From owner-proteins@net.bio.net Fri Aug 01 23:00:00 1997 Path: biosci!daresbury!uninett.no!stdio!news-feed.inet.tele.dk!europa.clark.net!169.207.30.81!newsfeeds.sol.net!newsspool.sol.net!newspump.wustl.edu!fas-news.harvard.edu!oitnews.harvard.edu!hsph.harvard.edu!lcchen From: Longchuan Chen Newsgroups: bionet.molbio.proteins Subject: Baculovirus expression system Date: Sat, 2 Aug 1997 10:11:08 -0400 Organization: Harvard University University Information Systems Lines: 9 Message-ID: NNTP-Posting-Host: hsph.harvard.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII Has anyone used the Bac-to-Bac baculovirus expression system from Life Technologies to overexpress recombinant proteins? Is it a better system as claimed? I couldn't find any publications in Medline using this system. You feedback is much appreciated. Longchuan Chen lcchen@hsph.harvard.edu From owner-proteins@net.bio.net Fri Aug 01 23:00:00 1997 Path: biosci!ALPHA.INCQS.FIOCRUZ.BR!ivano From: ivano@ALPHA.INCQS.FIOCRUZ.BR (Ivano de Filippis) Newsgroups: bionet.molbio.proteins Subject: Dendrogram softwares Date: 2 Aug 1997 13:23:53 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 31 Sender: daemon@net.bio.net Distribution: world Message-ID: <9708032033.AA05860@alpha.incqs.fiocruz.br> NNTP-Posting-Host: net.bio.net --=====================_870568117==_ Content-Type: text/plain; charset="us-ascii" Does anybody knows any site on the web where I could get softwares to build trees (dendrograms) from data obtained after electrophoresis, such as whole cell proteins patterns and isoenzyme electrophoresis? Thanks. Ivano --=====================_870568117==_ Content-Type: text/plain; charset="iso-8859-1" Content-Transfer-Encoding: quoted-printable Content-Disposition: attachment; filename="ASSINA1.TXT" Ivano de Filippis Fundacao Oswaldo Cruz - FIOCRUZ Instituto Nacional de Controle de Qualidade em Saude - INCQS Depto. de Microbiologia Lab. de Materiais de Refer=EAncia Av. Brasil, 4365 - Manguinhos Rio de Janeiro - 21045-900 - BRASIL Tel.: 55-21-598-4290/4291/4292/4293 FAX: 55-21-290-0915 E-mail: ivano@alpha.incqs.fiocruz.br --=====================_870568117==_-- From owner-proteins@net.bio.net Sat Aug 02 23:00:00 1997 From: "Johan Lennartsson" Subject: Detection of Tyrosine Phosphorylation Newsgroups: bionet.molbio.proteins Message-ID: <01bc9fdd$e98dbba0$9ff0ee82@johan> X-Newsreader: Microsoft Internet News 4.70.1161 NNTP-Posting-Host: johalenn.anst.uu.se Date: 3 Aug 97 07:30:53 GMT Lines: 23 Path: biosci!agate!newsgate.cuhk.edu.hk!news.glink.net.hk!uunet!in3.uu.net!134.222.90.2!EU.net!Norway.EU.net!uninett.no!newsfeed.sunet.se!news99.sunet.se!news99.sunet.se!newsfeed.uu.se!johalenn.anst.uu.se Hi I am currently working with the SCF receptor tyrosine kinase (c-Kit) and I have a problem with detecting tyrosine phosphorylation after ligand stimulation. I am also working with the PDGF receptor and there I have no problem detecting tyrosine phosphorylation. In both cases I use porcine aortic endothelial cells transfected with the receptors. I have checked the expression using Western Blots and it seems to be OK. Briefly this is the way I try detect tyrosine phosphorylation: 1) I Starve the cells for 24 h in serum free medium. 2) Incubate the cells with 100uM Na3VO4 for 1 h. 3) Stimulate the cells with SCF (100ng/ml) for 5 to 10 minutes. 4) Cool the cells quickly and lyse the cells in 20mM Tris, 150mM NaCl, 1% Triton- X100, 10% glycerol, 1mM PMSF, 1mM Na3VO4 and 1%trasylol. 5) Immunoprecipitate the receptor with polyclonal antibodies. 