From owner-proteins@net.bio.net Wed Apr 01 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.wli.net!news-peer-west.sprintlink.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!howland.erols.net!news.net.uni-c.dk!news.daimi.aau.dk!not-for-mail From: Per Mygind Newsgroups: bionet.molbio.proteins Subject: Re: LIC, any pitfalls to look out for? Date: Thu, 02 Apr 1998 15:37:40 +0100 Organization: DAIMI, Computer Science Dept. at Aarhus University Lines: 31 Message-ID: <3523A2B4.3BE6@medmicro.aau.dk> References: <35227AA0.B5ADE4E4@elroy.qci.bioch.bcm.tmc.edu> NNTP-Posting-Host: majestetix.medmicro.aau.dk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (X11; I; SunOS 5.5.1 sun4m) To: Brad Poland Brad Poland wrote: > > I have been thinking of trying the Ligation Independent Cloning from > Novagen, does > anyone have experience with this technique? If so what are some > pitfalls to look out > for? Thanks. > Its an exellent tool, apart from being ligation independant, it is also restriction-endonuclease independent, meaning that no restrictionsites are required. I personally cloned 4 different gene fragments for expression, in a matter of days....... (absolute no affiliation, but I regret I didn't invent the technique ;-) Regards Per Mygind ************************************************************************ If you are are not part of the solution, you are part of the precipitate ************************************************************************ Per Mygind, Cand.scient Department of Medical Microbiology and Immunology The Bartholin Building, University of Aarhus, Denmark phone : 89 42 17 47, fax : 86 19 61 28 ************************************************************************ It's hard work and great art to make life not so serious ************************************************************************ From owner-proteins@net.bio.net Wed Apr 01 23:00:00 1998 Path: biosci!agate!news-spur1.maxwell.syr.edu!news.maxwell.syr.edu!Cabal.CESspool!bofh.vszbr.cz!lyra.csx.cam.ac.uk!kaf28 From: kaf28@mole.bio.cam.ac.uk (Kevin A Floyd) Newsgroups: bionet.molbio.proteins Subject: cross-linking membrane proteins Date: 2 Apr 1998 20:54:45 GMT Organization: University of Cambridge, England Lines: 25 Message-ID: <6g0tul$q4f$1@lyra.csx.cam.ac.uk> NNTP-Posting-Host: mole.bio.cam.ac.uk X-Newsreader: TIN [version 1.2 PL2] Hi all! I am attempting without much success to cross-link a thylakoid protein in an attempt to identify proteins it is associated with. I would greatly appreciate any advice anybody has to offer. This is what I am doing: I am using the crosslinker BASED (Pierce chemicals), a crosslinker with a cleavable di-sulphide link with two photoactivated linking groups. the aim is to cross link the membrane proteins , run it on a acyrlamide gel and make a western blot using an antibody raised to my protein of intrest. After (succesfully!) cross linking I wish to excise the band, reduce the cross linker and blot onto PVDF membrane for N-terminal sequencing of any proteins associated with my one of intrest. I have tried altering the cross-linker and protein concentrations, the pH of buffers used, the solvent in which the crosslinker is made up in all to no avail. I would greatly appreciate it if you could mail me advice on kaf28@mole.bio.cam.ac.uk Thanks Kevin floyd From owner-proteins@net.bio.net Wed Apr 01 23:00:00 1998 Path: biosci!agate!news-spur1.maxwell.syr.edu!news.maxwell.syr.edu!cpk-news-hub1.bbnplanet.com!chicago-news-feed2.bbnplanet.com!news.bbnplanet.com!uwm.edu!msunews!not-for-mail From: Ivan Delgado Newsgroups: bionet.molbio.proteins Subject: X-ray crystal structure Date: Thu, 02 Apr 1998 15:52:16 -0500 Organization: Michigan State University Lines: 24 Message-ID: <3523F9F0.854A115@pilot.msu.edu> NNTP-Posting-Host: 35.8.197.128 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 4.03 (Macintosh; I; PPC) X-MD5: 1a105540ca5dc7a1392ce866c02405e1 Hi, I have successfully overexpressed a plant protein in bacteria and as far as I can tell it its functionally active. Due to the unique binding characteristics of this protein, I was interested in determining its structure. I have read about the different methods of how to obtain crystals and know a lot less about the procedure of crystal analysis itself. Could anybody guide me towards a way of obtaining a crystal and more importantly, of how to obtain a crystal structure? (such as an institute that provides a facility to do such experiments, or a collaboration). Thanks in advance for your help, Ivan ------------------------------------------------------------------------ Ivan J Delgado Orlic Lab: 517-353-3519 MSU-DOE Plant Research Laboratory delgadoi@pilot.msu.edu East Lansing, MI 48824-1312 http://www.msu.edu/user/delgadoi ------------------------------------------------------------------------ We hope that when the insects take over the world, they will remember with gratitude how we took them along on all our picnics. -Bill Vaughan From owner-proteins@net.bio.net Wed Apr 01 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news.algonet.se!feed1.news.luth.se!luth.se!Cabal.CESspool!bofh.vszbr.cz!lyra.csx.cam.ac.uk!funes.mrc-lmb.cam.ac.uk!user From: hdejonge@freemail.nl (H. de Jonge) Newsgroups: bionet.molbio.proteins Subject: Protein precipitation Date: 2 Apr 1998 13:32:04 GMT Organization: Utrecht University, The Netherlands Lines: 15 Message-ID: NNTP-Posting-Host: funes.mrc-lmb.cam.ac.uk X-Newsreader: Value-Added NewsWatcher 2.12d0+ Dear reader, I am a Dutch biology student working on a project that involves the separation of proteins. I found out that addition of heparin to my concentrated protein solution caused a precipitation of the protein. I think this can be explained by the polysaccharide binding to the positive charge on the surface of the protein making it less soluble. It seems to stay in solution if I lower the protein concentration and this confuses me. Can someone explain to me what the mechanism is? How can the protein concentration affect its solubility when it complexes with heparin (or other charged molecules)? Thank you. Hugo de Jonge. From owner-proteins@net.bio.net Wed Apr 01 23:00:00 1998 Path: biosci!agate!nntp-out.monmouth.com!newspeer.monmouth.com!newsfeed.direct.ca!newshub1.home.com!news.home.com!news.rdc1.ct.home.com!not-for-mail From: Menoret Newsgroups: bionet.molbio.proteins Subject: hsp-immunity Date: Thu, 02 Apr 1998 05:22:34 -0800 Organization: @Home Network Lines: 5 Message-ID: <3523911A.32591E9B@home.com> Reply-To: menoret@home.com NNTP-Posting-Host: c1003124-a.blfld1.ct.home.com Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.02 [en]C-AtHome0402 (Win95; U) The Center for Immunotherapy of Cancer and Infectious Diseases organize the first international conference on : Heat Shock Proteins in Immune Response. For more information visit ; http://www.hspimmunity.com From owner-proteins@net.bio.net Wed Apr 01 23:00:00 1998 Path: biosci!acdlabs.com!vic From: vic@acdlabs.com ("Victor Graziano") Newsgroups: bionet.molbio.proteins Subject: (Free Software Demo) Protein Manager Date: 2 Apr 1998 13:41:55 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 30 Sender: daemon@net.bio.net Distribution: world Message-ID: <000501bd5e80$00b79ce0$1ee9b0cf@T95ie.acdlabs.com> NNTP-Posting-Host: net.bio.net Dear Protein-Analysis subscribers, Advanced Chemistry Development Inc. (ACD), a leader in Cheminformatics software, has just released Protein Manager, the newest member of its extensive software family. This remarkable software package, coupled with the Swissprot, Prosite, PDB, ACD Restriction Enzyme and (soon to follow) ACD Regulatory Protein Databases, will dramatically accelerate peptide drug discovery. The primary functions of Protein Manager are protein analysis, data management and data publishing. Software operation is easy, simply load candidate proteins into the Protein Manager 'protein workbook' and perform a multitude of functions including: -Isoelectric point determination -Protein secondary structure prediction ( including Predator prediction method) -Multiple protein alignment with consensus matching -Enzymatic digestion of selected proteins (ACD enzyme database) -Protein scale determination (hydrophobicity, polarity, bulkiness...) -3D viewing of all available Protein Database (PDB) files Furthermore, proteins may be generated at random in Protein Manager, or modified from existing protein files. It's as easy as cut, copy and pasting. Please feel free to visit our web site and download a demo copy or product video preview, both available March 31st. Should you have any questions regarding Protein Manager, please feel free to contact one of our sales representatives. ACD thanks you for your time and hopes you enjoy Protein Manager. From owner-proteins@net.bio.net Wed Apr 01 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!dallas-news-feed2.bbnplanet.com!news.bbnplanet.com!newsfeed.gte.net!newsfeed1.earthlink.net!nntp.earthlink.net!usenet From: John Philo <"jphilo*NO SPAM12*"@earthlink.net> Newsgroups: bionet.molbio.proteins Subject: Re: Protein precipitation Date: Thu, 02 Apr 1998 08:03:48 -0800 Organization: Alliance Protein Laboratories Lines: 49 Message-ID: <3523B6E4.6C42@earthlink.net> References: Reply-To: jphilo*NO, SPAM12*@earthlink.net NNTP-Posting-Host: 207.217.142.66 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.03 (Win95; I) To: "H. de Jonge" H. de Jonge wrote: > > Dear reader, > > I am a Dutch biology student working on a project that involves the > separation of proteins. I found out that addition of heparin to my > concentrated protein solution caused a precipitation of the protein. I > think this can be explained by the polysaccharide binding to the positive > charge on the surface of the protein making it less soluble. It seems to > stay in solution if I lower the protein concentration and this confuses > me. Can someone explain to me what the mechanism is? How can the protein > concentration affect its solubility when it complexes with heparin (or > other charged molecules)? > > Thank you. > > Hugo de Jonge. Hugo, There are several mechanisms that may be at work here. The first is the one you allude to, that the binding of the highly negatively charged heparin neutralizes the positive charges on your protein. This in turn can reduce charge repulsion effects that tend to prevent protein molecules from coming together to aggregate and precipitate. Based on my own work with fibroblast growth factors, it is also likely that you have multiple protein molecules bound along the long heparin polymer like beads on a string. It is known for FGFs that the proteins pack very tightly together. These high molecular weight complexes tend to have low solubility. If this mechanism is correct, then you may be able to avoid precipitation by adding MORE heparin, since when there are more heparin molecules the number of proteins bound to any one heparin will be reduced. It is also known that binding to heparin can induce significant conformational changes in proteins. This may do something such as exposing hydrophobic groups which can then stick to other proteins and lead to precipitation. Whatever the mechanism, it is not surprising that you see less or no precipation at lower protein concentrations. You said it yourself--the protein/heparin complexes have low solubility, and thus will stay in solution up to a certain maximal concentration (the "solubility"). 'Hope this helps, John Philo, Alliance Protein Laboratories *** Remove "*NO SPAM12*" from return address before replying. *** From owner-proteins@net.bio.net Wed Apr 01 23:00:00 1998 Path: biosci!rutgers!nntp.upenn.edu!newshub.northeast.verio.net!uunet!in4.uu.net!news.or.intel.com!usenet From: layqkuplmakemoney@yuocanberich.net Newsgroups: bionet.molbio.proteins Subject: Add A Link....Make Money!!!!!!! Date: 2 Apr 1998 15:58:33 GMT Organization: HaveSomeFun Lines: 39 Message-ID: <6g0cj9$r3o@news.or.intel.com> NNTP-Posting-Host: qrlablinq02.ra.intel.com Mime-Version: 1.0 Content-Type: multipart/mixed; boundary="PART_BOUNDARY_YKNQEJWHLD" X-Newsreader: 2.0.14 --PART_BOUNDARY_YKNQEJWHLD Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit **************************************************************** * This Article was Posted By an unregistered version of: * * Newsgroup AutoPoster 95 * * Send email address for info! Fax: +46-31-470588 * **************************************************************** --PART_BOUNDARY_YKNQEJWHLD Content-Type: text/html; charset=us-ascii; name="test.html" Content-Transfer-Encoding: 7bit Content-Disposition: inline; filename="test.html" Content-Base: "file:///C|/test.html" --PART_BOUNDARY_YKNQEJWHLD Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit Make money just by adding a link to your homepage. http://members.spree.com/andyt/ From owner-proteins@net.bio.net Thu Apr 02 23:00:00 1998 Path: biosci!rutgers!rockyd.rockefeller.edu!news-pen-14.sprintlink.net!206.229.87.26!news-east.sprintlink.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!netnews.com!newspeer.monmouth.com!news-feed.inet.tele.dk!bofh.vszbr.cz!fci-se!fci!newsfeed.sunet.se!news99.sunet.se!news01.sunet.se!umdac!umu.se!tby From: tby@randbaek.chem.umu.se (Tomas Bystrom) Newsgroups: bionet.molbio.proteins Subject: Peptide-fluorinated solvent interactions? Date: 3 Apr 1998 11:56:17 GMT Organization: Me, myself and I... Lines: 6 Message-ID: NNTP-Posting-Host: randbaek.chem.umu.se X-Newsreader: slrn (0.9.3.2 UNIX) I wonder if it is studied how and why fluorinated hydrocarbons is so common in dissolving peptides? Is there any specific interactions between the fluorine atoms and the peptide? Any references would be very welcome! /Tomas Bystrom From owner-proteins@net.bio.net Thu Apr 02 23:00:00 1998 Newsgroups: bionet.molbio.proteins Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!chicago-news-feed2.bbnplanet.com!news.bbnplanet.com!news.xnet.com!ais.net!vixen.cso.uiuc.edu!uchinews!midway.uchicago.edu!aekentsi From: aekentsi@midway.uchicago.edu (alex kentsis) Subject: Re: Peptide stability 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: Fri, 3 Apr 1998 13:45:52 GMT Lines: 20 check that you used endotoxin and pyrogen free water to store this peptide. Dr.W.H. Colledge (whc23@cus.cam.ac.uk) wrote: : In general, how stable is a basic peptide if stored in solution at -20'C? : What might be expected to happen to a peptide stored under these : conditions? Could a peptide stored under these conditions change to : produce a substance that was toxic to mammalian cells? Many thanks. : -- : Dr. W.H. Colledge : Physiology : University of Cambridge : Cambridge : CB2 3EG : Tel. 01223-333881 : Fax. 01223-333840 From owner-proteins@net.bio.net Thu Apr 02 23:00:00 1998 Newsgroups: bionet.molbio.proteins Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.internetmci.com!128.174.5.49!vixen.cso.uiuc.edu!uchinews!midway.uchicago.edu!aekentsi From: aekentsi@midway.uchicago.edu (alex kentsis) Subject: Re: Protein precipitation 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: <3523B6E4.6C42@earthlink.net> Date: Fri, 3 Apr 1998 13:50:38 GMT Lines: 63 the effect of heparin and other polyhydric polymers on proteon solubility is largely physical in nature. these substances exclude water because of their large partial molar volume and as a result tend to increase effective protein concentration. thus, a protein solutions finds itself much closer to its solubility limit in the presence of these osmolytes. this is the reason for the dependence on protein cocentration. as you clearly point out, heparin binding to protein (which does not occur at these concentrations; you need to be in the molar range) cannot explain this. check out the work of tanford, ben-naim, and timasheff. John Philo ("jphilo*NOSPAM12*"@earthlink.net) wrote: : H. de Jonge wrote: : > : > Dear reader, : > : > I am a Dutch biology student working on a project that involves the : > separation of proteins. I found out that addition of heparin to my : > concentrated protein solution caused a precipitation of the protein. I : > think this can be explained by the polysaccharide binding to the positive : > charge on the surface of the protein making it less soluble. It seems to : > stay in solution if I lower the protein concentration and this confuses : > me. Can someone explain to me what the mechanism is? How can the protein : > concentration affect its solubility when it complexes with heparin (or : > other charged molecules)? : > : > Thank you. : > : > Hugo de Jonge. : Hugo, : There are several mechanisms that may be at work here. The first is the : one you allude to, that the binding of the highly negatively charged : heparin neutralizes the positive charges on your protein. This in turn : can reduce charge repulsion effects that tend to prevent protein : molecules from coming together to aggregate and precipitate. : Based on my own work with fibroblast growth factors, it is also likely : that you have multiple protein molecules bound along the long heparin : polymer like beads on a string. It is known for FGFs that the proteins : pack very tightly together. These high molecular weight complexes tend : to have low solubility. If this mechanism is correct, then you may be : able to avoid precipitation by adding MORE heparin, since when there are : more heparin molecules the number of proteins bound to any one heparin : will be reduced. : It is also known that binding to heparin can induce significant : conformational changes in proteins. This may do something such as : exposing hydrophobic groups which can then stick to other proteins and : lead to precipitation. : Whatever the mechanism, it is not surprising that you see less or no : precipation at lower protein concentrations. You said it yourself--the : protein/heparin complexes have low solubility, and thus will stay in : solution up to a certain maximal concentration (the "solubility"). : 'Hope this helps, : : John Philo, Alliance Protein Laboratories : *** Remove "*NO SPAM12*" from return address before replying. *** From owner-proteins@net.bio.net Thu Apr 02 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.wli.net!news-feed.inet.tele.dk!bofh.vszbr.cz!fu-berlin.de!jussieu.fr!univ-lyon1.fr!not-for-mail From: Jean-Christophe Roux Newsgroups: bionet.molbio.proteins Subject: RNA/protein isolation Date: Fri, 03 Apr 1998 14:54:43 +0200 Organization: UCBL Lines: 34 Message-ID: <3524DC12.B7770D7C@rockefeller.univ-lyon1.fr> Reply-To: jcroux@rockefeller.univ-lyon1.fr NNTP-Posting-Host: rockumr5578-4.univ-lyon1.fr Mime-Version: 1.0 Content-Type: multipart/alternative; boundary="------------77DB5B7476DEA2FA52A679D5" X-Mailer: Mozilla 4.01 [en] (Win95; I) X-Priority: 3 (Normal) --------------77DB5B7476DEA2FA52A679D5 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit Dear all. I would like to find articles about simultaneous isolation of RNA and proteins. My protocol of RNA extraction is guanidium/phenol /chloroforme and ineed to quantify RNA and protein from the same samples. Is it possible? Thank you very much --------------77DB5B7476DEA2FA52A679D5 Content-Type: text/html; charset=us-ascii Content-Transfer-Encoding: 7bit
    Dear all.
    I would like to find  articles about simultaneous isolation of RNA and proteins.
    My protocol of RNA extraction  is guanidium/phenol /chloroforme and ineed to quantify RNA and protein from the same samples.
    Is it possible?
    Thank you very much
 
