any other protein assays which are more sensitive than Bradford? martin Martin Offterdinger Internal Med.I,Dept. Oncology University of Vienna Austria E-Mail:a8803349.nospam@unet.univie.ac.at (remove nospam before mailing) From owner-proteins@net.bio.net Thu Feb 05 22:00:00 1998 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!europa.clark.net!4.1.16.34!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!portc02.blue.aol.com!pitt.edu!newsfeed.pitt.edu!pxpst2 From: pxpst2@vms.cis.pitt.edu (Peter Pediaditakis) Newsgroups: bionet.molbio.proteins Subject: Re: Iodo-beads iodination of peptides Date: Fri, 06 Feb 1998 09:55:15 -0500 Organization: University of Pittsburgh Lines: 47 Message-ID: References: <34DA7A3A.B512DBF7@hunnu.edu.cn> NNTP-Posting-Host: pelli.pathology.pitt.edu X-Newsreader: MT-NewsWatcher 2.3.5 In article <34DA7A3A.B512DBF7@hunnu.edu.cn>, zhuqi wrote: > ear Dr.Peter: > I am a Grad-student in China.I need iodinate a toxin (33 amino > acids)which have no Tyr but His.I want label it with 125I. I used mothod > > of Chloramine T.But the product is so little.So I think the the reason > > is this peptide have no Tyr.Do you have some useful method to improve > the product of Iodination?Would you mind give me some advice? > Thank you in advance. > Qi Zhu This is a tough one. I will look into they modifications of His that are possible but that will take some time(few hours) but off the top of my head I was wondering if you can add some tyrosines to the end of your protein via cloning techniques? If you can and it still has activity then this would be the best way. Alternatively, if you can place a cDNA for the protein into an in vitro expresion vector(Pex plasmid from Promega) then you can use some radiooactive amino acid and produce small amts of a VERY HOT protein. You could then use this as a tracer. Regards Peter Pediaditakis PS. I am a graduate student not a doctor but maybe soon :) -- "Don't you eat that yellow snow WAtch out where the Huskies go" --------------------------------------------------------------------- Let us not forget those marketing boys from SPAM CENTRAL bestrealtor@marketingmaster.com info@herbchew.com clickthru@timefreedom.com invite@onlinenow.net zippydj@nevwest.com Offer@shire.com empower@empowerlabs.com dynamarket@vaprnet.com root@mail.icongrp.com cashrewards@hotmail.com From owner-proteins@net.bio.net Thu Feb 05 22:00:00 1998 Path: biosci!agate!newsfeed.kornet.nm.kr!nntp.kreonet.re.kr!xfer.kren.nm.kr!news.maxwell.syr.edu!newsfeed.nacamar.de!fu-berlin.de!160.45.212.137!not-for-mail From: Isabell Witt Newsgroups: bionet.molbio.proteins Subject: Proteinrecovery from Microcon columns Date: Fri, 06 Feb 1998 15:26:36 +0100 Organization: Freie Universitaet Berlin Lines: 10 Message-ID: <34DB1D9B.B2A4D15D@zedat.fu-berlin.de> NNTP-Posting-Host: 160.45.212.137 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Access: 16 19 X-Trace: fu-berlin.de 886775220 20558 (none) X-Mailer: Mozilla 4.01 [de] (WinNT; I) X-Priority: 3 (Normal) Dear netters, do you have experiences with proteinrecovery from membranes of microcon (amicon) columns. When we concentrated a partially purified proteinsolution by this method we couldn`t recover our DNA binding activity. We assume that either the complex isn`t stable enough to survive this procedure or that the complex or part of it sticks to the membrane. Is it advisable to use detergents, and if so which one in what concentration? Thank you very much From owner-proteins@net.bio.net Thu Feb 05 22:00:00 1998 Path: biosci!daresbury!is.bbsrc.ac.uk!server3.netnews.ja.net!server4.netnews.ja.net!news.cc.ic.ac.uk!not-for-mail From: Koen De Smet Newsgroups: bionet.molbio.proteins Subject: inhibiting enzyme with antiserum Date: Fri, 06 Feb 1998 13:15:46 +0000 Organization: Department of Medical Microbiology Lines: 29 Message-ID: <34DB0D02.58D9@nospam.ic.ac.uk> Reply-To: k.desmet@nospam.ic.ac.uk NNTP-Posting-Host: cb-22.sm.med.ic.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.02 (Macintosh; I; PPC) I have some genes, which I presume to code for certain enzymes based on sequence homologies. I cloned the genes in His-tagging vectors to produce recombinant proteins. The recombinant proteins weee insoluble and denaturing/renaturing did not restore enzymatic activity. I produced antisera against the recombinant proteins The antisera recognise proteins of the expected size in cell free extracts. I can measure the enzyme activities in cell-free extracts. So while I have enzyme activities and genes, I have not yet demonstrated that they are linked. Now, to prove that these genes really encode these enzymes, I was wondering if it is possible to inhibit the enzyme activity by adding antiserum to the cell free extract. Does anybody have any experience with this? If I add antiserum and still measure enzyme activity, does that disprove my assumption, or can enzymes function while bound to antibodies? Or should I remove the enzyme by adding antiserum and then precipitate it with protein A? All commnents are much appreciated. Koen -- Koen De Smet ============================================================== ==>> To reply by email, remove "nospam." from the address <<== Imperial College School of Medicine at St Mary's http://www.sm.ic.ac.uk/medmicro/home. ============================================================== From owner-proteins@net.bio.net Thu Feb 05 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!news-peer.gip.net!news.gsl.net!gip.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!newsfeed.nacamar.de!uni-erlangen.de!winx03!wpxx02.toxi.uni-wuerzburg.de!krasel From: Cornelius Krasel Newsgroups: bionet.molbio.proteins Subject: Re: sequence search question Date: Fri, 6 Feb 1998 13:20:17 +0100 Organization: University of Wuerzburg, Germany Lines: 13 Message-ID: <16veb6.k63.ln@wpxx02.toxi.uni-wuerzburg.de> References: NNTP-Posting-Host: wpxx02.toxi.uni-wuerzburg.de X-Newsreader: TIN [UNIX 1.3 unoff BETA 970930; i486 Linux 2.0.32] Sean Davidson wrote: > I am trying to figure out a way to search the structural databases (i.e. > crystal structures) for proteins that have only one Tryptophan in the > sequence. If you have GCG, use FINDPATTERNS. --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 Thu Feb 05 22:00:00 1998 Path: biosci!agate!newsfeed.kornet.nm.kr!nntp.kreonet.re.kr!logbridge.uoregon.edu!scanner.worldgate.com!rover.ucs.ualberta.ca!gallin2.biology.ualberta.ca!wgallin From: wgallin@gpu.srv.ualberta.ca (Warren Gallin) Newsgroups: bionet.molbio.proteins Subject: Re: inhibiting enzyme with antiserum Date: Fri, 6 Feb 98 16:20:54 GMT Organization: University of Alberta Lines: 40 Message-ID: References: <34DB0D02.58D9@nospam.ic.ac.uk> NNTP-Posting-Host: gallin2.biology.ualberta.ca X-Trace: pulp.ucs.ualberta.ca 886781567 7942 (None) 129.128.185.130 X-Complaints-To: usenet@pulp.ucs.ualberta.ca X-Newsreader: VersaTerm Link v1.1.4 In Article <34DB0D02.58D9@nospam.ic.ac.uk>, Koen De Smet wrote: >I have some genes, which I presume to code for certain enzymes based on sequence >homologies. >I cloned the genes in His-tagging vectors to produce recombinant proteins. >The recombinant proteins weee insoluble and denaturing/renaturing did not restore >enzymatic activity. >I produced antisera against the recombinant proteins >The antisera recognise proteins of the expected size in cell free extracts. >I can measure the enzyme activities in cell-free extracts. > >So while I have enzyme activities and genes, I have not yet demonstrated that they >are linked. Now, to prove that these genes really encode these enzymes, I was >wondering if it is possible to inhibit the enzyme activity by adding antiserum to the >cell free extract. Does anybody have any experience with this? If I add antiserum and >still measure enzyme activity, does that disprove my assumption, or can enzymes >function while bound to antibodies? Or should I remove the enzyme by adding antiserum >and then precipitate it with protein A? Antibodies do not necessarily inhibit an enzyme, although if you have polyclonal antibodies it is a reasonable thing to try. I think that the best way to show what you want to show is to couple some purified Ig to a matrix like Sepharose and then do a simple absorption, appropriately controlled. Ideally you should be able to release the bound material and show that it has enzyme activity, or you might even have enzyme activity on the beads without releasing the enzyme. Warren Gallin Department of Biological Sciences University of Alberta Edmonton, Alberta T6G 2E9 Canada wgallin@gpu.srv.ualberta.ca From owner-proteins@net.bio.net Thu Feb 05 22:00:00 1998 Path: biosci!rutgers!nntp.upenn.edu!dsinc!pitt.edu!newsfeed.pitt.edu!pxpst2 From: pxpst2@vms.cis.pitt.edu (Peter Pediaditakis) Newsgroups: bionet.molbio.proteins Subject: Re: Proteinrecovery from Microcon columns Date: Fri, 06 Feb 1998 10:06:46 -0500 Organization: University of Pittsburgh Lines: 45 Message-ID: References: <34DB1D9B.B2A4D15D@zedat.fu-berlin.de> NNTP-Posting-Host: pelli.pathology.pitt.edu X-Newsreader: MT-NewsWatcher 2.3.5 In article <34DB1D9B.B2A4D15D@zedat.fu-berlin.de>, Isabell Witt wrote: > When we concentrated a partially purified > proteinsolution by this method we couldn`t recover our DNA binding > activity. We assume that either the complex isn`t stable enough to > survive this procedure or that the complex or part of it sticks to the > membrane. Is it advisable to use detergents, and if so which one in what > concentration? I have used the amicon dialysis apparatus/column for the purification of many proteins and can say that the technique is quite gental and rarely destroys proteins native structure. You must keep in mind that you will lose salts and trace elements(ie Mg,Ca,etc) which are reguired for binding activity. With that said, I would like to add that proteins can stick to the membrane. One protein(Hepatocyte Growth Factor)that I have worked with was notorius for sticking to any untreated glass,membranes, silca, and just about any surface. It created big problems. Detergents are a bad idea because you will almost certainly lose binding activity. The best way to prevent sticking is to pretreat your membranes with BSA(1mg/ml),overnight. This should block all your non specific binding sites on the membrane and on the plastic housing. Peter Pediaditakis -- "Don't you eat that yellow snow WAtch out where the Huskies go" --------------------------------------------------------------------- Let us not forget those marketing boys from SPAM CENTRAL bestrealtor@marketingmaster.com info@herbchew.com clickthru@timefreedom.com invite@onlinenow.net zippydj@nevwest.com Offer@shire.com empower@empowerlabs.com dynamarket@vaprnet.com root@mail.icongrp.com cashrewards@hotmail.com From owner-proteins@net.bio.net Thu Feb 05 22:00:00 1998 Path: biosci!bloom-beacon.mit.edu!news.kodak.com!news-pen-16.sprintlink.net!newsfeed.nysernet.net!news.nysernet.net!207.41.200.14!news-pen-14.sprintlink.net!206.229.87.26!news-east.sprintlink.