6) Separate the proteins with SDS-PAGE and do a Western blot using antibodies against phosphotyrosine. Can anyone tell me what might be wrong. //Johan Lennartsson From owner-proteins@net.bio.net Sat Aug 02 23:00:00 1997 Path: biosci!agate!newsfeed.kornet.nm.kr!howland.erols.net!newsxfer3.itd.umich.edu!oleane!in2p3.fr!univ-lyon1.fr!cri.ens-lyon.fr!news From: Stephane Ory Newsgroups: bionet.molbio.proteins Subject: Re: anti MYC antibody looking for supplier Date: Tue, 29 Jul 1997 11:49:14 +0100 Organization: Ecole Normale Superieure de Lyon Lines: 4 Message-ID: <33DDCAAA.638B@ens-lyon.fr> References: Reply-To: Stephane.Ory@ens-lyon.fr NNTP-Posting-Host: mac-lr5-139.ens-lyon.fr Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0 (Macintosh; I; 68K) I used anti-myc antibody from Oncogene Research Products. It works well in immunofluorescence, but I don't try by western blotting. Stephane From owner-proteins@net.bio.net Sun Aug 03 23:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!europa.clark.net!205.252.116.205!howland.erols.net!rill.news.pipex.net!pipex!join.news.pipex.net!pipex!iafrica.com!not-for-mail From: hoboes@iafrica.com (S. Rutherford) Newsgroups: bionet.molbio.proteins Subject: bacteriolytic peptides Date: Mon, 04 Aug 1997 18:32:59 GMT Organization: UUNET Internet Africa Lines: 3 Message-ID: <33e61feb.203021@dbn-news.iafrica.com> NNTP-Posting-Host: 196-31-98-37.iafrica.com X-Newsreader: Forte Free Agent 1.1/16.230 does anyone know of a bacteriolytic peptide/lysozyme which has a pH optimum in the region of 5 - 6? From owner-proteins@net.bio.net Sun Aug 03 23:00:00 1997 Path: biosci!TELENET.NET!geneh From: geneh@TELENET.NET (Gene Holowachuk) Newsgroups: bionet.molbio.proteins Subject: Bacterial Expression & ArgU Date: 4 Aug 1997 08:09:06 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 20 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net We are trying to overproduce a mammalian protein in E coli and are having very poor success. The sequence includes 7 arg codons (3-AGA & 4-AGG) which are 6% of the total codons. I was wondering if anyone could provide us with a pACYC-argU plasmid or similar &/or advice. The expression plasmid we are using is a derivative of pBR322 which would have no compatibility problems with pACYC plasmid. Please post direct to me as we dont subscribe to the list. MTIA. =========================================================================== + Gene Holowachuk, Ph.D. .***. .***. .***. + + Research Institute * | | | * * | | | * | | | * + + Mary Imogene Bassett Hospital *| | | * * | | | * * | | | * + + One Atwell Road * | | | * | | | * * | | | * + + Cooperstown, NY 13326-1394 '***' '***' '***' + + + + Email:"geneh@usa.net" OR "geneh@telenet.net" + + Voice: 607-547-3937 FAX: 607-547-3061 / 4904 + =========================================================================== From owner-proteins@net.bio.net Sun Aug 03 23:00:00 1997 Path: biosci!daresbury!uninett.no!stdio!news-feed.inet.tele.dk!news.maxwell.syr.edu!EU.net!sun4nl!sun4nl!rivm.nl!not-for-mail From: "J.M. Ossewaarde" Newsgroups: bionet.molbio.proteins Subject: Re: Baculovirus expression system Date: 4 Aug 1997 14:31:26 GMT Organization: RIVM Lines: 23 Message-ID: <01bca0e2$9f4789d0$cb0be083@tjaco2> References: NNTP-Posting-Host: liojo.rivm.nl Mime-Version: 1.0 Content-Type: text/plain; charset=ISO-8859-1 Content-Transfer-Encoding: 7bit X-Newsreader: Microsoft Internet News 4.70.1161 I think the Ref you're looking for is: Efficient Generation of Infectious Recombinant Baculoviruses by Site-Specific Transposon-Mediated Insertion of Foreign Genes into a Baculovirus Genome Propagated in Escherichia coli V.A. LUCKOW, S.C. LEE, G.F. BARRY, P.0. OLINS JOURNAL OF VIROLOGY 1993;67:4566-4579 Longchuan Chen wrote in article ... > > Has anyone used the Bac-to-Bac baculovirus expression system from Life > Technologies to overexpress recombinant proteins? Is it a better system > as