  --------------77DB5B7476DEA2FA52A679D5-- From owner-proteins@net.bio.net Thu Apr 02 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news-xfer.netaxs.com!news-nb.rutgers.edu!amenti.rutgers.edu!not-for-mail From: meton@rci.rutgers.edu (Daniel Gonzalez) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Green Fluorescent Protein Purificaton Short Course Date: 3 Apr 1998 13:29:51 -0500 Organization: Rutgers University Lines: 57 Message-ID: <6g39qv$kmc$1@amenti.rutgers.edu> NNTP-Posting-Host: amenti.rutgers.edu Summary: Hands-On Laboratory Course Using the Green-Fluorescent Protein Keywords: Green Fluorescent Protein Purificaton Short Course Xref: biosci bionet.molbio.methds-reagnts:66316 bionet.molbio.proteins:12600 I have been asked to announce the following, The State University of New Jersey Rutgers Campus at New Brunswick Center for Research and Education in Bioluminescence and Biotechnology Programs in Biotechnology, Presents: Protein Purification: Isolation, Analysis, and Characterization of GFP, A Five and One-Half Day Hands-On Laboratory Course Using the remarkable Green-Fluorescent Protein (GFP), A Novel Marker For Gene Expression, as the source material June 7-12, 1998, July 12-17, 1998,and August 2-7, 1997 More than 500 scientists from around the world have strongly recommended this intensive course as an opportunity to develop protein research and analytical skills in a retreat setting. Participants work hard, identify and solve problems in the lab and enjoy camaraderie and good food and beer with colleagues. This five-day laboratory course covers a wide variety of conventional methods for protein isolation, purification, and characterization. The course format integrates hands-on laboratory exercises with classroom lectures, demonstrations, study breaks, and short take-home assignments. A special feature of the course is that all laboratory work will be performed on the same starting sample (Aequorea GFP*), which will be purified from an exceedingly crude form to near homogeneity as judged by high performance liquid chromatography (HPLC), SDS gel electrophoresis, isoelectric focusing, and capillary zone electrophoresis. This feature provides a continuity of purpose, integrating dozens of preparative and analytical protein techniques in a way that few competing courses can match. A problem-solving approach will be used throughout the course. Under the guidance of experienced lab instructors, participants will work in groups of three to plan their own protocols, analyze data, and interpret results. A student-teacher ratio not greater than 8:1 will be maintained and the faculty coordinators will be present throughout the course. *Note: The use of GFP from a recombinant source (E. coli) is also being used as starting material due to its popularity within the scientific community. For further details you can reach us, by E-mail at: meton@rci.rutgers.edu by phone at: (908) 932-9071 extension 219 by FAX at: (908) 932-8965 for a brochure and further information please visit the GFP purification short course official Web site at: http://www.rci.rutgers.edu/~meton/protein.html From owner-proteins@net.bio.net Thu Apr 02 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.internetmci.com!192.220.251.22!netnews.nwnet.net!mule.fhcrc.org!news From: Clinical Division Newsgroups: bionet.molbio.proteins Subject: ? Methods for tissue total protein extraction ? Date: Fri, 03 Apr 1998 09:49:43 -0800 Organization: Marley Inc. Lines: 29 Message-ID: <35252137.44D91E77@nwlink.com> NNTP-Posting-Host: 140.107.60.65 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.03 [en] (Win95; I) Hello, I have recently needed to extract total proteins from mouse lung tissue. The proteins are then used to produce westerns for analysis. I am having problems with my protein extracts precipitating and am questioning my extraction protocol and am finding very little in the way of general protocols for total protein extractions from tissue. My current protocol is as follows: - Harvest lung tissue - Freeze in liquid nitrogen - Grind to powder with mortar and pestle - Add lysis buffer (50mM Tris-HCl, 10mM CaCl2, 2M Guanidine-HCl, 0.2% TritionX-100) - Vortex 2 minutes - Spin out particulate , 10k x g, rt, 60 seconds - Dialyze supernatant 48 hours (50mM Tris-HCl, 0.2% TritionX-100):(3,500 MWCO) - Storage -20C My resulting suspension of proteins tends to precipitate... Is there an easier method than this? Is the dialysis necessary? Would the 10mM CaCl2, 2M Guanidine-HCl interfere with western running? Any suggestions of alternate methods, or sources of alternate methods, would be greatly appreciated. Thanx From owner-proteins@net.bio.net Thu Apr 02 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!howland.erols.net!torn!nott!news.crc.ca!srv1.drenet.dnd.ca!newshost.dres.dnd.ca!cwbinc.dres.dnd.ca!user From: tdlaing@nospam.mailhost.dres.dnd.ca (T.D. Laing) Newsgroups: bionet.molbio.proteins Subject: Re: RNA/protein isolation Date: 3 Apr 1998 15:23:41 GMT Organization: Canada West Biosciences Lines: 35 Message-ID: References: <3524DC12.B7770D7C@rockefeller.univ-lyon1.fr> NNTP-Posting-Host: cwbinc.dres.dnd.ca In article <3524DC12.B7770D7C@rockefeller.univ-lyon1.fr>, jcroux@rockefeller.univ-lyon1.fr wrote: > --------------77DB5B7476DEA2FA52A679D5 > Content-Type: text/plain; charset=us-ascii > Content-Transfer-Encoding: 7bit > > Dear all. > I would like to find articles about simultaneous isolation of RNA > and proteins. > My protocol of RNA extraction is guanidium/phenol /chloroforme and > ineed to quantify RNA and protein from the same samples. > Is it possible? > Thank you very much You can use Trizol reagent (GIBCO/BRL) to isolate RNA, DNA and proteins from the same sample. Isolation by TRIzol is a guanidinium/phenol/chloroform extraction method. But it depends on whether you need active protein, or if you can use denatured protein for your studies. Good luck! Terrina -- T.D. Laing Canada West Biosciences c/o Defence Research Establishment Suffield PO Box 4000 Medicine Hat AB T1A 8K6 Canada tdlaing@mailhost.dres.dnd.ca Remove "nospam" from my e-mail address to reply. My opinions do not reflect those of my employers. From owner-proteins@net.bio.net Thu Apr 02 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!chicago-news-feed2.bbnplanet.com!news.bbnplanet.com!news.itis.com!news.doit.wisc.edu!Home From: klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: An interesting puzzle - Pierce's "Gentle Ag/Ab elution buffer"? Date: Sat, 04 Apr 1998 03:26:41 GMT Organization: UW-Madison Lines: 27 Message-ID: <6g499h$1ek_002@doit.wisc.edu> NNTP-Posting-Host: f183-060.net.wisc.edu X-Newsreader: News Xpress 2.01 X-No-Archive: Yes Xref: biosci bionet.molbio.methds-reagnts:66333 bionet.molbio.proteins:12603 I am trying to understand what that could be... Background: Pierce claims that this "magic" elution buffer is in every respect better than conventional low pH elution, giving better recoveries and much better preservation of antibody activity. This does not appear to be a pure marketing lie - another company, Sterogene, sells "Actistep" elution buffer and claims practically the same. Sterogene specializes in industrial applications so I tend to believe there is some truth to the claims. What is known: efficiently disrupts antigen/antibody interactions, pH is 6.5, is incompatible with phosphate, is not chaotropic agent, is dialyzable out (the latter told by Pierce tech support, so is not nesessarily correct :-)) . I am pulling my hairs out trying to understand what that could be... My best guess is some mild ionic detergent like CHAPSO and the likes but incompatibility with phosphate stumbles me... Your ideas, please. TIA, Dima From owner-proteins@net.bio.net Thu Apr 02 23:00:00 1998 Path: biosci!agate!newsfeed.kornet.nm.kr!howland.erols.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!worldnet.att.net!news.u.washington.edu!not-for-mail From: Uncle S Newsgroups: bionet.molbio.proteins Subject: Re: non-denaturing acrylamide gel electrophoresis Date: Fri, 03 Apr 1998 16:57:50 -0800 Organization: Interzon.com Lines: 35 Message-ID: <3525858E.41C6@interzon.com> References: <3524DF5A.FFB2F918@ualg.pt> NNTP-Posting-Host: frank.bmsc.washington.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: nntp4.u.washington.edu 891651471 10574 (None) 140.142.64.5 X-Complaints-To: help@cac.washington.edu X-Mailer: Mozilla 3.01SC-SGI (X11; I; IRIX 5.3 IP12) Cecilia Santos wrote: > > I would like to get some information on non-denaturing gel > electrophoresis such as running buffers and gel composition nondenaturing electrophoresis is mentioned briefly in: Laue-T-M and Rhodes-D-G (1990) Methods in Enzymology 182: 566-587, specifically pp 582-583. within that, there is further reference to Rodbard-D and Chrambach-A (1971) Anal Biochem. 40, 95 and Andrews-A-T (1986) _Eectrophoresis: Theory, Techniques, and Biochemical and Clinical Applications._ Clarendon Press, Oxford. Here's the short answer: You've got to figure out the pI of your protein and alter the pH of your buffers so that the protein is charged. expasy will calculate the pI if you know the sequence. http://expasy.hcuge.ch/ From there, you have to empirically determine the optimal concentration of acrylamide and salts for good separation. I just started with the standard lamelli system minus the SDS (which worked for my calculated pI) and tweaked it from there. Shamus -- The difference between love and hate is: hate lasts. -Bukowski From owner-proteins@net.bio.net Thu Apr 02 23:00:00 1998 Path: biosci!rutgers!rockyd.rockefeller.edu!news-pen-14.sprintlink.net!206.229.87.26!news-east.sprintlink.net!news-peer.sprintlink.net!news-peer-west.sprintlink.net!news.sprintlink.net!Sprint!newsfeed.wli.net!pln-w!extra.newsguy.com!lotsanews.com!ix.netcom.com!tor-nx1.netcom.ca!news.uunet.ca!not-for-mail From: "Victor Graziano" Newsgroups: bionet.molbio.proteins Subject: Free Protein Manager Demo Software Date: 3 Apr 1998 19:38:46 GMT Organization: Advanced Chemistry Design Lines: 40 Message-ID: <01bd5f38$2fee5e00$1ee9b0cf@T95ie.acdlabs.com> NNTP-Posting-Host: 207.176.233.30 X-Newsreader: Microsoft Internet News 4.70.1162 Advanced Chemistry Development Inc. (ACD), a leader in Cheminformatics software, has just released Protein Manager, the newest member of its extensive software family. This remarkable software package, coupled with the Swissprot, Prosite, PDB, ACD Restriction Enzyme and (soon to follow) ACD Regulatory Protein Databases, will dramatically accelerate peptide drug discovery. The primary functions of Protein Manager are protein analysis, data management and data publishing. Software operation is easy, simply load candidate proteins into the Protein Manager 'protein workbook' and perform a multitude of functions including: -Isoelectric point determination -Protein secondary structure prediction ( including Predator prediction method) -Multiple protein alignment with consensus matching -Enzymatic digestion of selected proteins (ACD enzyme database) -Protein scale determination (hydrophobicity, polarity, bulkiness...) -3D viewing of all available Protein Database (PDB) files Furthermore, proteins may be generated at random in Protein Manager, or modified from existing protein files. It's as easy as cut, copy and pasting. Please feel free to visit our web site and download a demo copy or product video preview, both available March 31st. Should you have any questions regarding Protein Manager, please feel free to contact one of our sales representatives. ACD thanks you for your time and hopes you enjoy Protein Manager. _______________________________________ Victor Graziano B.Sc., M.Sc. Advanced Chemistry Development, Inc. Marketing & Sales Manager (Biochemistry) P: (416) 368-3435 F: (416) 368-5596 US & Canada: (800) 304-3988 http://www.acdlabs.com From owner-proteins@net.bio.net Thu Apr 02 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.wli.net!news-peer-west.sprintlink.