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!cpk-news-hub1.bbnplanet.com!cam-news-feed1.bbnplanet.com!news.bbnplanet.com!nntp.neu.edu!lynx01.dac.neu.edu!rstrong From: rstrong@lynx01.dac.neu.edu (Richard Strong) Newsgroups: bionet.molbio.proteins Subject: Newbie needs help w/on-line protein sequence searches Date: 6 Feb 1998 22:47:47 GMT Organization: Northeastern University, Boston, MA. 02115, USA Lines: 31 Message-ID: <6bg3uj$4aq$1@isn.dac.neu.edu> NNTP-Posting-Host: lynx01.dac.neu.edu X-Newsreader: TIN [version 1.2 PL2] Greetings and I apologize if this isn't the proper newsgroup to post my inquiry on. I'm a Ph.D candidate in chemistry who is trying learn my way around the protein data bases located on the internet. Up to this point I've experienced a bit of difficulty getting sequence information on common proteins and would appreciate any help/advice that more experienced searchers could provide. For example, I've been trying to locate the primary sequence for bovine serum albumin; however, the prominent data bases that I've searched (SwissProt, Owl, etc.) only provided the sequence for bovine serum albumin precursor as it's closest match. Even though I pulled some BSA accession numbers from my literature references, I still could not find BSA in the data bases. Now granted that finding the primary structure for BSA wasn't that difficult when I put in a little effort at the library, but I would've expected that the structure for BSA would've been readily available on one of the on-line data bases. Is it that the primary structures for such well characterized proteins aren't included in these data bases because they are not overly difficult to find, or am I not looking in the right places on the internet? Any help would be appreciated. Thanks, Rick From owner-proteins@net.bio.net Thu Feb 05 22:00:00 1998 Path: biosci!GENOME.BIOTECH.WASHINGTON.EDU!eugene From: eugene@GENOME.BIOTECH.WASHINGTON.EDU (Eugene Kolker) Newsgroups: bionet.molbio.proteins Subject: Recomb98: student financial support Date: 6 Feb 1998 16:37:39 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 34 Sender: daemon@net.bio.net Distribution: world Message-ID: <199802070036.QAA15874@genome.biotech.washington.edu> NNTP-Posting-Host: net.bio.net Thanks to the generosity of our governmental and corporate sponsors, limited financial support for travel expenses will be available for students attending RECOMB 98. Support is offered on a competitive basis. In order to apply for financial support, you must: 1) be a graduate student or undergraduate student attending RECOMB 98, 2) register for RECOMB 98 including the submission of registration fees by Feb. 20, 1998, 3) submit a one page essay describing why you should receive financial support. The essay (up to *500* words) should include the following information: i) your name, ii) your email address, iii) the school you are attending, iv) the professor who is overseeing your research, v) a short description of your research, vi) the paper title if you have a paper at RECOMB 98, v) a short description of your research, vi) the paper title if you have a paper at RECOMB 98, vii) the poster title if you have a poster at RECOMB 98, viii) any other information that you think would be relevant. The essay should be sent via email to support@ecology.biomath.mssm.edu by Feb. 20, 1998. Those students selected for financial support will be notified by email on or before March 9, 1998. Early registration ends February 20, 1998! Hotel conference rate ends February 24, 1998! From owner-proteins@net.bio.net Thu Feb 05 22:00:00 1998 Path: biosci!agate!ihnp4.ucsd.edu!sdd.hp.com!vixen.cso.uiuc.edu!logbridge.uoregon.edu!howland.erols.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!newsfeed.internetmci.com!164.67.42.145!awabi.library.ucla.edu!164.67.80.81!news.ucla.edu!not-for-mail From: me@somehost.somedomain (Martin) Newsgroups: bionet.molbio.proteins Subject: Re: inhibiting enzyme with antiserum Date: 6 Feb 1998 23:11:23 GMT Organization: My Organization Lines: 58 Message-ID: <6bg5ar$mbc$1@uni.library.ucla.edu> References: <34DB0D02.58D9@nospam.ic.ac.uk> Reply-To: kmchow1@hotmail.com NNTP-Posting-Host: edgerton13.physci.ucla.edu Mime-Version: 1.0 Content-Type: Text/Plain; charset=US-ASCII X-Newsreader: WinVN 0.99.9 (Released Version) (16bit) You mentioned that they (recombinant proteins and those cell extracts) have simliar sizes, which indicates that you have run Western for them. And, I believe it is one of a good indication. As far as enzyme inhibition exp., I suggest that the antisera be first purified with DEAE (or a Protein A column, if your lab. can afford). Therfore, should any inhibition occurs, it must be due to the IgG fractions, not other unknown materials from the blood. Use your anitsera to purify more protein, i.e. make an affinity column. Then do a peptide map from them. Martin (norton@ucla.edu) In article <34DB0D02.58D9@nospam.ic.ac.uk>, k.desmet@nospam.ic.ac.uk says... > >I have some genes, which I presume to code for certain enzymes based on sequence >homologies. >I cloned the genes in His-tagging vectors to produce recombinant proteins. >The recombinant proteins weee insoluble and denaturing/renaturing did not restore >enzymatic activity. >I produced antisera against the recombinant proteins >The antisera recognise proteins of the expected size in cell free extracts. >I can measure the enzyme activities in cell-free extracts. > >So while I have enzyme activities and genes, I have not yet demonstrated that they >are linked. Now, to prove that these genes really encode these enzymes, I was >wondering if it is possible to inhibit the enzyme activity by adding antiserum to the >cell free extract. Does anybody have any experience with this? If I add antiserum and >still measure enzyme activity, does that disprove my assumption, or can enzymes >function while bound to antibodies? Or should I remove the enzyme by adding antiserum >and then precipitate it with protein A? > >All commnents are much appreciated. > >Koen > > >-- >Koen De Smet >============================================================== >==>> To reply by email, remove "nospam." from the address <<== > Imperial College School of Medicine at St Mary's > http://www.sm.ic.ac.uk/medmicro/home. >============================================================== From owner-proteins@net.bio.net Thu Feb 05 22:00:00 1998 Path: biosci!daresbury!uninett.no!Cabal.CESspool!bofh.vszbr.cz!news.maxwell.syr.edu!europa.clark.net!4.1.16.34!cpk-news-hub1.bbnplanet.com!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!news.bbnplanet.com!nih.gov!not-for-mail From: jhmiller@helix.nih.gov (Jay Miller) Newsgroups: bionet.molbio.proteins Subject: Re: sensitive protein assay Date: 6 Feb 1998 16:40:56 GMT Organization: National Institutes of Health Lines: 19 Distribution: world Message-ID: <6bfeeo$63j$1@light.nih.gov> References: <34dad398.4953740@news.univie.ac.at> Reply-To: jhmiller@helix.nih.gov NNTP-Posting-Host: ams06057.niams.nih.gov X-Newsreader: IBM NewsReader/2 2.0 While it's probably not as sensitive as fluorescence, you might look into Quantagold offered by Diversified Biotech (617-965-8557). It's fairly sensitive, altho i found that it didn't react with all proteins. In <34dad398.4953740@news.univie.ac.at>, a8803349.nospam@unet.univie.ac.at (Martin Offterdinger) writes: >Hi ! >Is there anyone who tried the nano orange protein assay from molecular >probes? I am currently using Bradford, but it is not sensitive enough >as I have only got 5-8ul of a very dilute sample and would like to get >an exact protein concentration. Are there any other protein assays >which are more sensitive than Bradford? >martin >Martin Offterdinger >Internal Med.I,Dept. Oncology >University of Vienna >Austria >E-Mail:a8803349.nospam@unet.univie.ac.at >(remove nospam before mailing) From owner-proteins@net.bio.net Fri Feb 06 22:00:00 1998 From: "Chris Bussineau" References: <6b587t$9db$1@gwsun.medinf.mu-luebeck.de> <6bavsl$pau$2@izvestia.its.unimelb.edu.au> Subject: Re: HPLC-Manager-Software Date: Sat, 7 Feb 1998 08:14:46 -0800 Lines: 29 X-Newsreader: Microsoft Outlook Express 4.71.1712.3 X-MimeOLE: Produced By Microsoft MimeOLE V4.71.1712.3 Message-ID: <#DGw8N#M9GA.359@upnetnews02.moswest.msn.net> Newsgroups: bionet.molbio.proteins Path: biosci!agate!newsfeed.kornet.nm.kr!nntp.kreonet.re.kr!news.maxwell.syr.edu!newsfeed.internetmci.com!207.68.152.12!upnetnews01!upnetnews02 If they "had" it they would have to support it, and vice versa. Petter wrote in message ... >In article <6bavsl$pau$2@izvestia.its.unimelb.edu.au>, >murphy_r@licre.ludwig.edu.au (Roger Murphy) wrote: > >>In article <6b587t$9db$1@gwsun.medinf.mu-luebeck.de>, "Lars Komorowski" > wrote: >>>Who can help me ? I lost data from HPLCManager 3.0 for DOS from Pharmacia >>>LKB. And I don't have the disks anymore (they were from 1991). Who can send >>>me the installation files or maybe newer ones if they work ? >>>Lars >>> >>> >>You should contact Pharmacia, I'm sure they'd have a copy somewhere! >> >> >> >>Roger Murphy, Ph.D. >>Biological Production Facility >>Ludwig Institute for Cancer Research >>Austin & Repatriation Medical Centre >>Studley Road, >>Heidelberg, Vic. 3084 >>Australia. From owner-proteins@net.bio.net Fri Feb 06 22:00:00 1998 Path: biosci!agate!ihnp4.ucsd.edu!sdd.hp.com!usc!howland.erols.net!news1.chicago.iagnet.net!qual.net!iagnet.net!195.74.75.15!fci-se!fci!newsfeed.sunet.se!news99.sunet.se!news01.sunet.se!130.239.8.18.MISMATCH!umdac!umu.se!not-for-mail From: Mariusz.Kowalczyk@genfys.slu.se Newsgroups: bionet.molbio.proteins Subject: Re: sensitive protein assay Date: 7 Feb 1998 21:03:39 GMT Organization: UMDAC Lines: 18 Message-ID: <6bii7b$ll2$1@studium.student.umu.se> References: <34dad398.4953740@news.univie.ac.at> NNTP-Posting-Host: b195.genfys.slu.se Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Newsreader: knews 1.0b.0 Hi, I have no idea if it will help :-) but you can check so-called "BCA method". Details are in following refs.: 1) Smith & co., Analalitical Biochem. 