claimed? I couldn't find any publications in Medline using this > system. You feedback is much appreciated. > > Longchuan Chen > lcchen@hsph.harvard.edu > > From owner-proteins@net.bio.net Sun Aug 03 23:00:00 1997 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news-peer.sprintlink.net!news-pull.sprintlink.net!news-in-east.sprintlink.net!news.sprintlink.net!Sprint!129.98.1.7!moonbeam.aecom.yu.edu!not-for-mail From: Alan Schoenfeld Newsgroups: bionet.molbio.proteins Subject: partial proteolysis in gel Date: Mon, 04 Aug 1997 10:43:22 +0000 Organization: Albert Einstein College of Medicine Lines: 9 Message-ID: <33E5B24A.38BA@aecom.yu.edu> NNTP-Posting-Host: darkstar.aecom.yu.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.02 (Macintosh; I; 68K) I have been trying to do a partial proteolysis (with V8 and chymotrypsin). The problem: I cut out the bands for my protein and digest in the gel. I am comparing it to an in vitro translated product (NOT cut out from gel). I get digest of the in vitro product but not the cut out bands. I suspect that there is more digestion in the liquid in vitro sample because the gel slices are not accessible (although I stop the gel for 1 hour while all products are still in the stacking gel). How can I get equal (and better) digestion? From owner-proteins@net.bio.net Sun Aug 03 23:00:00 1997 Path: biosci!agate!howland.erols.net!rill.news.pipex.net!pipex!join.news.pipex.net!pipex!server1.netnews.ja.net!lyra.csx.cam.ac.uk!drm21 From: drm21@mole.bio.cam.ac.uk (David Micklem) Newsgroups: bionet.molbio.proteins Subject: Re: anti MYC antibody looking for supplier Date: Mon, 04 Aug 1997 12:35:11 +0000 Organization: Wellcome/CRC Institute Lines: 147 Message-ID: References: <33DDCAAA.638B@ens-lyon.fr> NNTP-Posting-Host: drm-mac1.welc.cam.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=ISO-8859-1 Content-Transfer-Encoding: 8bit X-Newsreader: Yet Another NewsWatcher 2.3.0 The Hybridoma Data Bank (available at http://www.atcc.org/) list several suppliers of 9E10 anti-Myc antibody. I've pasted in the information below, along with the information about the cell-line, which is available from ATCC. FWIW, I've used both the Oncogene Science and Berkeley Ab Co ones. Both work fine for immunofluorscence. The stuff from Oncogene Science, if diluted as they suggested to me, would have been sufficient for ONE western blot - a bit expensive for me. The Babco one has worked OK for me on westerns with NBT/BCIP staining, but for some reason not at all with ECL (only two attempts. Re-probing with alk-phos Ab and NBT/BCIP showed clear bands). Neither work anywhere near as well as a batch of antibody I got off someone at the LMB in Cambridge. They made it and purified it themselves, and it gave great whole-mount stainings (fly ovaries) with very little background. Also fabulous on western blots. Too bad I used up my aliquot :-( David PS Sorry about the cryptic format of the info below - its exactly as provided by the hybridoma data bank.... 20555 RE 1.G>Homo sapiens 1.CN>human 1.CD>COLO 320 HSR 1.SN>c-myc AN 20555 DI P>BAbCo Berkeley Antibody Company, Inc. DI 1223 South 47th Street DI Richmond, CA 94804 DI 1-510-412-8930 DI 2.P>Cambridge Research Biochemicals DI Gadbrook Park DI Northwich DI Cheshire CW9 7RA, England, UK DI 44 606 41100 DI 665440 CRBLTD G DI 3.P>Genosys Biotechnologies, Inc. DI 1442 Lake Front Circle, Suite 185 DI The Woodlands, TX 77380-3600 USA DI 1-713-363-3693 DI 1-800-234-5362 (toll free USA) DI 4.P>Oncogene Research Products DI 84 Rogers Street DI Cambridge, MA 02142 USA DI 1-800-662-2616 (Toll free USA) DI 1-617-577-9333 DI 5.P>PharMingen DI 10975 Torreyana Road DI San Diego, CA 92121 USA DI 1-619-677-7737 DI 1-800-848-6227 (toll free USA) DE