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!EU.net!Portugal.EU.net!news.rccn.net!ualg.pt!not-for-mail From: Cecilia Santos Newsgroups: bionet.molbio.proteins Subject: non-denaturing acrylamide gel electrophoresis Date: Fri, 03 Apr 1998 14:08:42 +0100 Organization: Universidade do Algarve Lines: 3 Message-ID: <3524DF5A.FFB2F918@ualg.pt> NNTP-Posting-Host: 10.10.40.53 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.04 [en] (Win95; I) I would like to get some information on non-denaturing gel electrophoresis such as running buffers and gel composition From owner-proteins@net.bio.net Thu Apr 02 23:00:00 1998 Newsgroups: bionet.molbio.proteins Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.internetmci.com!128.174.5.49!vixen.cso.uiuc.edu!uchinews!midway.uchicago.edu!aekentsi From: aekentsi@midway.uchicago.edu (alex kentsis) Subject: Re: Peptide-fluorinated solvent interactions? 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: Fri, 3 Apr 1998 13:54:17 GMT Lines: 20 if you are talking about fluoroalkanes, which i doubt, the rationale would be the highly hydrophobic nature of the fluorine atom. being relatively large, its charge in the outer shell is delocalized, giving it very apolar character, and enabling it solubilize otherwise water-insoluble peptides. however, the hydrocarbonds you may be referring to are trifluoroethanol and/or hexafluoroisopropanol. in this case, these alcohols structure water and destabilize polypeptide backbone exposure in such a way as to induce formation of helical (and sometimes beta-sheet) structures. check out biochemistry two months from now for an article on the topic. Tomas Bystrom (tby@randbaek.chem.umu.se) wrote: : I wonder if it is studied how and why fluorinated hydrocarbons is so common : in dissolving peptides? Is there any specific interactions between the : fluorine atoms and the peptide? : Any references would be very welcome! : /Tomas Bystrom From owner-proteins@net.bio.net Fri Apr 03 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.internetmci.com!193.174.75.126!news-was.dfn.de!news-kar1.dfn.de!newsfeed.nacamar.de!uni-erlangen.de!cs.tu-berlin.de!news.uni-hamburg.de!not-for-mail From: behrends@plexus.uke.uni-hamburg.de (Soenke Behrends) Newsgroups: bionet.molbio.proteins Subject: species homologue? Date: Sat, 04 Apr 1998 17:19:09 GMT Organization: University of Hamburg -- Germany Lines: 18 Message-ID: <35266b6b.18061343@news.uni-hamburg.de> NNTP-Posting-Host: freud.uke.uni-hamburg.de X-Newsreader: Forte Free Agent 1.11/32.235 Dear netters, I have a new amino acid sequence and I am not sure whether it is the a species homologue (as I currently think) or maybe a new isoform (as other people tell me). Amino acid similarity as calculated by the MegAlign DNAstar program is 67% between human and rat. If you have a good citation for that kind of question I would be most grateful. Thanks Soenke Dr. Soenke Behrends Hamburg Germany From owner-proteins@net.bio.net Fri Apr 03 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!cam-news-hub1.bbnplanet.com!cam-news-feed2.bbnplanet.com!news.bbnplanet.com!bigboote.WPI.EDU!garden.WPI.EDU!mary From: Mary Devlin Newsgroups: bionet.molbio.proteins Subject: REQ: Protein transmembrane segment modeling Date: Sun, 5 Apr 1998 01:15:52 -0500 Organization: Worcester Polytechnic Institute Lines: 27 Message-ID: NNTP-Posting-Host: garden.wpi.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII I am an undergraduate at Worcester Polytechnic Institute researching the Na,K-ATPase. I am interested in the cation coordinating ability of oxy-containing amino acids in the inner, hydrophillic region of the transmebrane segments. I have done some point mutation/kenetics studies of the enzyme, but I am interested in molecular modeling and plan to complete my senior research project with a 3-d hypothetical model of the TMs I am studying. We have some crosslinking data, and I am looking for some sort of program for a PC or UNIX platform that could help me in positioning/visualizing the TM fragments. I have been using PCMODEL in my molecular modeling class, but am I would like something more specifically geared towards protein chemistry. Thank you, Mary Devlin --- Mary A. Devlin, Biochemistry '98 | Protein Chemistry, Nanotechnology, email: mary@wpi.edu | Skeptical Chemist, ADPlab Manager. url: http://www.wpi.edu/~mary | MasterPlan Control Center[tm] tel: GH06 x5731, ADPlab x5788 | We have the technology to rebuild you. From owner-proteins@net.bio.net Sat Apr 04 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!feeder.qis.net!news.umbc.edu!umbc7.umbc.edu!zhowar1 From: zaxxon Newsgroups: bionet.molbio.proteins Subject: Too high Tris-HCl in stacker SDS-PAGE a problem? Date: Sun, 5 Apr 1998 15:33:56 -0400 Organization: University of Maryland, Baltimore County Lines: 16 Message-ID: NNTP-Posting-Host: umbc7.umbc.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII Okay - here's my next question. Upon reviewing all of my solution-making notes, I found that I've been making up the stock solution for my stacker gel (for SDS-PAGE) with a higher than intended Tris-Cl concentration. (I missed this before) It should've been about 125 mM but I think it's actually ~150 mM. As I understand it, I understand the zone between the chloride and glycine ions 'sweeps' the polypeptides onto the resolving gel. Could the higher Tris-Cl concentration affect this? It wouldn't account for sample-to-sample differences - but it might account for the overall loss of protein I've been experiencing. My resolving gel appears to be made up correctly (with .375 Tris-Cl at pH 8.8). Thanks, Zach From owner-proteins@net.bio.net Sun Apr 05 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!chicago-news-feed2.bbnplanet.com!news.bbnplanet.com!uwm.edu!lll-winken.llnl.gov!fnnews.fnal.gov!nntp-server.caltech.edu!news From: Zeev Pancer Newsgroups: bionet.molbio.proteins,bionet.software,bionet.molbio.methds-reagnts Subject: Proteolytic map, freeware or web site? Date: Mon, 06 Apr 1998 19:01:06 -0700 Organization: Caltech Lines: 18 Message-ID: <352988DE.AD778EA7@cco.caltech.edu> Reply-To: zpancer@cco.caltech.edu NNTP-Posting-Host: zpancer-ppp.caltech.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.04 [en] (Win95; U) Xref: biosci bionet.molbio.proteins:12608 bionet.software:20851 bionet.molbio.methds-reagnts:66385 Hi, Can anyone refer me to a freeeware or web site that can search a protein sequence for clevage-sites of proteolytic enzymes? Thanks in advance. -- Zeev Pancer, Ph.D. Division of Biology 156-29 California Institute of Technology 1200 E. California Blvd., Pasadena, CA 91125, USA Tel: (626) 395-4940 Fax: (626) 583-8351 http://www.cco.caltech.edu/~zpancer/zev-mirsky-info.html From owner-proteins@net.bio.net Sun Apr 05 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.direct.ca!Supernews73!Supernews60!supernews.com!nntp.primenet.com!newsxfer3.itd.umich.edu!news.eecs.umich.edu!news.bu.edu!not-for-mail From: Ali Malay Newsgroups: bionet.molbio.proteins Subject: protein aggregation Date: Sun, 05 Apr 1998 02:02:02 -0500 Organization: BU Lines: 7 Message-ID: <35272C68.D87C2966@acs4.bu.edu> Reply-To: malay@acs4.bu.edu NNTP-Posting-Host: bio-mac2.bu.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 4.04 (Macintosh; I; 68K) Hello everyone, I am looking for protocols that will let me quantitate protein aggregation/precipitation in vitro. Your suggestions are appreciated! Ali From owner-proteins@net.bio.net Sun Apr 05 23:00:00 1998 Path: biosci!newshost.lanl.gov!awabi.library.ucla.edu!208.134.241.18!newsfeed.internetmci.com!204.59.152.222!news-peer.gip.net!news.gsl.net!gip.net!newsfeed.nacamar.de!chico.franken.de!news-nue1.dfn.de!news-lei1.dfn.de!news-ber1.dfn.de!news-ham1.dfn.de!news-han1.dfn.de!news.gwdg.de!not-for-mail From: Silke Beismann Newsgroups: bionet.molbio.proteins Subject: Imidazole glycerol phoshate Date: Tue, 07 Apr 1998 08:16:37 +0200 Organization: GWDG, Goettingen Lines: 5 Message-ID: <3529C4C5.448B@Uni-MolGen.gwdg.de> NNTP-Posting-Host: 134.76.70.64 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (WinNT; I) Hi, does anyone know where I can get imidazole glycerol phosphate? I need this substance for investigating an enzymatic reaction but I could not find a provider. Do I really have to produce IGP myself? Silke From owner-proteins@net.bio.net Sun Apr 05 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.direct.ca!news.uoregon.edu!news.ycc.yale.edu!not-for-mail From: "Miguel A. Talavera" Newsgroups: bionet.molbio.proteins Subject: Telomerase structure??? Date: Mon, 06 Apr 1998 23:53:41 -0400 Organization: Yale University Lines: 7 Message-ID: <3529A345.4AFA9CA3@yale.edu> NNTP-Posting-Host: net186-111.its.yale.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.04 [en] (Win95; I) Is the crystal structure of telomerase known? If anyone knows of either an article that has reported telomerase crystal structure or research groups working towards this structure , please email the bibliographic information to me. Thanks in advance. Miguel A. Talavera From owner-proteins@net.bio.net Mon Apr 06 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news-peer.sprintlink.net!news.sprintlink.net!Sprint!worldnet.att.net!news.u.washington.edu!not-for-mail From: Uncle S Newsgroups: bionet.molbio.proteins,bionet.software,bionet.molbio.methds-reagnts Subject: Re: Proteolytic map, freeware or web site? Date: Tue, 07 Apr 1998 18:20:31 -0700 Organization: Interzon.com Lines: 34 Message-ID: <352AD0DF.41C6@interzon.com> References: <352988DE.AD778EA7@cco.caltech.edu> <3529F689.CE150AF0@alderley.zeneca.com> NNTP-Posting-Host: clara.bmsc.washington.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: nntp4.u.washington.edu 891998432 38324 (None) 140.142.64.5 X-Complaints-To: help@cac.washington.edu X-Mailer: Mozilla 3.0Gold (X11; U; IRIX 5.3 IP22) Xref: biosci bionet.molbio.proteins:12618 bionet.software:20856 bionet.molbio.methds-reagnts:66406 > Zeev Pancer wrote: > > Can anyone refer me to a freeeware or web site that can search a protein > > sequence for clevage-sites of proteolytic enzymes? Mathew Woodwark wrote: > You might like to try ms-digest in proteinprospector. It is part of a > package that using mass spec peptide mass data t identify proteins and one > of the programs allows you to paste in a sequence and have it cut by a > number of proteolytic enzymes > > The address is > > http://prospector.ucsf.edu/ An excellent suggestion. If you're going to do a lot of digestion simulations, you might not want to do it all over the net. PAWS is a really handy mass spec program that you can put on a Wintel or Mac machine. Prospector is nice for chopping up proteins that you pull out of databases, but I do most of my mass spec work with PAWS. here's the site http://www.proteometrics.com/ be warned, it runs on java, so turn that language on in your browser. better yet, use one of the alternate pages: http://www.proteometrics.com/version2.html http://www.proteometrics.com/version1.html (bare bones) Shamus From owner-proteins@net.bio.net Mon Apr 06 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.internetmci.com!193.174.75.126!news-was.dfn.de!news-kar1.dfn.de!newsfeed.nacamar.de!univ-lyon1.fr!not-for-mail From: Jean-Christophe Roux Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: RNA/Protein isolation Date: Tue, 07 Apr 1998 19:18:55 +0200 Organization: UCBL Lines: 32 Message-ID: <352A5FFE.9D4B4EBB@rockefeller.univ-lyon1.fr> Reply-To: jcroux@rockefeller.univ-lyon1.fr NNTP-Posting-Host: rockumr5578-4.univ-lyon1.fr Mime-Version: 1.0 Content-Type: multipart/alternative; boundary="------------AE8B4783780AC384DF8D4414" X-Mailer: Mozilla 4.01 [en] (Win95; I) X-Priority: 3 (Normal) Xref: biosci bionet.molbio.methds-reagnts:66401 bionet.molbio.proteins:12614 --------------AE8B4783780AC384DF8D4414 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit Dear all. I would like to find scientific publications (references) about simultaneous isolation of RNA and proteins. My protocol of RNA extraction is guanidium/phenol /chloroforme and i need to quantify RNA and protein from the same samples. Thank you very much. --------------AE8B4783780AC384DF8D4414 Content-Type: text/html; charset=us-ascii Content-Transfer-Encoding: 7bit Dear all.
I would like to find  scientific publications (references) about simultaneous isolation of RNA and proteins.
My protocol of RNA extraction  is guanidium/phenol /chloroforme and i need to quantify RNA and protein from the same samples.