150/1985: 76-85, 2) Redinbaugh & Turley, Analitical Biochem 153/1986: 267-271 3) Hill & Straka, Analitical Biochem 170/1988: 203-208 All I can say is: it worked for me :-). Method is quite sensitive, (detects 1-10 microgram of protein per sample), the only problem might be with sulfhydryl reagents (and sugars probably)... Last reference in the list describes way around that problem, however I didn't test if that works. Regards, M.K. -- Greetz from Mariusz Kowalczyk, Ph.D. student| SLU, Inst. för skoglig gentet. och växtfys | .SiGnAtUrE ViRuS :-) From owner-proteins@net.bio.net Fri Feb 06 22:00:00 1998 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!128.223.220.30!logbridge.uoregon.edu!arclight.uoregon.edu!news.cc.ukans.edu!not-for-mail From: Mousheng Xu Newsgroups: bionet.molbio.proteins Subject: !!! HELP-SURVEY-BONUS: BSC 4.0 !!! Date: Sat, 07 Feb 1998 17:03:22 -0600 Organization: University of Kansas Computing Services Lines: 9 Message-ID: <34DCE83A.4DAA@eecs.ukans.edu> NNTP-Posting-Host: blizzard.rsl.ukans.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01Gold (X11; I; OSF1 V4.0 alpha) CC: mxu@eecs.ukans.edu Dear Scientists: Please see my last post titled as "UPDATE: Bioinformation Search Center 4.0" for introduction about 4.0. The one who finishes the survey of BSC will get the following bonus: 1. Core codes of the whole BSC system. 2. My thanks to you. -- Mousheng Xu From owner-proteins@net.bio.net Fri Feb 06 22:00:00 1998 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!128.223.220.30!logbridge.uoregon.edu!arclight.uoregon.edu!news.cc.ukans.edu!not-for-mail From: Mousheng Xu Newsgroups: bionet.molbio.proteins Subject: UPDATE: Bioinformation Search Center 4.0 Date: Sat, 07 Feb 1998 16:54:24 -0600 Organization: University of Kansas Computing Services Lines: 27 Message-ID: <34DCE620.ABD@eecs.ukans.edu> NNTP-Posting-Host: blizzard.rsl.ukans.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01Gold (X11; I; OSF1 V4.0 alpha) Dear Scientists: The Bioinformation Search Center (BSC) 4.0 (http://www.ittc.ukans.edu/~mxu/cgi-bin/thesis/bi.cgi) is now available to the public! Major new features of Version 4.0: 1. Short words are allowed for search. For example, in the previous versions of BSC, the "IX" of "human factor IX" was not used in search, but in BSC 4.0, it is used. 2. Keywords are highlighted in the results. Major known problem: Common words are not used for search in BSC. For the above example, both "human" and "factor" are so common that they are not used for search. In this case, it is bad. But a lot of common words such as "the", "a", "an" are surely bad in search of biological information. This problem can easily be solved, technically. But it takes programmer's time and the university's computer disk space. Please give this baby a try! Any comments are highly appreciated! Please see below for the brief introduction to BSC. Thanks a lot. Sincerely, Mousheng Xu From owner-proteins@net.bio.net Fri Feb 06 22:00:00 1998 Path: biosci!agate!ihnp4.ucsd.edu!sdd.hp.com!vixen.cso.uiuc.edu!howland.erols.net!news-peer.sprintlink.net!news-backup-west.sprintlink.net!news-in-west.sprintlink.net!news.sprintlink.net!Sprint!205.242.56.12!news.up.net!news From: "Daniel Burhop" Newsgroups: bionet.molbio.proteins Subject: Creatine Date: 8 Feb 1998 05:01:18 GMT Organization: up.net Lines: 4 Message-ID: <01bd344f$54efbbe0$23f110d0@oemcomputer> NNTP-Posting-Host: ssm1-35.up.net X-Newsreader: Microsoft Internet News 4.70.1161 My Anatomy & Physiology class is doing research on the athletic enhacer creatine. If anybody has any information on the negative side effects of this, will you please tell me. Thanks From owner-proteins@net.bio.net Sat Feb 07 22:00:00 1998 Path: biosci!agate!howland.erols.net!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newspeer.monmouth.com!uunet!in2.uu.net!news.optus.net.au!bunyip.cc.uq.edu.au!not-for-mail From: "Trent Munro" Newsgroups: bionet.molbio.proteins Subject: Re: Proteinrecovery from Microcon columns Date: Mon, 09 Feb 1998 08:46:30 +1000 Organization: University of Queensland Lines: 35 Message-ID: <6blcpd$j2r$1@nargun.cc.uq.edu.au> References: <34DB1D9B.B2A4D15D@zedat.fu-berlin.de> NNTP-Posting-Host: 130.102.116.50 Mime-Version: 1.0 Content-Type: text/plain; charset="US-ASCII" Content-Transfer-Encoding: 7bit X-Newsreader: Microsoft Outlook Express for Macintosh - 4.0a (190) First of all I assume you are talking about the spin columns, if not ignore this message. I have used these things alot over the last two years and found that results differ widely depending on your protein, in more cases than not I get about a five % recovery (measured by HPLC). So to cut a long boring story short I now only ever use chronatography for protein concentration. In saying all of that, you can boost recovery by bringing the protein back into solution with a strong buffer eg 1M tris etc, SDS (1%) and b-mercapto, however all those thing are subsequently difficult to get rid of so the whole process becomes complex and time consuming. The best use for them is for concentrating dilute protein samples for SDS PAGE as the loading buffer is excellent for bringing nearly all proteins back into solution. To answere your original question yes detergents (!% SDS) and heating (65 degrees) will help recovery but will it stuff the protein. Hope something in there helps Cheers Trent Munro ---------- In article <34DB1D9B.B2A4D15D@zedat.fu-berlin.de>, Isabell Witt wrote: Dear netters, do you have experiences with proteinrecovery from membranes of microcon (amicon) columns. When we concentrated a partially purified proteinsolution by this method we couldn`t recover our DNA binding activity. We assume that either the complex isn`t stable enough to survive this procedure or that the complex or part of it sticks to the membrane. Is it advisable to use detergents, and if so which one in what concentration? Thank you very much From owner-proteins@net.bio.net Sat Feb 07 22:00:00 1998 Path: biosci!agate!howland.erols.net!Supernews73!supernews.com!Supernews69!not-for-mail From: dalmiabk@phibred.com (bipin k. dalmia) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: protein solubilizing out of SDS-PAGE gel Date: Sun, 08 Feb 1998 19:53:03 GMT Organization: All USENET -- http://www.Supernews.com Lines: 17 Message-ID: <34de0c50.257493920@207.126.101.81> NNTP-Posting-Host: 18783@204.233.143.2 X-Newsreader: Forte Free Agent 1.1/32.230 Xref: biosci bionet.molbio.methds-reagnts:64762 bionet.molbio.proteins:12268 hello: i am having trouble detecting a protein on SDS-PAGE gels. this is a very very soluble protein and i think that it may be solubilizing out of the gel during the staining steps. i am fixing the gel with 10%acetic acid/45% methanol and staining with CBB. is there any other way to ensure that the protein stays in the gel? bip Bipin K. Dalmia, Ph.D. Protein Core Facility Pioneer Hi-Bred International, Inc. Johnston, Iowa 50131 dalmiabk@phibred.com From owner-proteins@net.bio.net Sat Feb 07 22:00:00 1998 Path: biosci!daresbury!uninett.no!Cabal.CESspool!bofh.vszbr.cz!howland.erols.net!fastnet!ptdnetP!newsgate.ptd.net!nntp.flash.net!news.mira.net.au!news.mel.aone.net.au!newsfeed-in.aone.net.au!inferno.mpx.com.au!news.unimelb.edu.au!ludwignt-4 From: murphy_r@licre.ludwig.edu.au (Roger Murphy) Newsgroups: bionet.molbio.proteins Subject: Re: Iodo-beads iodination of peptides Date: Mon, 09 Feb 98 06:03:28 GMT Organization: Ludwig Institute for Cancer Research Lines: 34 Message-ID: <6bm290$pi7$1@izvestia.its.unimelb.edu.au> References: <34DA7A3A.B512DBF7@hunnu.edu.cn> NNTP-Posting-Host: ludwignt-4.austin.unimelb.edu.au X-Newsreader: News Xpress 2.0 Beta #0 In my experience, the Iodo-beads method will label histidine if you conduct the reaction in borate buffer at pH 9. I've no experience of chloramine T as this has always proved too destructive of the peptides I have been labelling. Hope this helps, Roger In article <34DA7A3A.B512DBF7@hunnu.edu.cn>, zhuqi wrote: >ear Dr.Peter: > I am a Grad-student in China.I need iodinate a toxin (33 amino >acids)which have no Tyr but His.I want label it with 125I. I used mothod > >of Chloramine T.But the product is so little.So I think the the reason > >is this peptide have no Tyr.Do you have some useful method to improve >the product of Iodination?Would you mind give me some advice? > Thank you in advance. > >Sincerely Yours : > Qi Zhu > 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 Sat Feb 07 22:00:00 1998 Path: biosci!agate!howland.erols.net!Cabal.CESspool!bofh.vszbr.cz!fu-berlin.de!masternews.telia.net!news-stkh.gip.net!news.gsl.net!gip.net!news.kolumbus.fi!news.uninet.ee!kadri.ut.ee!not-for-mail From: Peeter Toomik Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: protein solubilizing out of SDS-PAGE gel Date: Mon, 09 Feb 1998 09:20:26 +0200 Organization: Tartu University Lines: 32 Message-ID: <34DEAE3A.33928D8@ut.ee> References: <34de0c50.257493920@207.126.101.81> NNTP-Posting-Host: biochem3.bio.ut.ee Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: kadri.ut.ee 887008585 13728 (None) 193.40.8.203 X-Complaints-To: usenet@kadri.ut.ee X-Mailer: Mozilla 4.04 [en] (Win95; I) Xref: biosci bionet.molbio.methds-reagnts:64777 bionet.molbio.proteins:12273 First, try fixing with 10-12% trichloroacetic acid. If it does not help, add some 5% of formaldehyde to CBB solution. No preliminary fixing! You can find the recipe in "Practical Protein Chemistry", A.Dabre (ed.), Wiley, 1986, which refers to S.Irie et al., Anal. Biochem. 