P>9E10 ;distributor DE P>MMS-150R-1000 ;distributor DE P>MMS-150R-500 ;distributor DE 2.C>9E10 ;distributor DE 2.P>OM-11-908 ;distributor DE 3.P>9E10 ;distributor DE 3.P>OM-11-908 ;distributor DE 3.P>OM-11-908A ;distributor DE 4.P>OP10 ;distributor DE 5.P>14851A ;distributor DE 5.P>9E10 ;distributor DO G>Mus musculus CN>mouse S>BALB/c O>spleen IP G>Mus musculus x Mus musculus CN>mouse x mouse O>spleen IP CE>B-lymphocyte CD>Sp2/0 PD ;IgG1 AS ;Western blot RE 1.G>Homo sapiens 1.CN>human 1.CD>COLO 320 HSR 1.SN>c-myc RE 1.a.CC>neoplasm 1.b.CC>protein AP ;Western blot ;diagnostic use ;immunofluorescence AP ;immunoprecipitation AV ;ascites ;2.purified ;3.lyophilized ;3.purified AV ;3.supernatant ;5.purified SD 14851A SD 9E10 SD MMS150R1000 SD MMS150R500 SD OM11908 SD OM11908A SD OP10 LD EUR ;BD FI>EUR0002332 EUR803.TXT EI DA>8803 CV>8809 CI ;catalog ATCC Number: CRL-1729 Name: MYC 1-9E10.2 Tissue: hybridoma; B cell; B lymphocyte Species: mouse (B cell); mouse (myeloma) Depositor: J.M. Bishop; G.I. Evan AnimalStrain: BALB/c (B cell); BALB/c (myeloma) Receptors: HeLaMarkers: no VirusSuscept: VirusResist: Tumorigenic: Oncogene: RevTranscript: Karyotype: Morphology: lymphoblast Isoenzymes: Isotype: IgG1 AntigenExp: Products: immunoglobulin; monoclonal antibody; against human myc (c-myc) protein Growth: suspension Contaminants: References: Molec. Cell. Biol. 5:3610-3616, 1985 PassageSub: ATCCMedium: ATCC Cat No. 30-2001 Medium: RPMI 1640 medium with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate, 90%; fetal bovine serum, 10% Price Code: J FreezeMedium: FluidRenewal: every 2 to 3 days Subculturing: Cultures can be maintained by addition or replacement of fresh medium. Start cultures at 2 X 10 exp5 cells/ml and maintain between 1 X 10 exp5 and 1 X 10 exp6 cells/ml SplitRatio: DoublingTime: Restrictions: the depositor has provided these cells for research purposes only and with the following restrictions: (1) no distribution should be made to third parties, (2) any proposed commercial use must be negotiated with the Hooper Foundation, NSW 1542, University of California, San Francisco, California, 94143, (3) all papers reporting any use of these or derived clones should make direct reference to the original publication (Molec. Cell. Biol. 4:2843-2850, 1984) Packing Class: 1 YearAccess: MinPDL: MaxPDL: BioSafety: Comments: mice were immunized with a synthetic peptide derived using the sequence of the human c-myc gene product; spleen cells were fused with Sp2/0-Ag14 myeloma cells; the antibody precipitates the p62 c-myc protein, but does not react with mouse or chicken c-myc; tested and found negative for ectromelia virus (mousepox) -- D.R.Micklem, Time flies like an arrow... Wellcome/CRC Institute, Fruit flies like a banana. Cambridge CB2 1QR, UK Email:drm21@mole.bio.cam.ac.uk Unsolicited mail will incur a US$100 processing charge. From owner-proteins@net.bio.net Mon Aug 04 23:00:00 1997 Path: biosci!NS1.NIMR.MRC.AC.UK!d-fesque From: d-fesque@NS1.NIMR.MRC.AC.UK (D Fesquet) Newsgroups: bionet.molbio.proteins Subject: pCYC-ARG Date: 5 Aug 1997 03:58:24 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 23 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net hi, the plasmid you are looking for is described in Biotechniques (1997) 22,82-85 and is available , simply email : endow@galactose.me.duke.edu (check that this is the correct email , perhaps it is mc instead of me?) good luck didier ****************************************************************************** Didier FESQUET Division of Yeast Genetics Medical Research Council The Ridgeway, MILL HILL London NW7 1AA Tel: 00-44-181-959-3666 extension 2241 ou 2244 Fax: 00-44-181-913-8536 secretariat labo Tel (home):00-44-181-343-2440 e-mail:d-fesque@nimr.mrc.ac.uk ****************************************************************************** From owner-proteins@net.bio.net Mon Aug 04 23:00:00 1997 Newsgroups: bionet.molbio.proteins Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!europa.clark.net!4.1.16.34!