Thank you very much.
 
  --------------AE8B4783780AC384DF8D4414-- From owner-proteins@net.bio.net Mon Apr 06 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news-peer.sprintlink.net!news.sprintlink.net!Sprint!worldnet.att.net!news.u.washington.edu!not-for-mail From: Uncle S Newsgroups: bionet.molbio.proteins,bionet.software,bionet.molbio.methds-reagnts Subject: Re: Proteolytic map, freeware or web site? Date: Tue, 07 Apr 1998 22:17:16 -0700 Organization: Interzon.com Lines: 27 Message-ID: <352B085C.41C6@interzon.com> References: <352988DE.AD778EA7@cco.caltech.edu> <3529F689.CE150AF0@alderley.zeneca.com> <352AD0DF.41C6@interzon.com> NNTP-Posting-Host: mozart.bmsc.washington.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: nntp4.u.washington.edu 892012637 24458 (None) 140.142.64.5 X-Complaints-To: help@cac.washington.edu X-Mailer: Mozilla 3.0Gold (X11; U; IRIX 5.3 IP12) Xref: biosci bionet.molbio.proteins:12619 bionet.software:20857 bionet.molbio.methds-reagnts:66411 Uncle S wrote: > > here's the site > http://www.proteometrics.com/ > > be warned, it runs on java, so turn that language on in your browser. Just to clarify, the default page at the proteometrics site is a java based menu system, PAWS isn't. [Well, they offer a netscape plug in version of PAWS that may well be, but that wasn't what I meant] > http://www.proteometrics.com/version2.html This one has no java, but does contain a table. > http://www.proteometrics.com/version1.html > (bare bones) This one is for old versions of lynx or mosaic. It rather reminds me of my homepage in its "15 minutes with HTML" simplicity. Shamus -- The difference between love and hate is: hate lasts. -Bukowski From owner-proteins@net.bio.net Mon Apr 06 23:00:00 1998 From: Cornelius Krasel Subject: Re: Imidazole glycerol phoshate Newsgroups: bionet.molbio.proteins References: <3529C4C5.448B@Uni-MolGen.gwdg.de> X-Newsreader: TIN [UNIX 1.3 unoff BETA 970930; i486 Linux 2.0.32] Date: Tue, 7 Apr 1998 19:06:48 +0200 Message-ID: <9fmdg6.jco.ln@wpxx02.toxi.uni-wuerzburg.de> NNTP-Posting-Host: wpxx02.toxi.uni-wuerzburg.de Lines: 36 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news-peer.gip.net!news.gsl.net!gip.net!newsfeed.nacamar.de!uni-erlangen.de!uni-wuerzburg.de!wpxx02.toxi.uni-wuerzburg.de!krasel Silke Beismann wrote: > does anyone know where I can get imidazole glycerol phosphate? I need > this substance for investigating an enzymatic reaction but I could not > find a provider. Do I really have to produce IGP myself? Altavista says: http://www.trc-canada.com/listofch.html D-erythro-IMIDAZOLEGLYCEROL PHOSPHATE MONOHYDRATE [IGP] Catalogue #: i35000 Molecular Weight: 256.15 Molecular Formula: C6H11N2O6P • H2O Comments: Saccharomyces ceresisiac cells, inhibited by aminotriazole, accumulate IGP, the natural intermediate in the biosynthesis of histidine in bacteria and fungi. Ames, B.N., et al: J. Gen. Microbiol., 22, 369 (1960), Ames, B.N., et al: J. of Biol. Chem., 236, 7, 2019 (1961) Structure: [INLINE] Please indicate how many units you would like to order: (All prices are in Canadian dollars) 1_ x10mg @$75.00 The company to order from is Toronto Research Chemicals, Inc., located (surprise!) in Canada. Hope that helps, --Cornelius. -- /* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */ /* D-97078 Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de SP4 */ /* "Science is the game we play with God to find out what His rules are." */ From owner-proteins@net.bio.net Mon Apr 06 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.wli.net!Supernews73!Supernews60!supernews.com!newshub1.home.com!news.home.com!news.rdc1.ct.home.com!not-for-mail From: Menoret Newsgroups: bionet.molbio.proteins Subject: immunity and hsp Date: Tue, 07 Apr 1998 21:35:54 -0700 Organization: @Home Network Lines: 6 Message-ID: <352AFEAA.A0EEFFAD@home.com> Reply-To: menoret@home.com NNTP-Posting-Host: c1003124-a.blfld1.ct.home.com Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.02 [en]C-AtHome0402 (Win95; U) Please note the International Congress on "Heat shock proteins in Immune Response "is to be held in Farmington (CT) USA, October 12-15, 1998. For information and registration details: www.hspimmunity.com Hope to see you there. From owner-proteins@net.bio.net Mon Apr 06 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!cam-news-hub1.bbnplanet.com!news.bbnplanet.com!feed1.news.luth.se!luth.se!news.kth.se!not-for-mail From: Arne Elofsson Newsgroups: bionet.molbio.proteins Subject: Re: transmembrane domains Date: 07 Apr 1998 16:36:34 +0200 Lines: 49 Message-ID: References: <6fhfl6$t07@gazette.bcm.tmc.edu> <3521E65B.62E7@neurochem.u-strasbg.fr> NNTP-Posting-Host: elof.biokemi.su.se Mime-Version: 1.0 (generated by tm-edit 7.105) Content-Type: multipart/mixed; boundary="Multipart_Tue_Apr__7_16:36:34_1998-1" Content-Transfer-Encoding: 7bit X-Newsreader: Gnus v5.4.37/XEmacs 19.15 --Multipart_Tue_Apr__7_16:36:34_1998-1 Content-Type: text/plain; charset=ISO-8859-1 Content-Transfer-Encoding: quoted-printable Pierre Hubert writes: > Simon Hoffenberg wrote: > > If there is more than 1 transmembrane domain in a protein, what are the > > combinations? For example, 7 domains in receptors coupled to G-proteins= or > > 12 in adenylate cyclase. Is it possible to have 2 or 8-9 domains? If th= ese > > numbers are not allowed by Mother Nature, does it mean that these prote= ins > > are in fact 1 and 7 transmembrane domain proteins, respectively? > > > > =09Have a look at Arkin, I. T., A. T. Brunger, & Engelman D.M. (1997). > =93Are there dominant membrane protein families with a given number of > helices?=94 Protein Struct Funct Genet 28(4): 465-466. > =09They have scanned four complete genome sequences for putative > transmembrane helices. > It looks like anything goes between 1 and 19 TM domains, with no obvious > domination of a particular family of membrane proteins with a given > number of helices. There study is not very careful. If you look more carefull into it, you find that there is a increased tendancy for 6 and 12 TM regions in multicellular organisms and 7 in unicellular organisms. But anything between 1 and ~14 (for helical proteins) exists. (Studies by Taylor & Jones (FEBS?) and Wallin & von Heijne (in press in prot sci) arne --Multipart_Tue_Apr__7_16:36:34_1998-1 Content-Type: text/plain; charset=US-ASCII --------------------------------------------------------------------------- The more I use Windows, the more I love Linux --------------------------------------------------------------------------- Arne Elofsson arne@biokemi.su.se http://www.biokemi.su.se/~arne/ Tel:+46(0)8-161553 Dept of Biochemistry, Stockholm University Fax:+46(0)8-153679 10691 Stockholm, Sweden --Multipart_Tue_Apr__7_16:36:34_1998-1-- From owner-proteins@net.bio.net Mon Apr 06 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newshub.northeast.verio.net!howland.erols.net!newsfeed.internetmci.com!204.59.152.222!news-peer.gip.net!news-lond.gip.net!news.gsl.net!gip.net!nntp.news.xara.net!xara.net!rill.news.pipex.net!pipex!bore.news.pipex.net!pipex!join.news.pipex.net!pipex!newshost.zeneca.co.uk!usenet From: Mathew Woodwark Newsgroups: bionet.molbio.proteins,bionet.software,bionet.molbio.methds-reagnts Subject: Re: Proteolytic map, freeware or web site? Date: Tue, 07 Apr 1998 10:48:57 +0100 Organization: Zeneca Lines: 42 Message-ID: <3529F689.CE150AF0@alderley.zeneca.com> References: <352988DE.AD778EA7@cco.caltech.edu> NNTP-Posting-Host: 192.168.3.214 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.04 [en] (Win95; I) Xref: biosci bionet.molbio.proteins:12611 bionet.software:20853 bionet.molbio.methds-reagnts:66389 Dear Zeev, You might like to try ms-digest in proteinprospector. It is part of a package that using mass spec peptide mass data t identify proteins and one of the programs allows you to paste in a sequence and have it cut by a number of proteolytic enzymes The address is http://prospector.ucsf.edu/ Might be worth a try... Cheers Mathew Woodwark Zeneca Usual disclaimers apply Zeev Pancer wrote: > Hi, > > Can anyone refer me to a freeeware or web site that can search a protein > sequence for clevage-sites of proteolytic enzymes? > > Thanks in advance. > > -- > Zeev Pancer, Ph.D. > Division of Biology 156-29 > California Institute of Technology > 1200 E. California Blvd., > Pasadena, CA 91125, USA > Tel: (626) 395-4940 > Fax: (626) 583-8351 > http://www.cco.caltech.edu/~zpancer/zev-mirsky-info.html From owner-proteins@net.bio.net Mon Apr 06 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.wli.net!ais.net!usenet.INS.CWRU.Edu!djt2 From: djt2@po.cwru.edu (Dennis Templeton) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: An interesting puzzle - Pierce's "Gentle Ag/Ab elution buffer"? Date: Tue, 07 Apr 1998 18:23:12 -0400 Organization: Case Western Reserve University Lines: 41 Message-ID: References: <6g499h$1ek_002@doit.wisc.edu> NNTP-Posting-Host: templeton.path.cwru.edu X-Newsreader: MT-NewsWatcher 2.4.4 Xref: biosci bionet.molbio.methds-reagnts:66404 bionet.molbio.proteins:12616 In article <6g499h$1ek_002@doit.wisc.edu>, klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) wrote: > I am trying to understand what that could be... > > Background: > > Pierce claims that this "magic" elution buffer is in every respect better than > conventional low pH elution, giving better recoveries and much better > preservation of antibody activity. This does not appear > to be a pure marketing lie - another company, Sterogene, sells > "Actistep" elution buffer and claims practically the same. Sterogene > specializes in industrial applications so I tend to believe there is some > truth to the claims. > > What is known: > > efficiently disrupts antigen/antibody interactions, pH is 6.5, is incompatible > with phosphate, is not chaotropic agent, is dialyzable out (the latter told by > Pierce tech support, so is not nesessarily correct :-)) . > > I am pulling my hairs out trying to understand what that could be... My best > guess is some mild ionic detergent like CHAPSO and the likes but > incompatibility with phosphate stumbles me... > > Your ideas, please. TIA, > > Dima Harlow and Lane recommend using organic reagents like Ethylene glycol or dioxane. They cite some refs, but I haven't tried this. =================================||================================== Dennis J Templeton, M.D., Ph.D. || Associate Professor || Department of Pathology Internet djt2@po.cwru.edu || 10900 Euclid Ave WWW http://templeton.cwru.edu || Cleveland, Ohio 44106 From owner-proteins@net.bio.net Tue Apr 07 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news-peer.gip.net!news-raspail.gip.net!news.gsl.net!gip.net!oleane!jussieu.fr!saphir.jouy.inra.fr!alzon.ensam.inra.fr!user From: Blanc@ensam.inra.fr (Stéphane Blanc) Newsgroups: bionet.molbio.proteins Subject: post-doc offer Date: 8 Apr 1998 08:32:47 GMT Organization: INRA-CNRS Lines: 54 Message-ID: NNTP-Posting-Host: alzon.ensam.inra.fr POST-DOCTORAL POSITION A post-doctoral position, starting between september and december 1998, is available with Stéphane Blanc at the Station de Recherches de Pathologie Comparée, Saint-Christol-lez-Alès, FRANCE. The Lab is currently working on the elucidation of the molecular mechanims of cauliflower mosaic virus (CaMV) aphid transmission. Several projects are in progress such as: (i) study of the interaction between the aphid transmission factor and cellular microtubules (Proc. Natl. Acad. Sci. USA, 93, 15158-15163); Possible applications of this property in plant pathology and beyond. (ii) Characterization of an additionnal (viral) factor involved in aphid transmission. Indeed virus particles and aphid transmssion factor are not sufficient for aphid transmission to occur. An additional viral gene product is now identified as a co-factor regulating the interactions between virus and vector. This is so far a unique phenomenon among viruses transmitted in a non-circulative, helper-dependent manner. etc....... The post-doc will be expected to develope a project related to either (i) or (ii) in agreement with Stéphane Blanc. Post-doctoral position is for 1 year with possible extension for a second year and the salary will be around 10 000FF a month, after tax. CV and applications are to be ready by june 1998 and the candidate will be selected during july or august. FOR FURTHER INFORMATIONS PLEASE CONTACT AS SOON AS POSSIBLE (E. Mail preferred) Stephane BLANC Station de Recherches de Pathologie Comparee INRA-CNRS 30 380 Saint Christol-lez-Ales FRANCE phone +33 04 66 78 37 15 Fax +33 04 66 52 46 99 E. Mail Blanc@ensam.inra.fr From owner-proteins@net.bio.net Tue Apr 07 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!nntp2.dejanews.com!nnrp1.dejanews.com!not-for-mail From: theodorn@medlib.georgetown.edu Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: An interesting puzzle - Pierce's "Gentle Ag/Ab elution buffer"? Date: Wed, 08 Apr 1998 08:43:13 -0600 Organization: Deja News - The Leader in Internet Discussion Lines: 58 Message-ID: <6gfuth$ts2$1@nnrp1.dejanews.com> References: <6g499h$1ek_002@doit.wisc.edu> NNTP-Posting-Host: 141.161.14.60 X-Article-Creation-Date: Wed Apr 08 13:43:13 1998 GMT X-Http-User-Agent: Mozilla/3.01-C-MACOS8 (Macintosh; I; PPC) Xref: biosci bionet.molbio.methds-reagnts:66417 bionet.molbio.proteins:12622 In article , djt2@po.cwru.edu (Dennis Templeton) wrote: > > In article <6g499h$1ek_002@doit.wisc.edu>, > klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) wrote: > > > I am trying to understand what that could be... > > > > Background: > > > > Pierce claims that this "magic" elution buffer is in every respect > better than > > conventional low pH elution, giving better recoveries and much better > > preservation of antibody activity. This does not appear > > to be a pure marketing lie - another company, Sterogene, sells > > "Actistep" elution buffer and claims practically the same. Sterogene > > specializes in industrial applications so I tend to believe there is some > > truth to the claims. > > > > What is known: > > > > efficiently disrupts antigen/antibody interactions, pH is 6.5, is > incompatible > > with phosphate, is not chaotropic agent, is dialyzable out (the latter > told by > > Pierce tech support, so is not nesessarily correct :-)) . > > > > I am pulling my hairs out trying to understand what that could be... My best > > guess is some mild ionic detergent like CHAPSO and the likes but > > incompatibility with phosphate stumbles me... > > > > Your ideas, please. TIA, > > > > Dima > > Harlow and Lane recommend using organic reagents like Ethylene glycol or > dioxane. They cite some refs, but I haven't tried this. > > =================================||================================== > Dennis J Templeton, M.D., Ph.D. || Associate Professor > || Department of Pathology > Internet djt2@po.cwru.edu || 10900 Euclid Ave > WWW http://templeton.cwru.edu || Cleveland, Ohio 44106 > The incompatibilty with phosphate could be due to Mg(++) salts, which are mildly chaotropic at high concentrations. I've also used the Pierce buffer in the past, and at that time I got the impression that there was also ethylene glycol in it, but I can't remember why I thought so at the time. My guess is that the gentle elution buffer is a neutral pH buffer with MgCl(2) and ethylene glycol. Nick Theodorakis Georgetown University Medical Center -----== Posted via Deja News, The Leader in Internet Discussion ==----- http://www.dejanews.com/ Now offering spam-free web-based newsreading From owner-proteins@net.bio.net Tue Apr 07 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.direct.ca!news.he.net!sws1.ctd.ornl.gov!not-for-mail From: Ying Xu Newsgroups: bionet.molbio.proteins Subject: CFP: JCO Special Issue on Computational Biology Date: Wed, 08 Apr 1998 11:41:35 -0400 Organization: Oak Ridge National Laboratory Lines: 94 Message-ID: <352B9AAE.AD5BDF@ornl.gov> NNTP-Posting-Host: aspen.lsd.ornl.gov Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.03 [en] (X11; I; SunOS 5.5.1 sun4m) SECOND CALL FOR PAPERS ====================== Journal of Combinatorial Optimization Special Issue on Computational Molecular Biology Guest Editors: Ying Xu, Satoru Miyano, Tom Head. Submission Deadline: August 15, 1998. The past ten years have witnessed the rapid development of a new discipline, computational molecular biology. Combinatorial optimization and algorithms have played a significant role in advancing this new discipline. The partnership between mathematics, in particular combinatorial optimization and algorithms, and molecular biology has greatly enriched both fields, leading to new ways of thinking and greater challenges to meet. The scope of this Special Issue includes all aspects of combinatorial optimization and algorithms in computational molecular biology. Original papers are solicited that describe research on combinatorial methods for problems arising from the following areas (nonexhaustive) of molecular biology: -- DNA sequencing -- DNA mapping -- recognition of genes and regulatory elements -- RNA/protein structure prediction -- molecular evolution -- combinatorial libraries and drug design -- bio-sequence analysis and comparison -- computing with biomolecules. Six hard copies of each submitted manuscript should be sent to one of the guest editors by August 15, 1998. Manuscripts must be prepared according to the normal submission requirements of the Journal of Combinatorial Optimization (see ). Each manuscript will be refereed by at least three anonymous reviewers. Notification of acceptance will be sent to the authors before November 30, 1998. The Special Issue is expected to be published by mid-1999. Addresses of the guest editors: Ying Xu Computational Biosciences Section Building 1060 COM Oak Ridge National Laboratory Oak Ridge, TN 37831-6480, USA Tel: 423-574-7263 Fax: 423-241-1965 Email: xyn@ornl.gov Web: Satoru Miyano Human Genome Center Institute of Medical Science University of Tokyo Shirokanedai 4-6-1, Minato-ku, Tokyo 108-8639, Japan. Tel.: +81-3-5449-5615 Fax: +81-3-5449-5442 Email: miyano@ims.u-tokyo.ac.jp Tom Head Department of Mathematics Binghamton University Binghamton, NY 13902-6000, USA Tel: 607-777-2278 Email: tom@math.binghamton.edu Web: -- ----------------------------------------------------------------------------- | Ying Xu | Office: 423-574-7263 | | Computational Biosciences Section | Fax: 423-241-1965 | | Life Sciences Division, ORNL | Home: 423-675-3783 | | Bldg 1060 COM, MS 6480 | Email: xyn@ornl.gov | | Oak Ridge, TN 37831-6480, USA | Web: http://compbio.ornl.gov/~xyn/ | ----------------------------------------------------------------------------- From owner-proteins@net.bio.net Tue Apr 07 23:00:00 1998 Path: biosci!acdlabs.com!vic From: vic@acdlabs.com ("Victor Graziano") Newsgroups: bionet.molbio.proteins Subject: Free Protein Manager demo software Date: 8 Apr 1998 12:34:12 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 45 Sender: daemon@net.bio.net Distribution: world Message-ID: <000601bd6325$1d25d3c0$3ee9b0cf@vic.acdlabs.com> NNTP-Posting-Host: net.bio.net Dear protein-analysis@net.bio.net subscribers, Advanced Chemistry Development Inc. (ACD), a leader in Cheminformatics software, has just released Protein Manager, the newest member of its extensive software family. This remarkable software package, coupled wit= h the Swissprot, Prosite, PDB, ACD Restriction Enzyme and (soon to follow) = ACD Regulatory Protein Databases, will dramatically accelerate peptide drug discovery. The primary functions of Protein Manager are protein analysis, data management and data publishing. Software operation is easy, simply load candidate proteins into the Protein Manager 'protein workbook' and perfor= m a multitude of functions including: -Isoelectric point determination -Batch Phys-Chem property predictions -Protein secondary structure prediction ( including Predator prediction method) -Multiple protein alignment with consensus matching -Enzymatic digestion of selected proteins (ACD enzyme database) -Protein scale determination (hydrophobicity, polarity, bulkiness...) -3D viewing of all available Protein Database (PDB) files Furthermore, proteins may be generated at random in Protein Manager, or modified from existing protein files. It's as easy as cut, copy and pasting. Please feel free to visit our web site and download a demo copy= or product video preview, both available March 31st. Should you have any questions regarding Protein Manager, please feel free to contact one of o= ur sales representatives. ACD thanks you for your time and hopes you enjoy Protein Manager. _______________________________________ Victor Graziano B.Sc., M.Sc. Advanced Chemistry Development, Inc. Marketing & Sales Manager (Biochemistry) T:=A0(416) 368-3435 F:=A0(416) 368-5596 US & Canada: (800) 304-3988 http://www.acdlabs.com From owner-proteins@net.bio.net Tue Apr 07 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!netnews.com!howland.erols.net!rill.news.pipex.net!pipex!server1.netnews.ja.net!hgmp.mrc.ac.uk!guardian.dcs.warwick.ac.uk!warwick!leicester!usenet From: "Dr E. Buxbaum" Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: An interesting puzzle - Pierce's "Gentle Ag/Ab elution buffer"? Date: Wed, 08 Apr 1998 11:11:40 -0700 Organization: University of Leicester (PCFS User) Lines: 19 Message-ID: <352BBDDC.64D7@le.ac.uk> References: <6g499h$1ek_002@doit.wisc.edu> NNTP-Posting-Host: pc80.msb.le.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Win16; I) Xref: biosci bionet.molbio.methds-reagnts:66416 bionet.molbio.proteins:12621 Dennis Templeton wrote: > > In article <6g499h$1ek_002@doit.wisc.edu>, > klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) wrote: > > Pierce claims that this "magic" elution buffer is in every respect > better than > > conventional low pH elution, giving better recoveries and much better > > preservation of antibody activity. This does not appear > > to be a pure marketing lie - another company, Sterogene, sells > > "Actistep" elution buffer and claims practically the same. Sterogene > > specializes in industrial applications so I tend to believe there is some > > truth to the claims. Dima > > Harlow and Lane recommend using organic reagents like Ethylene glycol or > dioxane. They cite some refs, but I haven't tried this. Diiodosalicylate is also commonly used for this purpose From owner-proteins@net.bio.net Tue Apr 07 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.internetmci.com!203.12.176.153!news.mel.aone.net.au!newsfeed-in.aone.net.au!news.mel.connect.com.au!munnari.OZ.AU!news.unimelb.edu.au!ludwignt-4 From: murphy_r@licre.ludwig.edu.au (Roger Murphy) Newsgroups: bionet.molbio.proteins Subject: metal chelate chromatography Date: Wed, 08 Apr 98 22:37:28 GMT Organization: Ludwig Institute for Cancer Research Lines: 28 Message-ID: <6gh165$98q$1@izvestia.its.unimelb.edu.au> NNTP-Posting-Host: ludwignt-4.austin.unimelb.edu.au X-Newsreader: News Xpress 2.0 Beta #0 Hi netters! I know that this subject has come up before, but what do people think about the two main products for metal chelate chromatography? I seem to remember a general consensus that the Qiagen product was better than the Pharmacia one (less leaching, higher loading, better affinity, etc), but here in Australia, the Qiagen resins are twice the price of the Pharmacia ones - is it really twice as good?? Confidential opinions can be sent to my email address if you're shy... Thanks in advance, Roger Roger Murphy, Ph.D. Biological Production Facility Ludwig Institute for Cancer Research Austin & Repatriation Medical Centre Studley Road, Heidelberg, Vic. 3084 Australia. Tel 61-3-94965463 Fax 61-3-94965436 Email murphy_r@licre.ludwig.edu.au From owner-proteins@net.bio.net Tue Apr 07 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeeds.sol.net!uwm.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!default From: klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: An interesting puzzle - Pierce's "Gentle Ag/Ab elution buffer"? <-- SOLVED Date: Wed, 08 Apr 1998 17:20:04 GMT Organization: UW-Madison Lines: 44 Message-ID: <6ggbkm$puk$1@news.doit.wisc.edu> References: <6g499h$1ek_002@doit.wisc.edu> NNTP-Posting-Host: martinlab.biochem.wisc.edu Mime-Version: 1.0 Content-Type: text/plain; charset=KOI8-R Content-Transfer-Encoding: 8bit X-Newsreader: News Xpress 2.01 X-No-Archive: Yes Xref: biosci bionet.molbio.methds-reagnts:66430 bionet.molbio.proteins:12625 In article <6g499h$1ek_002@doit.wisc.edu>, klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) wrote: :I am trying to understand what that could be... : : What is known: : :efficiently disrupts antigen/antibody interactions, pH is 6.5, is incompatible :with phosphate, is not chaotropic agent, is dialyzable out (the latter told by :Pierce tech support, so is not nesessarily correct :-)) . : :I am pulling my hairs out trying to understand what that could be... My best :guess is some mild ionic detergent like CHAPSO and the likes but :incompatibility with phosphate stumbles me... Thanks very much to everyone who replied, to the group and by email. Many people suggested it could be magnesium. Harlow & Lane "Antibodies" book lists 3.5 M MgCl2 pH 7.2 as one of the alternatives to acid elution. We ordered that stuff. It does not foam at all - so isn't detergent. It does form precipitate with phopshate, which is dissolved with EDTA but not EGTA, so it is Mg2+. By conductivity versus MgCl2 standards, it is 3.6 M solution. NaJO4 does not oxidize anything in this solution, so I don't think there is ethylene glycol present. Sooo, if anyone wants to make Pierce's magic solution, the recipie is: ~ 20 mM MES or PIPES, 3.6 M MgCl2, titrate pH to ~ 6.5 with NaOH Funny thing is that Pierce recommends to store this solution at 4C. Hehehe, like anything would grow in 3.5 M MgCl2! Or is it MgCl2 unstable? :-) 500 ml for $65 ... from Pierce or [when buying expensive MgCl2 from Sigma] 3.5 l for $104. Hmmm... Dima From owner-proteins@net.bio.net Tue Apr 07 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news-peer.gip.net!news-lond.gip.net!news.gsl.net!gip.net!nntp.news.xara.net!xara.net!server5.netnews.ja.net!server3.netnews.ja.net!server1.netnews.ja.net!bham!med1018.bham.ac.uk!user From: noone@cancer.bham.ac.uk (noone) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: An interesting puzzle - Pierce's "Gentle Ag/Ab elution buffer"? Date: Wed, 08 Apr 1998 17:09:45 +0100 Organization: noone Lines: 21 Message-ID: References: <6g499h$1ek_002@doit.wisc.edu> <6gfuth$ts2$1@nnrp1.dejanews.com> NNTP-Posting-Host: med1018.bham.ac.uk Xref: biosci bionet.molbio.methds-reagnts:66425 bionet.molbio.proteins:12624 > > The incompatibilty with phosphate could be due to Mg(++) salts, which are > mildly chaotropic at high concentrations. I've also used the Pierce buffer in > the past, and at that time I got the impression that there was also ethylene > glycol in it, but I can't remember why I thought so at the time. > > My guess is that the gentle elution buffer is a neutral pH buffer with MgCl(2) > and ethylene glycol. > I'm currently using this buffer (it works fairly well), one thing not mentioned is the fact that if you mix the buffer with a buffer containing DTT or beta-merc., a dark brown precipitate occurs (which makes it impossible to run PAGE directly after elution). I wonder whether this is maybe some metal like manganese, which has some similar properties to Mg but is very easily reduced. BTW, dialysis works fine. Peter From owner-proteins@net.bio.net Wed Apr 08 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!nntp.abs.net!ptdnetP!newsgate.ptd.net!newsfeed.nyu.edu!rockyd.rockefeller.edu!not-for-mail Message-ID: <352D1F8C.446B@rockvax.rockefeller.edu> From: Satish Nair X-Mailer: Mozilla 2.02 (X11; I; IRIX 5.3 IP22) MIME-Version: 1.0 Newsgroups: bionet.molbio.proteins Subject: Re: metal chelate chromatography References: <6gh165$98q$1@izvestia.its.unimelb.edu.au> Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit Lines: 13 Date: Thu, 09 Apr 1998 19:25:23 GMT NNTP-Posting-Host: skbind2.rockefeller.edu NNTP-Posting-Date: Thu, 09 Apr 1998 15:25:23 EST Zophonias O. Jonsson wrote: > > The Qiagen Ni-NTA holds more tightly onto the metal ion, but > usually the amount of protein lost by leaching is small and can be > compensated for by growing ‰10 ml more of culture. > Simpler yet, why not charge one column with the appropriate metal ion, have another column that is uncharged and hook up the 2 in series? The second column (the uncharged "catch column") will bind any ions (along with bound protein) that may leech of the first. From owner-proteins@net.bio.net Wed Apr 08 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!baron.netcom.net.uk!netcom.net.uk!nntp.news.xara.net!xara.net!server5.netnews.ja.net!server3.netnews.ja.net!server1.netnews.ja.net!bham!med1018.bham.ac.uk!user From: noone@cancer.bham.ac.uk (noone) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: An interesting puzzle - Pierce's "Gentle Ag/Ab elution buffer"? <-- SOLVED Date: Thu, 09 Apr 1998 08:56:59 +0100 Organization: noone Lines: 31 Message-ID: References: <6g499h$1ek_002@doit.wisc.edu> <6ggbkm$puk$1@news.doit.wisc.edu> NNTP-Posting-Host: med1018.bham.ac.uk Xref: biosci bionet.molbio.methds-reagnts:66444 bionet.molbio.proteins:12629 > > Thanks very much to everyone who replied, to the group and by email. > Many people suggested it could be magnesium. Harlow & Lane "Antibodies" > book lists 3.5 M MgCl2 pH 7.2 as one of the alternatives to acid > elution. > > We ordered that stuff. It does not foam at all - so isn't detergent. It > does form precipitate with phopshate, which is dissolved with EDTA but > not EGTA, so it is Mg2+. By conductivity versus MgCl2 standards, it is > 3.6 M solution. NaJO4 does not oxidize anything in this solution, so I > don't think there is ethylene glycol present. Sooo, if anyone wants to > make Pierce's magic solution, the recipie is: > > ~ 20 mM MES or PIPES, > 3.6 M MgCl2, > titrate pH to ~ 6.5 with NaOH > > Funny thing is that Pierce recommends to store this solution at 4C. > Hehehe, like anything would grow in 3.5 M MgCl2! > Or is it MgCl2 unstable? :-) > > 500 ml for $65 ... from Pierce > or [when buying expensive MgCl2 from Sigma] > 3.5 l for $104. Hmmm... OK, but what's the brown precipitate I get from this buffer when I add loading buffer (or any other DTT- or b-merc.