126, 350-354 (1982). Formaline concentration etc. may need some adjustment for particular protein. This method works O.K. in our lab. Peeter Toomik Tartu University Institute of Molecular and Cell Biology bipin k. dalmia wrote: > hello: > > i am having trouble detecting a protein on SDS-PAGE gels. this is a > very very soluble protein and i think that it may be solubilizing out > of the gel during the staining steps. i am fixing the gel with > 10%acetic acid/45% methanol and staining with CBB. is there any other > way to ensure that the protein stays in the gel? > > bip > > Bipin K. Dalmia, Ph.D. > Protein Core Facility > Pioneer Hi-Bred International, Inc. > Johnston, Iowa 50131 > > dalmiabk@phibred.com From owner-proteins@net.bio.net Sat Feb 07 22:00:00 1998 Path: biosci!bloom-beacon.mit.edu!news.kodak.com!news-pen-16.sprintlink.net!newsfeed.nysernet.net!news.nysernet.net!207.41.200.14!news-pen-14.sprintlink.net!206.229.87.26!news-east.sprintlink.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!portc02.blue.aol.com!pitt.edu!newsfeed.pitt.edu!pxpst2 From: pxpst2@vms.cis.pitt.edu (Peter Pediaditakis) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: protein solubilizing out of SDS-PAGE gel Date: Sun, 08 Feb 1998 18:17:46 -0500 Organization: University of Pittsburgh Lines: 35 Message-ID: References: <34de0c50.257493920@207.126.101.81> NNTP-Posting-Host: pelli.pathology.pitt.edu X-Newsreader: MT-NewsWatcher 2.3.5 Xref: biosci bionet.molbio.methds-reagnts:64764 bionet.molbio.proteins:12270 In article <34de0c50.257493920@207.126.101.81>, dalmiabk@phibred.com (bipin k. dalmia) wrote: > hello: > > i am having trouble detecting a protein on SDS-PAGE gels. this is a > very very soluble protein and i think that it may be solubilizing out > of the gel during the staining steps. i am fixing the gel with > 10%acetic acid/45% methanol and staining with CBB. is there any other > way to ensure that the protein stays in the gel? If your protein is abov about 10KD then stick the gel in a blotting apparatus and transfer it to a PVDF membrane. Millipore sells some that have smaller pores and are used for sequencing. Peter -- "Don't you eat that yellow snow WAtch out where the Huskies go" --------------------------------------------------------------------- Let us not forget those marketing boys from SPAM CENTRAL bestrealtor@marketingmaster.com info@herbchew.com clickthru@timefreedom.com invite@onlinenow.net zippydj@nevwest.com Offer@shire.com empower@empowerlabs.com dynamarket@vaprnet.com root@mail.icongrp.com cashrewards@hotmail.com From owner-proteins@net.bio.net Sun Feb 08 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!news-peer.gip.net!news.gsl.net!gip.net!newsfeed.internetmci.com!4.1.16.34!cpk-news-hub1.bbnplanet.com!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!news.bbnplanet.com!nih.gov!not-for-mail From: jhmiller@helix.nih.gov (Jay Miller) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: protein solubilizing out of SDS-PAGE gel Date: 9 Feb 1998 14:29:54 GMT Organization: National Institutes of Health Lines: 30 Distribution: world Message-ID: <6bn3t2$cji$1@light.nih.gov> References: <34de0c50.257493920@207.126.101.81> Reply-To: jhmiller@helix.nih.gov NNTP-Posting-Host: ams06057.niams.nih.gov X-Newsreader: IBM NewsReader/2 2.0 Xref: biosci bionet.molbio.methds-reagnts:64784 bionet.molbio.proteins:12279 I have a method for detecting peptides on gels using sulphosalicylic acid. The ref. is: BBA,701,395-404,'82, Hollecker & Creighton. Here's another one from, what used to called Bethesda Research Labs: 0.1% Coomassie Brilliant Blue, 25% isopropanol, 10% HOAc, 0.1% CuAc. It's the last that improves detection of peptides, or at least proteins such as insulin. And lastly, fix in 5% glutaraldehyde for about 2 hrs. and follow with CBB or maybe Ag. I'd try this one first. What you want is a method that will prevent the peptide from being washed out of the gel. The standard MeOH/HOAc won't prevent this leaching from the gel. The glutaraldehyde will prevent it. In <34de0c50.257493920@207.126.101.81>, dalmiabk@phibred.com (bipin k. dalmia) writes: >hello: > >i am having trouble detecting a protein on SDS-PAGE gels. this is a >very very soluble protein and i think that it may be solubilizing out >of the gel during the staining steps. i am fixing the gel with >10%acetic acid/45% methanol and staining with CBB. is there any other >way to ensure that the protein stays in the gel? > >bip > > >Bipin K. Dalmia, Ph.D. >Protein Core Facility >Pioneer Hi-Bred International, Inc. >Johnston, Iowa 50131 > >dalmiabk@phibred.com From owner-proteins@net.bio.net Sun Feb 08 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!news-peer.gip.net!news.gsl.net!gip.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!newsfeed.nacamar.de!news-kar1.dfn.de!news-stu1.dfn.de!news-mue1.dfn.de!news.gsf.de!news From: "Michael Rosemann" Newsgroups: bionet.molbio.proteins Subject: Unspecific signals produced by secondary Ab in Western Blot Date: 9 Feb 1998 11:28:35 GMT Organization: GSF Forschungszentrum fuer Umwelt und Gesundheit Lines: 34 Message-ID: <01bd354d$e697e4b0$df236b92@pc2090> NNTP-Posting-Host: pc2090.gsf.de X-Newsreader: Microsoft Internet News 4.70.1161 We have tried to get Western Blots for P53 and Bax working and encountered the following problem: The secondary antibody (Amersham's rabbit anti-mouse IgG, HRP conjugated) alone produced a lot of unspecific bands,especially a very prominent one at approx. 56kD. We already tried to get rid of it by mixing the secondary antibody with the protein extract or with BSA, but with little or no success. Even more surprising was the observation, that another secondary antibody (Santa Cruz' Donkey anti-goat IgG, HRP conjugated) produced an unspecific signal at the same position on the blot. We could justify that no endogenous peroxidases are responsible for this effect and that none of the reagents were contaminated. For the detection we use Amersham's ECL system. Somebody proposed, that the Bromphenolblue containing loading buffer (Laemmli's buffer with DTT) might be the reason, since other people using a recepie with Coomassie blue containing loading buffer (+ Thioglycolic Acid instead of DTT) told me that they could get rid of unspecific signals, however they could not tell me whether they came from the first or the secondary Ab. I would like to hear from anybody who can give advise or simply tell me of similar observations. Thanks in advance Michael (rosemann@gsf.de) From owner-proteins@net.bio.net Sun Feb 08 22:00:00 1998 Path: biosci!GENOME.BIOTECH.WASHINGTON.EDU!eugene From: eugene@GENOME.BIOTECH.WASHINGTON.EDU (Eugene Kolker) Newsgroups: bionet.molbio.proteins Subject: Recomb98: Deadlines & Preliminary Program Date: 9 Feb 1998 11:25:15 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 356 Sender: daemon@net.bio.net Distribution: world Message-ID: <199802091924.LAA08580@genome.biotech.washington.edu> NNTP-Posting-Host: net.bio.net Early registration ends February 20, 1998! Hotel conference rate ends February 24, 1998! For more details please visit our web site: http://www.mssm.edu/biomath/recomb98.html ---------------------------------------------------------------------------- RECOMB 98 SCHEDULE Saturday, March 21, 1998 7:00 pm - 10:00 pm Welcome Reception Renaissance Room Sunday, March 22, 1998 8:45 am Opening Remarks Embassy Ballroom Pavel Pevzner, RECOMB 1998 Program Committee Chair Gary Benson, RECOMB 1998 Conference Chair Sorin Istrail, RECOMB 1999 Program Committee Chair 9:00 am Distinguished New Technologies Lecture Pavel Pevzner, Chair David Cox The Human Genetic Variation: Oligonucleotide Chips and Human Disease 10:00 am Break Session 1, Chair: Martin Vingron 10:10 am Identifying Satellites in Nucleic Acid Sequences M. Sagot, G. Meyers 10:35 am Genome Analysis Using Clusters of Orthologous Groups (COGs) E. Koonin, R. Tatusov, M. Y. Galperin, M. N. Rozanov 11:00 am Break Session 2, Chair: Gene Myers 11:15 am Non-Adjoining Correlations Within Signals in DNA P. Agarwal and V. Bafna 11:40 am An Algorithm for Finding Tandem Repeats of Unspecified Pattern Size G. Benson 12:05 pm Lunch 1:30 pm Invited Lecture Session Sorin Istrail, Chair Charles Cantor Enhancements in Sequence Analysis With DNA Arrays 2:30 pm Break Session 3, Chair: Gary Benson 2:40 pm Motif Discovery in Biological Sequences Without Alignment or Enumeration I. Rigoutsos, A. Floratos 3:05 pm An Algorithm for Finding Novel Gapped Motifs in DNA Sequences E. Rocke, M. Tompa 3:30 pm A Polyhedral Approach to RNA Sequence Structure Alignment H. Lenhof, K. Reinert, M. Vingron 3:55 pm Break 4:10 pm Stanislav Ulam Computational Biology Address Michael