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!baron.netcom.net.uk!netcom.net.uk!uknet!lyra.csx.cam.ac.uk!server1.netnews.ja.net!warwick!bris.ac.uk!ssa.bris.ac.uk!ogsdw From: "S.D. Wainwright" Subject: Deglycosylation of proteins with tunicamycin X-Nntp-Posting-Host: ssa.bris.ac.uk Content-Type: TEXT/PLAIN; charset=US-ASCII Message-ID: Sender: usenet@fsa.bris.ac.uk (Usenet) Organization: University of Bristol, England Mime-Version: 1.0 Date: Tue, 5 Aug 1997 13:47:56 GMT Lines: 17 Dear All I would like to deglycosylate a protein that I am immunoprecipitating from cells grown in culture. I would like to do this by growing the cells in tunicamycin. I would be grateful if someone could email me their method. I would also like to know how to make a stock solution of tunicamycin and how you store the stock solution. Thanks in advance Shane D Wainwright From owner-proteins@net.bio.net Mon Aug 04 23:00:00 1997 Path: biosci!daresbury!uninett.no!Norway.EU.net!EU.net!news.maxwell.syr.edu!news.apfel.de!news-fra1.dfn.de!news-koe1.dfn.de!news.ruhr-uni-bochum.de!not-for-mail From: "Thorsten Schmidt" Newsgroups: bionet.molbio.proteins Subject: Sörensen´s PBS ??? Date: 5 Aug 1997 19:29:24 GMT Organization: Ruhr-Universitaet Bochum, Rechenzentrum Lines: 19 Message-ID: <01bca1d5$e1f88d80$11019386@schmidtdw.rz.ruhr-uni-bochum.de> NNTP-Posting-Host: dialppp-1-17.rz.ruhr-uni-bochum.de X-Newsreader: Microsoft Internet News 4.70.1155 Dear reader! I use Sörensen´s PBS containing 0,08 M Na2HPO4 0,02 M NaH2PO4 0,15 M NaCl pH 7,4 This buffer is widely used for in situ and histological staining. I need now for a publication the exact reference for this buffer! In which paper was this buffer published for the first time? Can you help me? Thank you very much in advance for your answer! Thorsten Schmidt From owner-proteins@net.bio.net Mon Aug 04 23:00:00 1997 Path: biosci!daresbury!lyra.csx.cam.ac.uk!server1.netnews.ja.net!server5.netnews.ja.net!server3.netnews.ja.net!baron.netcom.net.uk!netcom.net.uk!news-peer!btnet!infeed2.internetmci.com!newsfeed.internetmci.com!newsfeed.direct.ca!hub.org!news.zsu.edu.cn!usenet From: Liu XiaoSong Newsgroups: bionet.molbio.proteins Subject: inquire about E2 protein of Hpv Date: Tue, 05 Aug 1997 17:05:05 +0800 Organization: MRI Lines: 1 Message-ID: <33E6ECC1.75D1@fosu.edu.cn> Reply-To: lsx@fosu.edu.cn NNTP-Posting-Host: 202.192.168.205 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0 (Win95; I) Who know articles about E2 protein of HPV From owner-proteins@net.bio.net Mon Aug 04 23:00:00 1997 Path: biosci!daresbury!uninett.no!news-feed.inet.tele.dk!europa.clark.net!4.1.16.34!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!firehose.mindspring.com!cssun.mathcs.emory.edu!news.service.emory.edu!news From: Brett Burkholder Newsgroups: bionet.molbio.yeast,bionet.molbio.proteins Subject: Codon Bias Effect on Protein Expression Date: Tue, 05 Aug 1997 09:52:59 +0000 Organization: Department of Genetics, Emory University Lines: 22 Message-ID: <33E6F7FC.4CD3@gmm.gen.emory.edu> Reply-To: burkhold@gmm.gen.emory.edu NNTP-Posting-Host: djdquad.gen.emory.