-containing solution)? Peter From owner-proteins@net.bio.net Wed Apr 08 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.internetmci.com!193.174.75.126!news-was.dfn.de!news-fra1.dfn.de!news-ber1.dfn.de!news-ham1.dfn.de!news.mu-luebeck.de!not-for-mail From: "Lars Komorowski" Newsgroups: bionet.molbio.proteins Subject: Re: metal chelate chromatography Date: Thu, 9 Apr 1998 08:58:36 +0100 Organization: Med. Universitaet zu Luebeck Lines: 27 Message-ID: <6ghrqc$nnf$1@gwsun.medinf.mu-luebeck.de> References: <6gh165$98q$1@izvestia.its.unimelb.edu.au> 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 Roger Murphy schrieb in Nachricht <6gh165$98q$1@izvestia.its.unimelb.edu.au>... >Hi netters! > >I know that this subject has come up before, but what do people think about >the two main products for metal chelate chromatography? I seem to remember a >general consensus that the Qiagen product was better than the Pharmacia one >(less leaching, higher loading, better affinity, etc), but here in Australia, >the Qiagen resins are twice the price of the Pharmacia ones - is it really >twice as good?? > >Confidential opinions can be sent to my email address if you're shy... > >Thanks in advance, > >Roger If you have access to Clontech products try out their Talon resin. Lars From owner-proteins@net.bio.net Wed Apr 08 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.wli.net!news-peer-west.sprintlink.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!newsfeed.internetmci.com!206.103.240.185!newsfeed.infinet.com!usenet.INS.CWRU.Edu!djt2 From: djt2@po.cwru.edu (Dennis Templeton) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: An interesting puzzle - Pierce's "Gentle Ag/Ab elution buffer"? Date: Thu, 09 Apr 1998 09:25:21 -0400 Organization: Case Western Reserve University Lines: 34 Message-ID: References: <6g499h$1ek_002@doit.wisc.edu> <6gfuth$ts2$1@nnrp1.dejanews.com> NNTP-Posting-Host: templeton.path.cwru.edu X-Newsreader: MT-NewsWatcher 2.4.4 Xref: biosci bionet.molbio.methds-reagnts:66455 bionet.molbio.proteins:12633 In article , noone@cancer.bham.ac.uk (noone) wrote: > > > > The incompatibilty with phosphate could be due to Mg(++) salts, which are > > mildly chaotropic at high concentrations. I've also used the Pierce buffer in > > the past, and at that time I got the impression that there was also ethylene > > glycol in it, but I can't remember why I thought so at the time. > > > > My guess is that the gentle elution buffer is a neutral pH buffer with MgCl(2) > > and ethylene glycol. > > > > > I'm currently using this buffer (it works fairly well), one thing not > mentioned is the fact that if you mix the buffer with a buffer containing > DTT or beta-merc., a dark brown precipitate occurs (which makes it > impossible to run PAGE directly after elution). I wonder whether this is > maybe some metal like manganese, which has some similar properties to Mg > but is very easily reduced. > BTW, dialysis works fine. > Nickel makes a brown precipitate with DTT, as anyone who's mistakenly used DTT in a Nickle-his tag column can testify. (you can restore these columns successfully with a wash in 0.1% H2O2 until the color disappears). Dennis =================================||================================== Dennis J Templeton, Internet djt2@po.cwru.edu WWW http://templeton.cwru.edu From owner-proteins@net.bio.net Wed Apr 08 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!news.bbnplanet.com!news1.best.com!newsxfer3.itd.umich.edu!news.eecs.umich.edu!Cabal.CESspool!bofh.vszbr.cz!pegasus.csx.cam.ac.uk!lyra.csx.cam.ac.uk!powercenter-604.physiol.cam.ac.uk!user From: whc23@cus.cam.ac.uk (Dr.W.H. Colledge) Newsgroups: bionet.molbio.proteins,bionet.cellbiol,bionet.molbio.methds-reagnts Subject: Lac Z staining Date: Thu, 09 Apr 1998 13:31:02 +0100 Organization: University of Cambridge Lines: 13 Message-ID: NNTP-Posting-Host: powercenter-604.physiol.cam.ac.uk Xref: biosci bionet.molbio.proteins:12632 bionet.cellbiol:9203 bionet.molbio.methds-reagnts:66452 Does anyone know which mammalian enzyme is responsible for the background blue staining of some mammalian tissues when staining for b-galactosidase activity using X-gal? Thanks in advance. -- Dr. W.H. Colledge Physiology University of Cambridge Cambridge CB2 3EG Tel. 01223-333881 Fax. 01223-333840 From owner-proteins@net.bio.net Wed Apr 08 23:00:00 1998 Date: Thu, 09 Apr 1998 13:58:30 +0200 From: zjons@vetbio.unizh.ch (Zophonias O. Jonsson) Newsgroups: bionet.molbio.proteins Subject: Re: metal chelate chromatography Message-ID: References: <6gh165$98q$1@izvestia.its.unimelb.edu.au> Organization: Universitat Zurich-Irchel NNTP-Posting-Host: 130.60.120.27 Lines: 35 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.internetmci.com!209.95.128.196!news-nyc.telia.net!masternews.telia.net!feed1.news.luth.se!luth.se!news-ge.switch.ch!news-zh.switch.ch!rzunews1.unizh.ch!NewsWatcher!user In article <6gh165$98q$1@izvestia.its.unimelb.edu.au>, murphy_r@licre.ludwig.edu.au (Roger Murphy) wrote: > Hi netters! > > I know that this subject has come up before, but what do people think about > the two main products for metal chelate chromatography? I seem to remember a > general consensus that the Qiagen product was better than the Pharmacia one > (less leaching, higher loading, better affinity, etc), but here in Australia, > the Qiagen resins are twice the price of the Pharmacia ones - is it really > twice as good?? > > Confidential opinions can be sent to my email address if you're shy... Hi Netter! Basically, they all work but if you have an FPLC and a protein that expresses half-way decently I would recomment the Pharmacia Hi-Trap Chelating columns. They can be cleaned and re-used more than 10 times without problems and they are fast. You can also play around with different metals (Co++, Ni++, Cu++ etc.) to optimize the purification of you protein. The Qiagen Ni-NTA holds more tightly onto the metal ion, but usually the amount of protein lost by leaching is small and can be compensated for by growing ‰10 ml more of culture. Zophonias _____________________________________________________________________ Zophonias O. Jonsson Institut fur Veterinarbiochemie Tel: (41-1)-635-54-75 Universitat Zurich-Irchel Fax: (41-1)-635-68-16 Winterthurerstrasse 190 CH-8057 Zurich Switzerland _____________________________________________________________________ From owner-proteins@net.bio.net Wed Apr 08 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!news.bbnplanet.com!logbridge.uoregon.edu!europa.clark.net!207.114.4.11!nntp.abs.net!Cabal.CESspool!bofh.vszbr.cz!serra.unipi.it!news.caspur.it!news.unina.it!not-for-mail From: "Roberto Gualtieri" Newsgroups: bionet.molbio.proteins Subject: CELL SURFACE GLYCOPROTEINS Date: Thu, 9 Apr 1998 10:20:46 +0200 Organization: Centro di Servizi Didattico Scientifico Lines: 15 Message-ID: <6gi07n$j2a$1@news.unina.it> NNTP-Posting-Host: 143.225.252.106 Mime-Version: 1.0 Content-Type: text/plain; charset="iso-8859-1" Content-Transfer-Encoding: 7bit X-Newsreader: Microsoft Outlook Express 4.72.2106.4 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.2106.4 Hello! Does anyone know good basic reviews on cell surface glycoproteins/proteoglycans? I would like to get informations about their mode of association with the cell membrane and the glycocalix, types of saccharide chains, modes of enrichment/isolation for SDS-PAGE and lectin blotting analysis. Sicerely Dr. Roberto Gualtieri E mail: Gualtier@dgbm.unina.it From owner-proteins@net.bio.net Wed Apr 08 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!news.bbnplanet.com!logbridge.uoregon.edu!news.uoregon.edu!not-for-mail From: Ted Michelini Newsgroups: bionet.molbio.proteins Subject: Q: best western membrane? Date: Thu, 09 Apr 1998 21:43:46 -0800 Organization: UO Lines: 16 Message-ID: <352DB18E.9B2C4D05@molbio.uoregon.edu> Reply-To: tedm@molbio.uoregon.edu NNTP-Posting-Host: fp1-chemistry-26.uoregon.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-Trace: pith.uoregon.edu 892186712 10393 (None) 128.223.22.246 X-Complaints-To: usenet@news.uoregon.edu X-Mailer: Mozilla 4.03 (Macintosh; U; PPC) Fellow Bionetters, What is your prefered membrane for western detection? We're using std HRP chemiluminescent detection with both mono and poly clonal antibodies on proteins from 10kDa to ~100kDa, usually from small BioRad gels. Are the immobilon-NC (HAHY) better than the PVDF type? Or are other brands prefered? Please only reply if you have strong opinions! Email me directly as I have little time recently to visit this newsgroup. thanks in advance. Ted Michelini Institute of Molecular Biology University of Oregon tedm@molbio.uoregon.edu From owner-proteins@net.bio.net Wed Apr 08 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news-peer.gip.net!news-lond.gip.net!news.gsl.net!gip.net!nntp.news.xara.net!xara.net!server5.netnews.ja.net!daresbury!not-for-mail From: Tim Hubbard Newsgroups: bionet.molbio.proteins Subject: Announcement: Structure Prediction Euroconference Date: 9 Apr 1998 15:11:46 +0100 Lines: 70 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <6gikv2$2ui@mserv1.dl.ac.uk> X-Sender: th@pop.sanger.ac.uk Original-To: bionews@dl.ac.uk, proteins@dl.ac.uk, xtal-log@dl.ac.uk, str-nmr@dl.ac.uk, pdb-l@pdb.pdb.bnl.gov ISI - Institute for Scientific Interchange foundation Euroconference on: PROTEIN FOLDING AND STRUCTURE PREDICTION Torino - Villa Gualino, June 8-20, 1998 Scientific Program ------------------ The conference is addressed to young scientists from different backgrounds (physics, biochemistry, biology, genetics, chemistry) and a strong interest in the fields of protein function and structure prediction, and folding simulations. These subjects are of outstanding interest for the development of biotechnologies and for progress in pharmaceutical and medical sciences. The purpose of the conference is to inform the participants of the latest developments in these rapidly evolving and interdisciplinary fields and to provide them with an overview of the many facets of the underlying theoretical issues and experimental techniques. Lecturers --------- R. Bazzo, M. Bolognesi, C. Chothia, M. Delarue, E. Domany, S. Fuller, T. Gibson, P. Grassberger, T. Hubbard, D. Jones, A. M. Lesk, A. Maritan, H. Orland, C. Peterson, D. Rizzi, C. Sander, L. Serrano, E. Shakhnovich, M. Sippl, A Tramontano, G. Vriend. Grants ------ About 35 grants will be available for European Scientists under 35. Specific forms for application (to be found at http://www.isi.it/events/1998/pfsp.html) have to be sent by e-mail or fax to the ISI Foundation by April 20. A selection will be made on the basis of the EU criteria. Organising Committee -------------------- E. Domany, (Weizman Inst., Israel), H. Orland (CEA, Saclay), C. Peterson (Lund, Sweden), A. Tramontano (IRBM, Rome) Support Team ------------ P. Bruscolini, V. Morea Organisation ------------ ISI, Villa Gualino, Viale Settimio Severo 65, 10133 Torino, Italy Phone: +39 11 6603090 Fax: +39 11 6600049 E-mail: isi@isi36a.isi.it http://www.isi.it This conference is supported by the European Community -------------------------------------------------------------------------------- From owner-proteins@net.bio.net Thu Apr 09 23:00:00 1998 Path: biosci!THOMAS.BUTLER.EDU!rkarn From: rkarn@THOMAS.BUTLER.EDU (Karn Robert) Newsgroups: bionet.molbio.proteins Subject: mouse submaxillary cDNA library Date: 10 Apr 1998 05:32:46 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 7 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Hello out there, I need a copy of a mouse submaxillary cDNA library. Does anyone know where I can find either a commercial or private source for one? Thanks in advance, Bob Karn karn@butler.edu From owner-proteins@net.bio.net Thu Apr 09 23:00:00 1998 Path: biosci!MOFFITT.USF.EDU!tabibzadeh From: tabibzadeh@MOFFITT.USF.EDU ("Siamak Tabibzadeh, MD") Newsgroups: bionet.molbio.proteins Subject: Frontiers in Bioscience, A Journal and Virtual library Date: 10 Apr 1998 14:28:56 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 162 Sender: daemon@net.bio.net Distribution: world Message-ID: <000701bd64c7$281cfda0$31b0f783@104648.moffitt.usf.edu> NNTP-Posting-Host: net.bio.net This is a multi-part message in MIME format. ------=_NextPart_000_0004_01BD64A5.A0E9CBE0 Content-Type: text/plain; charset="iso-8859-1" Content-Transfer-Encoding: quoted-printable Frontiers in Bioscience A Journal and Virtual library THE BEST BIOSCIENCE HAS TO OFFER Cited by MEDLINE, Index Medicus, BIOSIS and Chemical Abstracts Frontiers in Bioscience (FBS) is a virtual journal and library bringing = the most up-to-date data in life sciences and medicine to the desktop of = every scientist. FBS offers distinct advantages over traditional print = journals including a rapid review process, free world-wide access, = electronic reprint, use of multimedia, and links to diverse databases = including MEDLINE. FBS is read by over 75,000 readers internationally = and is accessed over 200,000 times every month in the US alone. With its = mirror sites, over 700 editors and nearly 100 managing editors, FBS is = truly an international entity and a valuable platform for scientific = communication. The virtual library provides numerous databases, valuable = information as well as forms and links to thousands of Web sites of = interest to researchers and physicians. Visit this scientific treasure = on the Web. Team up with distinguished scientists who contribute to FBS: HIV-1 Nef and host cell protein kinases=20 Kalle Saksela=20 [Vol 2, pp d606-618. December 15, 1997] PubMed No: 9388166 Human immunodeficiency virus type I as a target for gene=20 therapy=20 Magn=FAs Gottfredsson and Paul R. Bohjanen=20 [Vol 2, pp d619-634, December 15, 1997] PubMed No: 9388165 Fibronectin-integrin interactions S. Johansson, G. Svineng, K. Wennerberg, A. Armulik and L.=20 Lohikangas [Vol 2 pp d126-146, March 1, 1997] PubMed No: 9159220 URLs: USA: http://www.bioscience.org Israel: http://bioinfo.weizmann.ac.il/bioscience France: http://vega.crbm.cnrs-mop.fr/bioscience=20 Frontiers in Bioscience is a non-profit organization dedicated to = fostering science, education and research ------=_NextPart_000_0004_01BD64A5.A0E9CBE0 Content-Type: text/html; charset="iso-8859-1" Content-Transfer-Encoding: quoted-printable