Waterman, Chair Joshua Lederberg 8:00 pm-10:00 pm Business Meeting Renaissance Room Monday, March 23, 1998 Embassy Ballroom 9:00 am Distinguished Conference Lecture Richard Karp, Chair Ron Davis Whole Genome Analysis: Expression, Replication, Recombination, Allelic Variation, and Drug Discovery Function 10:00 am Break Session 4, Chair: David Searls 10:10 am Assessment of Ab Initio Protein Structure Prediction A. M. Lesk 10:35 am A Self-Consistent Field Optimization Approach to Build Energetically and Geometrically Correct Lattice Models of Proteins B. A. Reva, A. V. Finkelstein, J. Skolnick 11:00 am Break 11:15 am Modeling DNA Shuffling F. Sun 11:40 am Multiple Genome Rearrangements D. Sankoff, M. Blanchette 12:05 pm Lunch 1:30 pm Invited Lecture Session Eugene Koonin, Chair Ruben Abagyan Challenges of biomolecular structure prediction 2:30 pm Break Session 5, Chair: Martin Farach 2:40 pm From Four-taxon Trees to Phylogenies: The Case of Mammalian Evolution A. Ben-Dor, B. Chor, D. Graur, R. Ophir, D. Pelleg 3:05 pm On Reconstructing Species Trees From Gene Trees In Term of Duplications B. Ma, M. Li, L. Zhang 3:30 pm The Ordinal Quartet Method P. E. Kearney 3:55 pm Break 4:10 pm Invited Lecture Session Bonnie Berger, Chair Michael Levitt Bridging the Gap Between Sequence and Structure 6:00 pm-10:00 pm Poster Session Renaissance Room Tuesday, March 24, 1998 Embassy Ballroom 9:00 am Invited Lecture Session Ron Shamir, Chair David Schwartz New Approaches to Genomic Analaysis Using Single Molecules 10:00 am Break Session 6, Chair: Minoru Kanehisa 10:10 am Algorithms for Optical Mapping R. M. Karp, R. Shamir 10:35 am Estimation for Restriction Sites Observed by Optical Mapping Using Reversible-jump Markov Chain Monte Carlo J. K. Lee, V. Dancik, M. S. Waterman 11:00 am Break 11:15 am Partitioning K Clones: Hardness Results and a Practical Solution to the K-Popultaion Problem L. Parida, B. Mishra 11:40 am Constructing Maps Using the Span and Inclusion Relations D. Fasulo, T. Jiang, R. M. Karp, N. Sharma 12:05 pm Lunch 1:30 pm Invited Lecture Session Webb Miller, Chair John Yates Mass spectrometry and the proteome 2:30 pm Break Session 7, Chair: Thomas Lengauer 2:40 pm How Fast a Protein Chain Can Fold to Its Most Stable Structure? A. V. Finkelstein, A. Ya. Badretdinov 3:00 pm Algorithmic Determination of Core Positions in the VL and VH Domains of Immunoglobulin Molecules I. Gelfand, A. Kister, C. Kulikowski, O. Stoyanov 3:30 pm Alignments Without Low-Scoring Regions Z. Zhang, P. Berman, W. Miller 3:55 pm Dynamic Programmimg Alignment Accuracy I. Holmes and R. Durbin 4:20 pm Break 4:30 pm Invited Lecture Session David Haussler, Chair Klaus Gubernator Evolutionary drug discovery Wednesday, March 25, 1998 Embassy Ballroom Session 8, Chair: Dan Gusfield 8:30 am Protein Folding in the Hydrophobic-Hydrophilic (HP) Model is NP-Complete B. Berger, T. Leighton 8:55 am On the Complexity of Protein Folding P. Crescenzi, D. Goldman, C. Papadimitriou, A. Piccolboni, M. Yannakakis 9:20 am A New Method for Modeling and Solving the Protein Fold Recognition Problem Y. Xu, D. Xu, E. C. Uberbacher 9:45 am Optimal Detection of Sequence Similarity by Local Alignment T. Hwa, M. Lassig 10:10 am Break Session 9, Chair: Terry Speed 10:20 am A Formally Exact Method to Numerically Analyze Local Denaturation in Superhelical DNA R. M. Fye, C. J. Benham 10:45 am The Theoretical Limits of DNA Sequence Discrimination of Polyamides W. L. Walker, D. S. Goodsell, E. M. Landaw 11:10 am Maxwell Demon and Topology Simplification by Type II Topoisomerases A. Vologodskii 11:35 am A Unified Approach to Word Statistics M. Regnier 12:00 pm Lunch Session 10, Chair: Phil Green 1:00 pm Regression Analysis of Multiple Protein Structures T. D. Wu, S. C. Schmidler, T. Hastie, D. L. Brutlag 1:25 pm Better Methods for Solving Parsimony and Compatiblity M. Bonet, M. Steel, T. Warnow, S. Yooseph 1:50 pm Beyond Mutation Matrices: Physical-Chemistry Based Evolutionary Models J. M. Koshi, D. P. Mindell, R. A. Goldstein 2:15 pm A Structure Based Similarity Measure for Nucleic Acid Sequence Comparison R. Liu, T. W. Blackwell and D. J. States 2:40 pm Break Session 11, Chair: David Benson 2:55 pm The Hierarchical Organization of Molecular Structure Computations C. C. Chen, J. P. Singh, R. B. Altman 3:20 pm Estimaiton of Allele Frequencies From Color-Multiplexed Electropherograms D. G. Politte, D. R. Maffitt, D. J. States 3:45 pm Analysis of the Position Dependent Amino Acid Probabilities and its Applicationts to the Search for Remote Homologues S. R. Sunyaev, I. V. Rodchenkov, F. Eisenhaber, E. N. Kuznetsov 4:10 pm Family-based Homology Detection via Pairwise Sequence Comparison W. N. Grundy End of RECOMB 98 From owner-proteins@net.bio.net Sun Feb 08 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!ais.net!howland.erols.net!fu-berlin.de!194.94.5.175!not-for-mail From: Axel Schumacher Newsgroups: bionet.molbio.proteins Subject: Re: Unspecific signals produced by secondary Ab in Western Blot Date: Mon, 09 Feb 1998 19:39:31 -0800 Organization: =?iso-8859-1?Q?Robert=2DR=F6ssle=2DKlinik?=, Humboldt-University Berlin Lines: 40 Message-ID: <34DFCBF3.4685@rrk-berlin.de> References: <01bd354d$e697e4b0$df236b92@pc2090> Reply-To: aschumacher@rrk-berlin.de NNTP-Posting-Host: 194.94.5.175 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Access: 16 1395 X-Trace: fu-berlin.de 887049587 20916 (none) X-Mailer: Mozilla 3.01 (Win16; I) Michael Rosemann wrote: > > We have tried to get Western Blots for P53 and Bax working and encountered > the following problem: The secondary antibody (Amersham's rabbit anti-mouse > IgG, > HRP conjugated) alone produced a lot of unspecific bands,especially a very > prominent one at approx. 56kD. ... > For the detection we use Amersham's ECL system. > ... > I would like to hear from anybody who can give advise or simply tell me of > similar observations. > > Thanks in advance Michael (rosemann@gsf.de) > Michael, We do have similar problems that whenever we use polyclonal sera as either primary or HRPconjugated secondary antibodies we do se background plus nonspecific bands. Therefore we use a triple sandwich method: primary ab (monoclonal if possible) goat anti mouse bioitinylated (Vector Labs) streptavidin-HRP (Amersham) Although the secondary goat serum is polyclonal we have little to no background and few if any nonspecific bands. One reason might be the additional signal amplification which leads to a better signal to noise ratio. We use Laemmli buffer with bromophenol blue with no problems. Hope this might help. Axel Schumacher aschumacher@rrk-berlin.de P.S. Should you find an anti-bax antibody that works, let me know. Ours so far detect either nonsense or nothing at all. From owner-proteins@net.bio.net Sun Feb 08 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!europa.clark.net!206.246.194.8!newsxfer.visi.net!rill.news.pipex.net!pipex!server1.netnews.ja.net!dundee.ac.uk!bute!jm6 From: Asteras Amaliadas Newsgroups: bionet.molbio.proteins,bionet.molbio.methds-reagnts Subject: Test (please ignore)... Date: Mon, 9 Feb 1998 15:18:38 +0000 Organization: The University, Dundee, DD1 4HN, Scotland, UK. Lines: 2 Message-ID: Reply-To: Asteras Amaliadas NNTP-Posting-Host: bute.st-and.ac.uk Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII X-Sender: jm6@st-andrews.ac.uk Xref: biosci bionet.molbio.proteins:12280 bionet.molbio.methds-reagnts:64787 From owner-proteins@net.bio.net Sun Feb 08 22:00:00 1998 Path: biosci!agate!howland.erols.net!fastnet!ptdnetP!newsgate.ptd.net!fci-se!fci!newsfeed.sunet.se!news99.sunet.se!news01.sunet.se!130.239.8.18.MISMATCH!umdac!umu.se!not-for-mail From: Mariusz.Kowalczyk@genfys.slu.se (kovi) Newsgroups: bionet.molbio.proteins Subject: Re: protein solubilizing out of SDS-PAGE gel Date: 9 Feb 1998 10:38:05 GMT Organization: UMDAC Lines: 20 Message-ID: <6bmmad$5rc$1@studium.student.umu.se> References: <34de0c50.257493920@207.126.101.81> NNTP-Posting-Host: b195.genfys.slu.se Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Newsreader: knews 1.0b.0 Hi, bipin k. dalmia writes: > i am having trouble detecting a protein on SDS-PAGE gels. this is a > very very soluble protein and i think that it may be solubilizing out > of the gel during the staining steps. i am fixing the gel with > 10%acetic acid/45% methanol and staining with CBB. is there any other > way to ensure that the protein stays in the gel? I think you can try 10% glutaraldehyde fixation after AcH/MetOH step. I am doing that overnight, but probably shorter time will be OK as well. Then you'll have to wash gel with water (several changes)... Probably you should try with silver stain. CBB stains different proteins in a different way, so it might be that your protein simply does not stain well with CBB. Regards, M.K. -- Greetz from Mariusz Kowalczyk, Ph.D. student| SLU, Inst. för skoglig gentet. och växtfys | From owner-proteins@net.bio.net Sun Feb 08 22:00:00 1998 Path: biosci!agate!newsfeed.kornet.nm.kr!newsfeed.dacom.co.kr!newsfeed.direct.ca!portc01.blue.aol.com!audrey02.news.aol.com!not-for-mail From: researchd@aol.com (ResearchD) Newsgroups: bionet.molbio.proteins Subject: Re: Unspecific signals produced by secondary Ab in Western Blot Date: 9 Feb 1998 14:20:22 GMT Lines: 12 Message-ID: <19980209142001.JAA15804@ladder02.news.aol.com> NNTP-Posting-Host: ladder02.news.aol.com X-Admin: news@aol.com References: <01bd354d$e697e4b0$df236b92@pc2090> Organization: AOL http://www.aol.com X-Newsreader: AOL Offline Reader -have you tried using just the secondary antibody (no primary)? If bands still apear, the antibody may be reacting with other serum proteins. Suggest trying an affinity absorbed secondary antibody (ideally absorbed against BSA and human serum proteins) see some of our secondary antibodies at: http://www.researchd.com/rdiabs/2ndabs.htm Sincerely, Research Diagnostics inc email:ResearchD@aol.com web: http://www.researchd.com From owner-proteins@net.bio.net Sun Feb 08 22:00:00 1998 Path: biosci!agate!howland.erols.net!news.idt.net!nntp2.cerf.net!moon.pepperdine.edu!usenet From: zhuqi Newsgroups: bionet.molbio.proteins Subject: Re: Iodo-beads iodination of peptides Date: Mon, 09 Feb 1998 16:18:44 +0800 Organization: Pepperdine University Lines: 9 Message-ID: <34DEBBE4.4A1D885C@hunnu.edu.cn> References: <34DA7A3A.B512DBF7@hunnu.edu.cn> <6bm290$pi7$1@izvestia.its.unimelb.edu.au> NNTP-Posting-Host: 202.197.120.114 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.03 [en] (Win95; I) To: Roger Murphy Dear Roger: Thank you for your advice. How can I make the Borate Buffer(PH9.0)? Regards Qi,Zhu From owner-proteins@net.bio.net Sun Feb 08 22:00:00 1998 Path: biosci!agate!howland.erols.net!news.idt.net!nntp2.cerf.net!moon.pepperdine.edu!usenet From: zhuqi Newsgroups: bionet.molbio.proteins Subject: Re: Iodo-beads iodination of peptides Date: Mon, 09 Feb 1998 16:20:57 +0800 Organization: Pepperdine University Lines: 10 Message-ID: <34DEBC69.EFCFB152@hunnu.edu.cn> References: <34DA7A3A.B512DBF7@hunnu.edu.cn> <6bm290$pi7$1@izvestia.its.unimelb.edu.au> NNTP-Posting-Host: 202.197.120.114 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.03 [en] (Win95; I) To: Roger Murphy Dear Roger: Thank you for your advice. How can I make the Borate Buffer(PH9.0)? Would you please tell me the paper or your work which have details on this matter? Regards Qi,Zhu From owner-proteins@net.bio.net Mon Feb 09 22:00:00 1998 Path: biosci!bloom-beacon.mit.edu!newsxfer3.itd.umich.edu!news-peer.gip.net!news.gsl.net!gip.net!peerfeed.ncal.verio.net!news.walltech.com!news.his.com!news3.his.com!news.cs.jhu.edu!news.jhu.edu!welchlink!stebby From: stebby@welchlink.welch.jhu.edu (Stephen C. Dahl) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: protein solubilizing out of SDS-PAGE gel Followup-To: bionet.molbio.methds-reagnts,bionet.molbio.proteins Date: 9 Feb 1998 07:40:28 GMT Organization: HCF - Johns Hopkins University, Baltimore, Maryland, USA Lines: 18 Message-ID: <6bmbtc$i98@news.jhu.edu> References: <34de0c50.257493920@207.126.101.81> NNTP-Posting-Host: 128.220.59.78 X-Newsreader: TIN [version 1.2 PL2] Xref: biosci bionet.molbio.methds-reagnts:64823 bionet.molbio.proteins:12285 bipin k. dalmia (dalmiabk@phibred.com) wrote: : hello: : i am having trouble detecting a protein on SDS-PAGE gels. this is a : very very soluble protein and i think that it may be solubilizing out : of the gel during the staining steps. i am fixing the gel with : 10%acetic acid/45% methanol and staining with CBB. is there any other : way to ensure that the protein stays in the gel? Can't say I've ever come across a protein *that* soluble, but if you think this is the case, run the gel and immediately blot it onto .22 nitrocellulose and stain with amido black, india ink, ponceau or whatever is your fancy. Unless its a really small molecule you should see it there. Good luck, Steve Dahl From owner-proteins@net.bio.net Mon Feb 09 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!news.maxwell.syr.edu!nntp.news.xara.net!xara.net!server6.netnews.ja.net!dundee.ac.uk!bute!jm6 From: Asteras Amaliadas Newsgroups: bionet.molbio.proteins,bionet.molbio.methds-reagnts Subject: Solubilisation of GST-fusion proteins? Date: Tue, 10 Feb 1998 15:45:27 +0000 Organization: The University, Dundee, DD1 4HN, Scotland, UK. Lines: 13 Message-ID: References: Reply-To: Asteras Amaliadas NNTP-Posting-Host: bute.st-and.ac.uk Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII X-Sender: jm6@st-andrews.ac.uk In-Reply-To: Xref: biosci bionet.molbio.proteins:12283 bionet.molbio.methds-reagnts:64816 Hi there, I am pretty sure that I do not ask you anything new in this newsgroup: I try to purify a GST-fusion protein in E. coli, but it seems it is completely insoluble. Any suggestions? Thanks in advance, Yannis From owner-proteins@net.bio.net Mon Feb 09 22:00:00 1998 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!204.59.152.222!news-peer.gip.net!news.gsl.net!gip.net!news.new-york.net!news.columbia.edu!news-not-for-mail From: Baohe Shen Newsgroups: bionet.molbio.proteins Subject: Re: Phosphoinositol 3,4,5-P3, To the Webmaster : What is wrong with the date? Date: Fri, 06 Feb 1998 10:25:54 -0500 Organization: Columbia University Lines: 4 Message-ID: <34DB2B82.1151@columbia.edu> References: <34CE2D17.6F6B@columbia.edu> NNTP-Posting-Host: phosphoimager2.bio.columbia.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.02Gold (WinNT; I) Hi, I posted the original message yesterday afternoon (Febrary 5, 1998), but why does it turn out 1/27/98 on this newsgroup? From owner-proteins@net.bio.net Mon Feb 09 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!news.maxwell.syr.edu!cam-news-hub1.bbnplanet.com!news.bbnplanet.com!Gamma.RU!srcc!Radio-MSU.net!news-ham1.dfn.de!news.mu-luebeck.de!not-for-mail From: "Lars Komorowski" Newsgroups: bionet.molbio.proteins Subject: E. coli signal sequences Date: Tue, 10 Feb 1998 17:40:45 +0100 Organization: Med. Universitaet zu Luebeck Lines: 5 Message-ID: <6bq067$btl$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 Who can help me with E. coli signal sequences ? Especially those which are responsible for delivering the protein to the periplasm. Lars Komorowski From owner-proteins@net.bio.net Mon Feb 09 22:00:00 1998 Path: biosci!email.msn.com!intein-intron From: intein-intron@email.msn.com ("Isidro Savillo") Newsgroups: bionet.molbio.proteins Subject: Prions Date: 10 Feb 1998 22:16:37 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 22 Sender: daemon@net.bio.net Distribution: world Message-ID: <0fae62714060b28UPIMSSMTPUSR04@email.msn.com> NNTP-Posting-Host: net.bio.net I have been following the biology of prions for sometime. Well we always believe about the central dogma. So we expect that the proteins at least if we root the source though we may be passing a series of pathways, indirect or direct comes from (a) gene(s). Considering the changes in protein conformation, I would suggest that there are 'pathogenic enzymes' which are responsible for the abnormal change. And prion's ability to transform normal to abnormal forms would suggest that prions are acting as enzymes themselves. If the source is human DNA then it may be due to a mutation, not in the genetic level but probably during the biosynthesis.. etc or whatever. In this stage we could hypothesize different ways which would end up to a prion. Update me! ISAVILLO California From owner-proteins@net.bio.net Tue Feb 10 22:00:00 1998 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!206.229.87.25!news-peer.sprintlink.net!news-pen-16.sprintlink.net!newsfeed.nysernet.net!news.nysernet.net!news.sprintlink.net!Sprint!129.98.1.7!news.aecom.yu.edu!skylark.aecom.yu.edu!user From: fengli@aecom.yu.edu (Li Feng) Newsgroups: bionet.molbio.proteins Subject: N-terminal blocked, is it normal? Date: Wed, 11 Feb 1998 15:38:03 -0500 Organization: Albert Einstein College of Medicine Lines: 10 Message-ID: NNTP-Posting-Host: skylark.aecom.yu.edu Mime-Version: 1.0 Content-Type: text/plain; charset=US-ASCII Content-Transfer-Encoding: 7bit X-Newsreader: Value-Added NewsWatcher 2.1d3+ Hello everyone, our lab is trying to identify a recombinant protein expressed in baculovirus/insect sell (Sf9) system. The N-terminal sequnecing (not resolvable) showed that it was blocked. My question is: is it normal for eukaryotic cell expression to have blocked N-terminal? What is the common block (eg. acetylation, etc.)? How to identify the N-terminal block without MS? Thanks. Li Feng Liver Center AECOM From owner-proteins@net.bio.net Tue Feb 10 22:00:00 1998 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!europa.clark.net!205.252.116.205!howland.erols.net!news.idt.net!nntp2.cerf.net!nntp3.cerf.net!newsfeed.san.rr.com!not-for-mail From: Michael Skidmore Newsgroups: bionet.molbio.gene-express,bionet.molbio.proteins Subject: EColi vector to express 2 proteins? Date: Wed, 11 Feb 1998 01:48:13 -0800 Organization: Road Runner Lines: 34 Message-ID: <34E173C5.A43EE646@san.rr.com> Reply-To: mskidmo1@san.rr.com NNTP-Posting-Host: dt063n7b.san.rr.com Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii; x-mac-type="54455854"; x-mac-creator="4D4F5353" Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.04 (Macintosh; U; PPC) Does anyone know of an E Coli expression vector (preferably T7 based for use in BL21DE3's with a his tag) that will express two proteins? I have a dimeric enzyme with a 65 and 37 KDa subunit that I want to express in 10 to 100mg amounts/ 20 liter prep. The 65k subunit expresses well in coli but the 37k is insoluble. Trying insect cells didn't help for dual expression (very low yield when using two separate viruses, high yield with both alone). I can combine the two subunits post purification but for various reasons I don't want to go that route, mainly yield and manhours problems. I read in Maniatis and the Red Book that one cannot use two expression vectors in the same bug if both have the same origin of replication. For example, I can't put the 65 k in pET19b and the 37k in another pET19b like I wanted to. Since just about every expression vector carries the Col1 ori, I am out of luck, unless I find another vector that isn't from the pBR322 / pUC family. We have a vector in the lab that expresses heavy and light chains of IgG's but they are secreted and there isn't a convient site to remove the pel signals. One suggestion I got was subclone the subunits back to back with a stop signal in between. The RNAP would make the first subunit and then hopefully not detach yet before the next subunit's coding region started. But they would not come out as a single peptide. I have no idea if that will work but am open to suggestions. Looking in the common catalogues hasn't come up with anything unless I make a new baculovirus vector with both. TIA. From owner-proteins@net.bio.net Tue Feb 10 22:00:00 1998 Path: biosci!LAUNCHMASTER.COM!launch From: launch@LAUNCHMASTER.COM Newsgroups: bionet.molbio.proteins Subject: Your Web Site Re-Launch Date: 11 Feb 1998 00:09:56 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 83 Sender: daemon@net.bio.net Distribution: world Message-ID: <199802110810.DAA12707@ns.LAUNCHMASTER.COM> Reply-To: launch@LAUNCHMASTER.COM NNTP-Posting-Host: net.bio.net To: proteins@net.bio.net Need a re-launch? Launchmaster will re-launch your web site to 50 search engines and indexes for $95. Satisfaction guaranteed! Millions of people use search engines to find web sites every day. But if your site isn't listed, no one will find it. Launchmaster will re-launch your site by submitting it to the top 50 search engines for $95. Here's how to do it: 1. Complete the form below and return it to us. 2. 