Frontiers in Bioscience

A Journal and Virtual library

THE BEST BIOSCIENCE HAS TO OFFER

Cited by MEDLINE, Index Medicus, BIOSIS and Chemical=20 Abstracts

Frontiers in Bioscience (FBS) is a virtual journal and library = bringing the=20 most up-to-date data in life sciences and medicine to the desktop of = every=20 scientist. FBS offers distinct advantages over traditional print = journals=20 including a rapid review process, free world-wide access, electronic = reprint,=20 use of multimedia, and links to diverse databases including MEDLINE. FBS = is read=20 by over 75,000 readers internationally and is accessed over 200,000 = times every=20 month in the US alone. With its mirror sites, over 700 editors and = nearly 100=20 managing editors, FBS is truly an international entity and a valuable = platform=20 for scientific communication. The virtual library provides numerous = databases,=20 valuable information as well as forms and links to thousands of Web = sites of=20 interest to researchers and physicians. Visit this scientific treasure = on the=20 Web.

Team up with distinguished scientists who contribute = to=20 FBS:

 

HIV-1 Nef and host cell protein kinases

Kalle Saksela

[Vol 2, pp d606-618. December 15, 1997] PubMed No: = 9388166

Human immunodeficiency virus type I as a target for = gene

therapy

Magnús Gottfredsson and Paul R. Bohjanen =

[Vol 2, pp d619-634, December 15, 1997] PubMed No: = 9388165

Fibronectin-integrin interactions

S. Johansson, G. Svineng, K. Wennerberg, A. Armulik = and L.

Lohikangas

[Vol 2 pp d126-146, March 1, 1997] PubMed No: = 9159220

 

URLs:

USA: http://www.bioscience.org

Israel: http://bioinfo.weizmann.ac.il/bioscience

France: http://vega.crbm.cnrs-mop.fr/bioscience

Frontiers in Bioscience is a non-profit organization = dedicated=20 to fostering science, education and=20 research