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(This information will be posted to the search engines/indexes): Contact name: Company Name: Address: City: State/Prov: Zip/Postal Code: Telephone: Fax: Email address: Contact e-mail address (in case we have questions about this order): URL: http:// Site Title: Description (250 characters): Key words (250 characters, in descending order of importance): If billing a different address, please complete the following: Addressee: Company Name: Address: City: State/Prov: Zip/Postal Code: Telephone: Fax: Email address: TERMS Terms are net 15 days from date of invoice. ______________________________________________________________________ Launchmaster, Inc. 12 Godfrey Place Wilton CT 06897 Phone: (203) 834-5823 Fax: (203) 834-1897 E-mail: launch@launchmaster.com From owner-proteins@net.bio.net Tue Feb 10 22:00:00 1998 Path: biosci!daresbury!uninett.no!news.maxwell.syr.edu!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!portc02.blue.aol.com!pitt.edu!newsfeed.pitt.edu!pxpst2 From: pxpst2@vms.cis.pitt.edu (Peter Pediaditakis) Newsgroups: bionet.molbio.proteins Subject: Re: Prions Date: Wed, 11 Feb 1998 08:59:47 -0500 Organization: University of Pittsburgh Lines: 44 Distribution: world Message-ID: References: <0fae62714060b28UPIMSSMTPUSR04@email.msn.com> NNTP-Posting-Host: pelli.pathology.pitt.edu X-Newsreader: MT-NewsWatcher 2.3.5 In article <0fae62714060b28UPIMSSMTPUSR04@email.msn.com>, intein-intron@email.msn.com ("Isidro Savillo") wrote: > I have been following the biology of prions for sometime. Well we always > believe about the central dogma. So we expect that the proteins at least if > we root the source though we may be passing a series of pathways, indirect > or direct comes from (a) gene(s). Considering the changes in protein > conformation, I would suggest that there are 'pathogenic enzymes' which are > responsible for the abnormal change. And prion's ability to transform normal > to abnormal forms would suggest that prions are acting as enzymes > themselves. > If the source is human DNA then it may be due to a mutation, not in the > genetic level but probably during the biosynthesis.. etc or whatever. In > this stage we could hypothesize different ways which would end up to a > prion. Well this is possible but has NOT been seen experimentally. No enzymatic activity has been attributed to prions. It is not such a strech to see how one protein can modify the structure of another protein. Although not talked about, chaparone proteins inside the cell modify the structure of other proteins without enzymatically acting on the other proteins. Although I am not saying that prions are chaparones, they do act in a similar manner as chaparones. Peter -- "Don't you eat that yellow snow WAtch out where the Huskies go" --------------------------------------------------------------------- Let us not forget those marketing boys from SPAM CENTRAL bestrealtor@marketingmaster.com info@herbchew.com clickthru@timefreedom.com invite@onlinenow.net zippydj@nevwest.com Offer@shire.com empower@empowerlabs.com dynamarket@vaprnet.com root@mail.icongrp.com cashrewards@hotmail.com From owner-proteins@net.bio.net Tue Feb 10 22:00:00 1998 From: dekimpe@inwchem.rug.ac.be Subject: native 2 D electrophoresis of plasma proteins Date: Wed, 11 Feb 1998 11:21:20 -0600 Reply-To: dekimpe@inwchem.rug.ac.be Message-ID: <887217282.193396738@dejanews.com> Newsgroups: bionet.cellbiol,bionet.molbio.proteins,bionet.general Organization: Deja News Posting Service Path: biosci!bloom-beacon.mit.edu!newsxfer3.itd.umich.edu!news-out.internetmci.com!newsfeed.internetmci.com!128.230.129.106!news.maxwell.syr.edu!nntp2.dejanews.com!grunt.dejanews.com!not-for-mail X-Article-Creation-Date: Wed Feb 11 17:14:42 1998 GMT X-Authenticated-Sender: dekimpe@inwchem.rug.ac.be X-Http-User-Agent: Mozilla/2.0 (compatible; MSIE 3.01; Windows 3.1) X-Originating-IP-Addr: 157.193.54.112 (inwch600.rug.ac.be) Lines: 20 Xref: biosci bionet.cellbiol:8813 bionet.molbio.proteins:12291 bionet.general:29334 hello, We're a group working on identification of metal - protein binding. In the past we mainly used chromatography for separation of metalloproteins in various tissues and body fluids. Now we've decided to give native 2 D electrophoresis a try. As 2D has much higher resolving power than protein chromatography. Could anybody out there provide us with a protocol for native 2 D electropho- resis of plasma proteins ? We're using the Multiphor System of Amershan-Pharmacia-Biotech. For the moment we're especially interrested in the identification of cis-platinum proteins. Jurgen De Kimpe and Sonnke Lustig dekimpe@inwchem.rug.ac.be or soennke.lustig@rug.ac.be Laboratory for Analytical Chemistry, Institute of Nuclear Sciences, University of Ghent, Proeftuinstraat 86, Ghent, East-Flanders, Belgium tel: 0032(0)92646624 or 0032(0)92646603 -------------------==== Posted via Deja News ====----------------------- http://www.dejanews.com/ Search, Read, Post to Usenet From owner-proteins@net.bio.net Tue Feb 10 22:00:00 1998 Path: biosci!bloom-beacon.mit.edu!eecs-usenet-02.mit.edu!news.sgi.com!sdd.hp.com!usc!howland.erols.net!newsfeed.internetmci.com!198.82.160.249!news.vt.edu!solaris.cc.vt.edu!news.cc.ukans.edu!not-for-mail From: Mousheng Xu Newsgroups: bionet.molbio.proteins Subject: UPDATE: Bioinformation Search Center 5.0!!! Date: Wed, 11 Feb 1998 22:29:55 -0600 Organization: University of Kansas Computing Services Lines: 25 Message-ID: <34E27AC3.167E@eecs.ukans.edu> NNTP-Posting-Host: blizzard.rsl.ukans.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01Gold (X11; I; OSF1 V4.0 alpha) Dear Scientists: The Bioinformation Search Center (BSC) 5.0 (http://www.ittc.ukans.edu/~mxu/cgi-bin/thesis/bi.cgi) is now available to the public! Major new features of Version 5.0: 1. The results of type "possibly related entries" are broadened -- BSC returns Class I results in addition to the results returned in BSC 4.0 (class II results). Major known problem: Common words are not used for search in BSC. For the above example, both "human" and "factor" are so common that they are not used for search. In this case, it is bad. But a lot of common words such as "the", "a", "an" are surely bad in search of biological information. This problem can easily be solved, technically. But it takes programmer's time and the university's computer disk space. Please give this baby a try! Any comments are highly appreciated! Thanks a lot. Sincerely, Mousheng Xu From owner-proteins@net.bio.net Wed Feb 11 22:00:00 1998 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsxfer3.itd.umich.edu!newsxfer.itd.umich.edu!news.itd.umich.edu!not-for-mail From: "Larry \"Harris\" Taylor, Ph.D." Subject: Tri-basic cleavage Newsgroups: bionet.molbio.proteins Message-ID: <01bd37c8$a7edac40$85bad68d@wakil239> X-Newsreader: Microsoft Internet News 4.70.1161 Lines: 11 Date: Thu, 12 Feb 1998 15:08:11 GMT NNTP-Posting-Host: host-133.subnet-186.med.umich.edu NNTP-Posting-Date: Thu, 12 Feb 1998 10:08:11 EST Any suggestions on tri-basic cleavage for in vivo processing of a large protein to smaller neuropeptides in vertebrates? The specific sequence is -----QSSQ RRR LLHQN------- Di basic cuts are common, but what occurs at a tri-basic site? Is the tribasic site cut out, or is there a series of peptides resul;ting from a dibasic cut? Any insights would be appreciated Larry Taylor lpt@umich.edu I From owner-proteins@net.bio.net Wed Feb 11 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!news1.chicago.iagnet.net!qual.net!iagnet.net!195.74.75.15!fci-se!fci!newsfeed.sunet.se!news99.sunet.se!news01.sunet.se!130.239.8.18.MISMATCH!umdac!umu.se!not-for-mail From: Mariusz.Kowalczyk@genfys.slu.se (kovi) Newsgroups: bionet.molbio.proteins Subject: Re: native 2 D electrophoresis of plasma proteins Date: 12 Feb 1998 11:29:28 GMT Organization: UMDAC Lines: 20 Message-ID: <6bumeo$bls$1@studium.student.umu.se> References: <887217282.193396738@dejanews.com> NNTP-Posting-Host: b195.genfys.slu.se Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Newsreader: knews 1.0b.0 Hej, dekimpe@inwchem.rug.ac.be writes: > Could anybody out there provide us with a protocol for native ^^^^^^ > 2 D electrophoresis of plasma proteins ? Standard protocol for 2De involves both "native" (IEF) and "non-native" (SDS-PAGE) steps (check: http://expasy.hcuge.ch/ch2d/technical-info.html). It is possible to run preparative 2De and then eluate protein from the SDS-PAGE gel and refold it, but it's quite unefficient way to purify proteins... I don't know if there is a protocol for 2De that involves two "native" electrophoretical steps :-) Regards, M.K: -- Greetz from Mariusz Kowalczyk, Ph.D. student| SLU, Inst. för skoglig gentet. och växtfys | From