------=_NextPart_000_0004_01BD64A5.A0E9CBE0-- From owner-proteins@net.bio.net Sat Apr 11 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!ix.netcom.com!news From: Mark Atlas Newsgroups: bionet.molbio.proteins Subject: The Internets safest way to buy used lab equipment HPLC ,GC and more Date: Sun, 12 Apr 1998 18:31:16 GMT Organization: Netcom Lines: 59 Message-ID: <6gr1ai$4r2@dfw-ixnews2.ix.netcom.com> NNTP-Posting-Host: sjc-ca3-18.ix.netcom.com X-NETCOM-Date: Sun Apr 12 1:31:46 PM CDT 1998 X-Newsreader: NETCOMplete/4.0 Going, Going...Sold! http://www.going-going-sold.com The Internets safest way to buy used lab equipment. All auction sales are given an in lab evaluation through an escrow which allows you to be certain that you will receive the items as described on our site. Leasing is available along with expert professional guidance from neutral partys which will assist you in your purchase. Below are some of our upcoming deals with the opening prices. Waters Alliance systems used, you bet opening bid $26,500 A functional Atomic absorbtion spec from Varian. It is a vintage model but you will certainly get your moneys worth at 100$ opening bids The following Items are scheduled to go on Auction over the next few weeks. ***************************************************** Sale Closing : 4/15/98 Opening Prices 1. Sorvall RC 3B and Rotor $6,995.00 2. Tissue Processor $1,000.00 3. ATD-400 with MCS-16 $22,000.00 4. Waters GPC system $10,500.00 5. Olympus Microscope $1,500.00 6. LCM-1 + Baseline software 810 w/ A/D SIM Box $12,000.00 7. Waters Baseline 810 Chromatography Software $1,100.00 ***************************************************** Sale Closing : 4/17/98 Opening Prices 1. Centra 4B $1,395.00 2. Tangential Flow System $15,000.00 3. 4' Biological Safety Cabinet $2,500.00 4. Beckman L5-75 Centrifuge $6,500.00 5. Beckman DU-62 $3,800.00 6. Beta Scint. 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Waters 717 autosampler $4,750.00 9. LAC/E box for Expert Ease $1,000.00 ***************************************************** (c) 1997, Internet Auctioneers International From owner-proteins@net.bio.net Sat Apr 11 23:00:00 1998 Path: biosci!agate!howland.erols.net!nntp.abs.net!feeder.qis.net!news.umbc.edu!haven.umd.edu!news.cs.jhu.edu!news.jhu.edu!welchlink!stebby From: stebby@welchlink.welch.jhu.edu (Stephen C. Dahl) Newsgroups: bionet.molbio.proteins Subject: Re: Q: best western membrane? Date: 10 Apr 1998 18:51:57 GMT Organization: HCF - Johns Hopkins University, Baltimore, Maryland, USA Lines: 18 Message-ID: <6glpod$4vu@news.jhu.edu> References: <352DB18E.9B2C4D05@molbio.uoregon.edu> NNTP-Posting-Host: 128.220.59.78 X-Newsreader: TIN [version 1.2 PL2] : Please only reply if you have strong opinions! : Email me directly as I have little time recently to visit this : newsgroup. Well, call me an A-hole, but this kind of post always gets me fuming... Like the rest of us have nothing better to do than sit on our fat butts and be the Dear Abbys of biology. Jeez, how much longer can it take to read a single thread in a newsgroup vs. read one's e-mail? So here's my opinion, posted to the group so others can benefit, modify, or critique... My favorite for hrp chemiluminescence is plain old nitrocellulose. Not the supported stuff because I've found too much variability. Just plain nitrocellulose. Low backgrounds, easy to Ponceau stain to visualize bands prior to use. Now as for which supplier, welllllllll...I'm currently retesting because my favorite has been sending me crap recently. From owner-proteins@net.bio.net Sun Apr 12 23:00:00 1998 Path: biosci!news.Stanford.EDU!Cabal.CESspool!bofh.vszbr.cz!howland.erols.net!news.pagesat.net!news.itis.com!news.doit.wisc.edu!default From: klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: An interesting puzzle - Pierce's "Gentle Ag/Ab elution buffer"? <-- SOLVED Date: Mon, 13 Apr 1998 17:42:59 GMT Organization: UW-Madison Lines: 37 Message-ID: <6gtiqi$c1a$1@news.doit.wisc.edu> References: <6g499h$1ek_002@doit.wisc.edu> <6ggbkm$puk$1@news.doit.wisc.edu> NNTP-Posting-Host: martinlab.biochem.wisc.edu Mime-Version: 1.0 Content-Type: text/plain; charset=KOI8-R Content-Transfer-Encoding: 8bit X-Newsreader: News Xpress 2.01 X-No-Archive: Yes Xref: biosci bionet.molbio.methds-reagnts:66498 bionet.molbio.proteins:12644 In article , noone@cancer.bham.ac.uk (noone) wrote: : :> :> Thanks very much to everyone who replied, to the group and by email. :> Many people suggested it could be magnesium. Harlow & Lane "Antibodies" :> book lists 3.5 M MgCl2 pH 7.2 as one of the alternatives to acid :> elution. :> :> We ordered that stuff. It does not foam at all - so isn't detergent. It :> does form precipitate with phopshate, which is dissolved with EDTA but :> not EGTA, so it is Mg2+. By conductivity versus MgCl2 standards, it is :> 3.6 M solution. NaJO4 does not oxidize anything in this solution, so I :> don't think there is ethylene glycol present. Sooo, if anyone wants to :> make Pierce's magic solution, the recipie is: :> :> ~ 20 mM MES or PIPES, :> 3.6 M MgCl2, :> titrate pH to ~ 6.5 with NaOH :> :> Funny thing is that Pierce recommends to store this solution at 4C. :> Hehehe, like anything would grow in 3.5 M MgCl2! :> Or is it MgCl2 unstable? :-) :> :> 500 ml for $65 ... from Pierce :> or [when buying expensive MgCl2 from Sigma] :> 3.5 l for $104. Hmmm... : : :OK, but what's the brown precipitate I get from this buffer when I add :loading buffer (or any other DTT- or b-merc.-containing solution)? Per your message, I mixed Pierce's solution with 1 M DTT (yes, that's 1000 mM!) and NO brown precipitate or any precipitate formed. So I have now idea what buffer you have. Dima From owner-proteins@net.bio.net Sun Apr 12 23:00:00 1998 Path: biosci!internet!biosci!not-for-mail From: biohelp (BIOSCI Administrator) Newsgroups: bionet.molbio.proteins Subject: BIOSCI/bionet miniFAQ & Fundraiser Date: 13 Apr 1998 02:00:12 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 233 Sender: daemon@net.bio.net Distribution: world Message-ID: <199804130900.CAA04386@net.bio.net> NNTP-Posting-Host: net.bio.net (LAST REVISION: 30-JUL-95) This BIOSCI "miniFAQ" is designed to answer the questions that come up the *most frequently*. The main BIOSCI FAQ (Frequently Asked Questions) is accessible on the World Wide Web at URL http://www.bio.net/. If you can not find an answer to your question in this or other documentation, the BIOSCI technical support staff answers e-mail queries sent to biosci-help@net.bio.net We can only answer questions about the use of the newsgroups and mailing lists. We unfortunately do not have the staff to do Internet information searches or answer scientific questions. Please post those to the appropriate BIOSCI/bionet newsgroups. Contents: -------- 0) BIOSCI NEEDS YOUR SUPPORT!! 1) Using the WWW to access the BIOSCI/bionet newsgroups. 2) What to do about "spams," i.e., junk mail, ads, etc. 3) Examples of subscribing and unsubscribing to the mailing lists. 4) The BIOSCI user address and research interest directory. 0) BIOSCI NEEDS YOUR SUPPORT!! ------------------------------ BIOSCI's government funding has been expended, and we are now operating solely from advertising revenue that we have raised from our Web site at http://www.bio.net/. We need just a few minutes of your time to help us serve you. You can do two important things which will take very little time for you individually and will immensely help us continue to help you. First, please use our WWW system at http://www.bio.net/ to access the archives. You can post or reply to messages via your Web browser as described in item #1 below. Your usage helps attract sponsors. If you contact any of our sponsors, please be sure to thank them for supporting BIOSCI. It is critical for them to get this feedback if they are to continue their sponsorship for the long term. Second, if you work for a company or organization that provides products or services of interest to the biology community, please pass this message on to your marketing or marketing communications department or other appropriate group. Please ask them to help support BIOSCI by sponsoring our Web site and explain the uses and benefits of the system to the biology community. If they are interested, they can then contact us for further information at our tech support address, biosci-help@net.bio.net. 1) Using the WWW to access the BIOSCI/bionet newsgroups. -------------------------------------------------------- As of 10 December 1995, all BIOSCI/bionet full newsgroups are accessible through the World Wide Web (WWW) at URL http://www.bio.net. One can read and reply publicly or privately to both recent postings and archived messages through one's Web browser if it is configured properly to send e-mail. Each newsgroup is equipped with its own WAIS index. The main BIOSCI home page also has access to the BIO-JOURNALS Table of Contents database WAIS index and the BIOSCI user address database described in another item further below. 2) What to do about "spams," i.e., junk mail, ads, etc. ------------------------------------------------------- BIOSCI is a set of parallel USENET newsgroups (the "bionet" groups), mailing lists, and a hypermail archive at URL http://www.bio.net/. The same postings are distributed on all media (except for a small number of mailing-list-only groups at net.bio.net). Unfortunately it is becoming a despicable practice on the Internet (by a few people out to make a fast buck) to do automated mass postings to thousands of newsgroups and mailing lists. These attempts to grab free advertising are refered to as "spams" in the usual, somewhat boneheaded, net terminology. USENET is more susceptible to this practice, and many spams originate on the USENET groups and then are passed on to the mailing lists. However, spammers also get lists of mailing addresses and hit these too, so neither medium is immune. What should you do personally if you get junk mail? --------------------------------------------------- Just delete it and move on without reading it further. Filing a protest is becoming increasingly useless because spammers are often disguising the addresses where the messages are sent from. Unless you really understand Internet mail systems, your attempt at protest by sending replies to the message will often end up being sent to the address of an innocent person that the spammer is victimizing. What can BIOSCI/bionet do to protect its newsgroups? ---------------------------------------------------- The only solution currently available is to moderate the newsgroup. If this newsgroup is already moderated, then you are in good shape. Moderation protects the USENET distribution from about 95% of the spams that are being sent to date and protects the mailing lists completely. Moderation means, however, that someone has to take the time to review each message before it goes out. We have set up software here that simply allows the moderator to forward to an address at net.bio.net messages that (s)he wishes to have distributed. This takes no more time than that needed to read the message and pass it on, say about 1 min. per message. Most newsgroups currently have a discussion leader who is responsible for their newsgroup. The discussions leaders and their e-mail addresses are listed in the BIOSCI Information Sheet which is available on the Web at http://www.bio.net/. If a newsgroup is being hit with too many junk postings, please contact the discussion leader for that group and see if there is interest in moderating the group. Please do not assume that by simply posting a complaint to the newsgroup itself, anyone on the BIOSCI staff will act on your complaint. With close to 100 newsgroups to run, the BIOSCI staff has to rely on the discussion leaders of each newsgroup to report problems directly to us at biosci-help@net.bio.net. We will moderate any of our newsgroups if the discussion leader tells us that the readership of the group wishes to do so and if a moderator is willing to do the work. For most BIOSCI/bionet groups, this entails only a few minutes of work each day. Moderating a newsgroup will resolve probably 95% of the junk postings on the USENET distribution. Unfortunately there are easy ways for determined spammers to override the moderation mechanism on USENET, but we can protect our e-mail subscribers from unwanted postings if the newsgroup is moderated. You can also access our newsgroups over the WWW at URL http://www.bio.net. While this Web interface will not stop spammers from trying to post to the groups, this will give you yet another way, besides using USENET news, to keep the junk out of your personal mail files. For those of you with local USENET news systems, the Web interface will also give you faster access to new newsgroups and recent postings. 3) Examples of subscribing and unsubscribing to the mailing lists. ------------------------------------------------------------------ PLEASE NOTE: The BIOSCI management does NOT act on subscription/unsubscription requests that are posted improperly to the newsgroups and mailing lists. People who do this only bother everyone on the lists to no avail. Please be sure to follow the proper procedures below. Gory details are in the BIOSCI Information sheets on the Web at http://www.bio.net. Below we give an example utilizing the METHODS-AND-REAGENTS list at both of our two BIOSCI sites: Users in the Americas and Pacific Rim countries who use the BIOSCI ------------------------------------------------------------------ node at computer net.bio.net: ---------------------------- A) Determine the "listname" which is the <=8 character mail address ^^^^^^^^^^^^^ for the group. These can be found in the BIOSCI Info. Sheet. For the METHODS-AND-REAGENTS group the mailing address is methods@net.bio.net. The listname is the portion of the address to the left of the @ sign, i.e., "methods". The listname is used with the "subscribe" and "unsubscribe" commands illustrated below. B) Mail all commands in the body of a mail message addressed to biosci-server@net.bio.net. Do NOT send commands to the newsgroup posting addresses! 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Because of our limited personnel resources, we ask that you resubmit a *complete* form to revise your entry; we only replace complete entries and do not have resources to edit old forms. From owner-proteins@net.bio.net Mon Apr 13 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.wli.net!news-spur1.maxwell.syr.edu!news.maxwell.syr.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!Home From: klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins Subject: Re: Q: best western membrane? Date: Tue, 14 Apr 1998 14:20:20 GMT Organization: UW-Madison Lines: 24 Message-ID: <6gvrb4$3b4_002@doit.wisc.edu> References: <352DB18E.9B2C4D05@molbio.uoregon.edu> <6glpod$4vu@news.jhu.edu> NNTP-Posting-Host: t-cssc-4-1-p1.dialup.wisc.edu X-Newsreader: News Xpress 2.01 X-No-Archive: Yes In article <6glpod$4vu@news.jhu.edu>, stebby@welchlink.welch.jhu.edu (Stephen C. Dahl) wrote: >: Please only reply if you have strong opinions! >: Email me directly as I have little time recently to visit this >: newsgroup. > > >Well, call me an A-hole, but this kind of post always gets me fuming... >Like the rest of us have nothing better to do than sit on our fat butts >and be the Dear Abbys of biology. Jeez, how much longer can it take to >read a single thread in a newsgroup vs. read one's e-mail? Agreed! >My favorite for hrp chemiluminescence is plain old nitrocellulose. Not >the supported stuff because I've found too much variability. Just plain >nitrocellulose. Low backgrounds, easy to Ponceau stain to visualize bands >prior to use. Now as for which supplier, welllllllll...I'm currently >retesting because my favorite has been sending me crap recently. Agreed again. Plain ole stuff works best here. Bio-Rad 0.45 um is what I recommend but I am sure there are many others just as good. Dima From owner-proteins@net.bio.net Mon Apr 13 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.internetmci.com!130.185.14.35!torn!resunix.sickkids.on.ca!NewsWatcher!user From: neil.pomroy@utorontoNOSPAM.ca (Neil Pomroy) Newsgroups: bionet.molbio.proteins Subject: Chelators and amino acid analysis Date: Tue, 14 Apr 1998 16:02:12 -0400 Organization: The Hospital for Sick Children Lines: 35 Message-ID: NNTP-Posting-Host: 142.20.52.32 Hello all, I am working with an protease that requires Zn++ as a cofactor. To stop the enzyme, I was planning on using EDTA at a reasonable (ie mM) concentration and analyze the peptide substrate by amino acid analysis. However, the people at the place I get my amino acid analysis done use PICT derivitization, and are complaining that the amino groups of EDTA may make their analyses wildly inaccurate. Now, aside from the tedious and expensive task of calibrating their analyses with varying EDTA concentrations, does anyone know of any quick and easy way to avoid these difficulties? A chelator of Zn++ with no amino groups? An easy way to precipitate EDTA? An alternate form of amino acid derivitization that doesn't use amino groups? I am unwilling to run the mixture down a column of any sort as I am concerned about aggregation and loss of my peptide substrate with local concentration changes that might occur on a column. I am somewhat restricted in that I'd like to keep the pH of my experiments near 7, though perhaps immediately before analysis that could be altered if it would help get rid of EDTA. Any ideas will be appreciated, TIA, -- Neil Pomroy The Hospital for Sick Children and University of Toronto To reply, remove the 'NOSPAM' from the return address. If you are adding me to your bulk e-mail list, please also add the chairman of the Federal Communications Commission at: wkennard@fcc.gov From owner-proteins@net.bio.net Tue Apr 14 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!feed2.news.erols.com!erols!news.mindspring.net!cssun.mathcs.emory.edu!cronkite.cc.uga.edu!mac200.ccrc.uga.edu!user From: rmerkle@uga.cc.uga.edu (Roberta K. Merkle) Newsgroups: bionet.molbio.proteins Subject: CARBOHYDRATE LABORATORY COURSES Date: 15 Apr 1998 13:31:06 GMT Organization: CCRC, Univ. Georgia Lines: 16 Message-ID: NNTP-Posting-Host: mac200.ccrc.uga.edu Techniques for Characterization of Complex Carbohydrates Four courses will be offered in 1998 at the Complex Carbohydrate Research Center (CCRC) of the University of Georgia: 1. Separation and Characterization of Glycoprotein Oligosaccharides (June 1-5), 2. Structural Analysis of Oligosaccharides (June 8-12), 3. Mass Spectrometry and MS/MS Analysis of Glycoconjugates (June 15-19), 4 NMR of Carbohydrates (July 22-26). Courses will consist of hands-on laboratory work, demonstrations and lectures; lab manual including selected analytical techniques and references will be provided. More details can be seen at the CCRC web site at http://www.ccrc.uga.edu/web/training/courses.html For further information and application form contact Dr. Roberta K. Merkle, CCRC, 220 Riverbend Road, The University of Georgia, Athens, Georgia 30602-4712. Phone: 706-542-4402. FAX: 706-542-4412. E-mail: rmerkle@uga.cc.uga.edu From owner-proteins@net.bio.net Tue Apr 14 23:00:00 1998 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!nyd.news.ans.net!newsfeeds.ans.net!bignews.mediaways.net!news-fra1.dfn.de!news-ber1.dfn.de!news-ham1.dfn.de!news.mu-luebeck.de!not-for-mail From: "Lars Komorowski" Newsgroups: bionet.molbio.proteins Subject: Protease assay Date: Tue, 14 Apr 1998 14:18:52 +0100 Organization: Med. Universitaet zu Luebeck Lines: 5 Message-ID: <6h1lp1$8fe$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 knows a reliable simple protease assay. It should work at high temperatures and low pH. Lars From owner-proteins@net.bio.net Tue Apr 14 23:00:00 1998 Path: biosci!news.Stanford.EDU!Cabal.CESspool!bofh.vszbr.cz!newsfeed.eris.dera.gov.uk!delos!server1.netnews.ja.net!hgmp.mrc.ac.uk!guardian.dcs.warwick.ac.uk!warwick!leicester!usenet From: "Dr E. Buxbaum" Newsgroups: bionet.molbio.proteins Subject: Re: Q: best western membrane? Date: Wed, 15 Apr 1998 13:29:14 -0700 Organization: University of Leicester (PCFS User) Lines: 20 Message-ID: <3535189A.614E@le.ac.uk> References: <352DB18E.9B2C4D05@molbio.uoregon.edu> <6glpod$4vu@news.jhu.edu> NNTP-Posting-Host: pc47.msb.le.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Win16; I) Stephen C. Dahl wrote: > My favorite for hrp chemiluminescence is plain old nitrocellulose. Not > the supported stuff because I've found too much variability. Just plain > nitrocellulose. Low backgrounds, easy to Ponceau stain to visualize bands > prior to use. Now as for which supplier, welllllllll...I'm currently > retesting because my favorite has been sending me crap recently. This is interesting, because I tried nitrocellulose once for dot-bloting (vacuum dot blotter of course, not hand spotting) and quantitation by HRP chemoluminescence and found that nitrocellulose, but not PVDF, quenched the chemoluminescence. Nitrocellulose did work with DAB/Ni detection, but not with chemoluminescence. I also found that protein recovery after dot bloting is higher with PVDF than with NC (95 vs 60% of the I-125 labeled Hsc70 bound to the membrane), and that PVDF gives a lower background signal. Additionally, I used to be frustrated by NC membranes breaking into pieces after 2 days on the rotary shaker, a problem which does not occur with PVDF. So on balance I would come out in favour of PVDF membranes like Immobilon P, despite their higher price tag. From owner-proteins@net.bio.net Tue Apr 14 23:00:00 1998 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.wli.net!news-peer-west.sprintlink.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!newsfeed.internetmci.com!193.174.75.126!news-was.dfn.de!news-kar1.dfn.de!hades.rz.uni-sb.de!ukmhg03.med-hg.uni-sb.de!user From: hgsdoo@med-rz.uni-sb.de (Matthias Engel) Newsgroups: bionet.molbio.proteins Subject: did phage-display cDNA screening ever work? Date: 15 Apr 1998 14:03:51 GMT Organization: University of Saarland, Computing Center, Germany. Lines: 29 Message-ID: NNTP-Posting-Host: ukmhg03.med-hg.uni-sb.de Hi everyone! Please excuse if you find this message to be posted to the wrong newsgroup, but the topic is certainly between the disciplines. I am trying to isolate cDNA clones from a phage-display library bought from Maximbiotech (as far as I know, the only commercial source of such cDNA libraries, since Clontech has stopped to sell them). I have tried panning against my bait protein either coupled to microtiter plates or to paramagnetic beads for over 5 rounds. The recombinant phagemid vectors that I isolated carry only tiny inserts (between 30 and 80 base pairs) and also show a nice consensus sequence - unfortunately without affinity to my target protein. I also detected 2 (!!!) mutations in the vector sequence, one producing an additional stop codon 5´ to the inserts. Did anybody ever isolate something useful out of Maximbiotech´s phage display cDNA libraries? Or was it impossible to get more than tiny inserts without a target sequence? Any help is welcome. Matthias -- Matthias Engel University of Saarland Dept. of Human Genetics Homburg, Germany From owner-proteins@net.bio.net Tue Apr 14 23:00:00 1998 Path: biosci!news.Stanford.EDU!Cabal.CESspool!bofh.vszbr.cz!newscore.univie.ac.at!fu-berlin.de!news-ber1.dfn.de!news-ham1.dfn.de!news.mu-luebeck.de!not-for-mail From: "Lars Komorowski" Newsgroups: bionet.molbio.proteins Subject: Starch detectio with iodine Date: Wed, 15 Apr 1998 19:25:47 +0100 Organization: Med. Universitaet zu Luebeck Lines: 7 Message-ID: <6h2qqc$3jf$1@gwsun.medinf.mu-luebeck.de> NNTP-Posting-Host: 141.83.167.168 X-Newsreader: Microsoft Outlook Express 4.71.1712.3 X-MimeOLE: Produced By Microsoft MimeOLE V4.71.1712.3 I want to detect starch on agar plates (as the sole carbon source) with iodine. Has anyone got an idea of how high the starch concentration has to be in order to identify plaques around a colony ? Or is there a better way to detect starch degradation on plates ? Lars From owner-proteins@net.bio.net Tue Apr 14 23:00:00 1998 Path: biosci!PUBLIC.BTA.NET.CN!shangzd F