owner-proteins@net.bio.net Wed Feb 11 22:00:00 1998 Path: biosci!email.msn.com!intein-intron From: intein-intron@email.msn.com ("Isidro Savillo") Newsgroups: bionet.molbio.proteins Subject: Prions Date: 12 Feb 1998 00:29:41 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 8 Sender: daemon@net.bio.net Distribution: world Message-ID: <08fe35427080c28UPIMSSMTPUSR03@email.msn.com> NNTP-Posting-Host: net.bio.net Recent findings have shown the presence of splicing activity in some translated proteins. Would it not be possible for "peptide recombination or insertion" to take place? This could modify all the structures of the protein. From owner-proteins@net.bio.net Wed Feb 11 22:00:00 1998 Path: biosci!agate!newsgate.duke.edu!news.vt.edu!solaris.cc.vt.edu!fci-se!fci!news-out.internetmci.com!newsfeed.internetmci.com!199.60.229.5!newsfeed.direct.ca!ix.netcom.com!news From: "Scott Shanes" Newsgroups: bionet.molbio.proteins Subject: US-NJ-BIOCHEMISTRY SCIENTIST NEEDED Date: 12 Feb 1998 13:31:42 GMT Organization: Netcom Lines: 15 Message-ID: <01bd37b8$f85e9460$2e79d6ce@scott-s-armada> NNTP-Posting-Host: trn-nj1-14.ix.netcom.com X-NETCOM-Date: Thu Feb 12 5:31:42 AM PST 1998 X-Newsreader: Microsoft Internet News 4.70.1161 I am looking for a Scientist who will be responsible for the isolation and characterization of endogenous ligands for orphan receptors. The candidate should have proven expertise in the purification of trace level bioactive substances from native sources. This person will be conversant in a broad spectrum of purification and analytical methods appropriate for the study of peptide or small molecule transmitters, hormones, or trace level metabolites. This person should possess a Ph.D. in the sciences with 1+ years post-doctoral experience. We are a leading bio-tech firm with research facilities in Northern, New Jersey and can provide excellent benefits (health insurance, dental, and vision plan, paid vacation and more). A high impact, high profile position with excellent opportunity for advancement. Please contact Scott Shanes by phone at 609-584-8733 Ext. 218, fax CV and cover letter to 609-584-9575 or E-Mail to sis@diedremoire.com. From owner-proteins@net.bio.net Wed Feb 11 22:00:00 1998 Newsgroups: bionet.molbio.proteins Path: biosci!bloom-beacon.mit.edu!news.kodak.com!news-pen-16.sprintlink.net!newsfeed.nysernet.net!news.nysernet.net!207.41.200.14!news-pen-14.sprintlink.net!206.229.87.26!news-east.sprintlink.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!vixen.cso.uiuc.edu!uchinews!midway.uchicago.edu!aekentsi From: aekentsi@midway.uchicago.edu (alex emil kentsis) Subject: Re: N-terminal blocked, is it normal? X-Nntp-Posting-Host: howard-nfs.uchicago.edu Message-ID: Sender: news@midway.uchicago.edu (News Administrator) Organization: The University of Chicago X-Newsreader: TIN [version 1.2 PL2] References: Date: Thu, 12 Feb 1998 15:50:07 GMT Lines: 16 many neuropeptides have n-terminal caps, usually acetyl or methyl. i am not sure about means to identify them. a better way to check for terminal capping (than sequencing) is to use an exopeptidase. Li Feng (fengli@aecom.yu.edu) wrote: : Hello everyone, our lab is trying to identify a recombinant protein : expressed in baculovirus/insect sell (Sf9) system. The N-terminal : sequnecing (not resolvable) showed that it was blocked. My question is: : is it normal for eukaryotic cell expression to have blocked N-terminal? : What is the common block (eg. acetylation, etc.)? How to identify the : N-terminal block without MS? Thanks. : Li Feng : Liver Center : AECOM From owner-proteins@net.bio.net Wed Feb 11 22:00:00 1998 Newsgroups: bionet.molbio.proteins Path: biosci!agate!howland.erols.net!vixen.cso.uiuc.edu!uchinews!midway.uchicago.edu!aekentsi From: aekentsi@midway.uchicago.edu (alex emil kentsis) Subject: Re: Prions X-Nntp-Posting-Host: howard-nfs.uchicago.edu Message-ID: Sender: news@midway.uchicago.edu (News Administrator) Organization: The University of Chicago X-Newsreader: TIN [version 1.2 PL2] References: <08fe35427080c28UPIMSSMTPUSR03@email.msn.com> Date: Thu, 12 Feb 1998 15:47:47 GMT Lines: 20 check out feb 6 and jan 3 issues of science. an article in the former conclusively demonstrates that conversion to the scrapie (beta) form of prp is not necessary for neurodegeneration. an article in the latter suggests that gag expansion may occur by way of rna editing. in any case, it would be inaccurate to call prp an enzyme because conversion occurs from the interacton of the meta-stable form of prp(c) with the scrapie form, converting c->scrapie, and forming an insoluble oligomer between the two. Isidro Savillo (intein-intron@email.msn.com) wrote: : Recent findings have shown the presence of splicing activity in some : translated proteins. Would it not be possible for "peptide recombination or : insertion" to take place? This could modify all the structures of the : protein. From owner-proteins@net.bio.net Wed Feb 11 22:00:00 1998 Path: biosci!bloom-beacon.mit.edu!news.kodak.com!news-pen-16.sprintlink.net!newsfeed.nysernet.net!news.nysernet.net!207.41.200.14!news-pen-14.sprintlink.net!206.229.87.26!news-east.sprintlink.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!woodstock.news.demon.net!demon!dispose.news.demon.net!demon!peer.news.zetnet.net!zetnet.co.uk!user From: nigel.osborn@zetnet.co.uk (Nigel J.Osborn) Newsgroups: bionet.molbio.proteins Subject: Fourier transform mass spectrometry Date: Thu, 12 Feb 1998 20:21:45 -0100 Organization: Amersham, Bucks Message-ID: NNTP-Posting-Host: man-057.dialup.zetnet.co.uk Lines: 9 Does anyone out there know of any contract labs able to analyse proteins by fourier tranform mass spectrometry (FTCIMS)? Ideally I'd like them to be in the U.K. but failing that anywhere in the world. Nigel Osborn From owner-proteins@net.bio.net Wed Feb 11 22:00:00 1998 Path: biosci!bmg.bhs.uab.edu!GMEACHAM From: GMEACHAM@bmg.bhs.uab.edu Newsgroups: bionet.molbio.proteins Subject: Caspase Inhibitors Date: 12 Feb 1998 06:25:36 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 9 Sender: daemon@net.bio.net Distribution: world Message-ID: <114F415C3EB3@bmg.bhs.uab.edu> NNTP-Posting-Host: net.bio.net To all: I am looking for inhibitors to the 'Group III' family of caspases. These include caspase-3 (Mch2), caspase-8 (MACH,Mch5), and caspase-9 (ICE-LAP6, Mch6). These proteases recognize the motif (I/L/V)ExD. Any help would be much appreciated. Thanks in advance. Geoff Meacham UAB Dept. of Cell Biology From owner-proteins@net.bio.net Wed Feb 11 22:00:00 1998 Path: biosci!GENOME.BIOTECH.WASHINGTON.EDU!eugene From: eugene@GENOME.BIOTECH.WASHINGTON.EDU (Eugene Kolker) Newsgroups: bionet.molbio.proteins Subject: Urgent RECOMB 98 registration deadlines approaching! Date: 12 Feb 1998 15:23:47 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 7 Sender: daemon@net.bio.net Distribution: world Message-ID: <199802122322.PAA06717@genome.biotech.washington.edu> NNTP-Posting-Host: net.bio.net Early registration ends February 20, 1998! Hotel conference rate ends February 24, 1998! For more details please visit our web site: http://www.mssm.edu/biomath/recomb98.html From owner-proteins@net.bio.net Wed Feb 11 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!news-peer.gip.net!news.gsl.net!gip.net!news-peer.sprintlink.net!news-pen-15.sprintlink.net!newsfeed.nysernet.net!news.nysernet.net!news.sprintlink.net!Sprint!129.98.1.7!news.aecom.yu.edu!post!yilu From: Yi Lu Newsgroups: bionet.molbio.proteins Subject: Re: Caspase Inhibitors Date: Thu, 12 Feb 1998 17:19:06 -0500 Organization: Albert Einstein College of Medicine Lines: 31 Message-ID: References: <114F415C3EB3@bmg.bhs.uab.edu> NNTP-Posting-Host: post.aecom.yu.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII X-Sender: yilu@post To: GMEACHAM@bmg.bhs.uab.edu In-Reply-To: <114F415C3EB3@bmg.bhs.uab.edu> HI, I think z-VAD-fmk or z-DEVD-fmk may be suitable for you. z-VAD-fmk is kind of general inhibitor for maybe all the caspases (3,4,6,8,9), while z-DEVD-fmk is a relatively specific inhibitor for caspase-3. The Enzyme Systems Products sell these caspases. Z-VAD-fmk works for me. If you get a chance to use these caspases, could you tell me whether they work for you? BTW, these caspase is used for in vivo assay, if you only need to do an in vitro assay, DEVD-CHO is good enough. Good luck. Louie Albert Einstein College of Medicine Department of Pathology On 12 Feb 1998 GMEACHAM@bmg.bhs.uab.edu wrote: > To all: > I am looking for inhibitors to the 'Group III' family of caspases. These > include caspase-3 (Mch2), caspase-8 (MACH,Mch5), and caspase-9 > (ICE-LAP6, Mch6). These proteases recognize the motif (I/L/V)ExD. > Any help would be much appreciated. Thanks in advance. > > Geoff Meacham > UAB Dept. of Cell Biology > > > From owner-proteins@net.bio.net Wed Feb 11 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!news.maxwell.syr.edu!xfer.kren.nm.kr!newsfeed.kornet.nm.kr!usenet.kornet.nm.kr!not-for-mail From: "À¯Àü »ýÈ­ÇÐ ¿¬±¸½Ç" Newsgroups: bionet.molbio.proteins Subject: testing Date: Fri, 13 Feb 1998 16:18:22 +0900 Organization: korea univ Lines: 4 Message-ID: <34E3F3BD.5CF08403@kuccbx.korea.ac.kr> NNTP-Posting-Host: clone.korea.ac.kr Mime-Version: 1.0 Content-Type: text/plain; charset=EUC-KR Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.03 [ko] (Win95; I) Very sorry for this testing. Good bye. From owner-proteins@net.bio.net Thu Feb 12 22:00:00 1998 Path: biosci!internet!biosci!not-for-mail From: biohelp (BIOSCI Administrator) Newsgroups: bionet.molbio.proteins Subject: BIOSCI/bionet miniFAQ & Fundraiser Date: 13 Feb 1998 02:00:08 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 233 Sender: daemon@net.bio.net Distribution: world Message-ID: <199802131000.CAA15754@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