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0 (Macintosh; I; 68K) Xref: biosci bionet.molbio.yeast:7472 bionet.molbio.proteins:11321 I have been trying, unsuccessfully, to express two human proteins in yeast. A third human protein expresses very well. I am using a GAL 1-10 promoter for all three, which should be giving huge overexpression. I have previously considered that the lack of an effective RBS could be the culprit. This inquiry resulted in the tip that A/T rich sequences just upstream of the start site help in expression. Now, I am wondering how much codon bias can affect protein expression. If there are a number of ill-favored codons in a row (say ones used less than 10% in normal yeast proteins), is that enough to halt or severely restrict the amount of protein translated? I've come across a couple of articles on the effect in E. coli, but are there any references on the effect of codon bias in yeast? Thanks, Brett Burkholder Department of Genetics Emory University Atlanta, GA From owner-proteins@net.bio.net Mon Aug 04 23:00:00 1997 Path: biosci!MIS.FINCHCMS.EDU!muellerd From: muellerd@MIS.FINCHCMS.EDU (David Mueller) Newsgroups: bionet.molbio.proteins Subject: Re: Codon Bias Effect on Protein Expression Date: 5 Aug 1997 09:06:20 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 44 Sender: daemon@net.bio.net Distribution: world Message-ID: <199708051604.LAA28824@mis.finchcms.edu> NNTP-Posting-Host: net.bio.net Why not try this question on the yeast discussion group? yeast@net.bio.net David Mueller At 09:52 AM 8/5/97 +0000, you wrote: >I have been trying, unsuccessfully, to express two human proteins in >yeast. A third human protein expresses very well. I am using a GAL >1-10 promoter for all three, which should be giving huge overexpression. > >I have previously considered that the lack of an effective RBS could be >the culprit. This inquiry resulted in the tip that A/T rich sequences >just upstream of the start site help in expression. > >Now, I am wondering how much codon bias can affect protein expression. >If there are a number of ill-favored codons in a row (say ones used less >than 10% in normal yeast proteins), is that enough to halt or severely >restrict the amount of protein translated? > >I've come across a couple of articles on the effect in E. coli, but are >there any references on the effect of codon bias in yeast? > >Thanks, > >Brett Burkholder >Department of Genetics >Emory University >Atlanta, GA > > <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<< >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> --------------------------------------------------------------------------- David Mueller Department of Biological Chemistry The Chicago Medical School 3333 Greenbay Rd. North Chicago, IL 60064 Tel: 847-578-8606 FAX: 847-578-3240 From owner-proteins@net.bio.net Tue Aug 05 23:00:00 1997 Path: biosci!agate!hammer.uoregon.edu!vixen.cso.uiuc.edu!howland.erols.net!gatech!newsfeed.pitt.edu!cbpw13.physio.pitt.edu!user From: none@pitt.ewdu (Lynn Wang) Newsgroups: bionet.molbio.proteins Subject: Re: Tris/tricine SDS-PAGE Date: Wed, 06 Aug 1997 16:52:22 +0100 Organization: Dept. of Pathology Lines: 16 Message-ID: References: <199706250257.LAA189646@plaza.snu.ac.kr> <5orive$6j0@falcon.le.ac.uk> <5pgpf9$6pb$2@light.nih.gov> <33E0AC88.519E1ED1@rz.ruhr-uni-bochum.de> NNTP-Posting-Host: cbpw13.physio.pitt.edu In article <33E0AC88.519E1ED1@rz.ruhr-uni-bochum.de>, Reinhard Depping wrote: . > > .> > >I am trying to detect the low molecular weight(8KD) protein using .> > >Tris-Tricine SDS-PAGE. But when I dried the gel, the gel was easily .> > >crashed. .> > .> we had the same problem with 15 % gels. We put it in 10 % glycerine for .> 30 min and then dried it. .> Reinhard You can also place glycerol 4g/30ml in the gel and that will keep it from cracking Peter From owner-proteins@net.bio.net Tue Aug 05 23:00:00 1997 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news.apfel.de!fu-berlin.de!news-ber1.dfn.de!news-ham1.dfn.de!news-han1.dfn.de!news.gwdg.de!not-for-mail From: Your Name Newsgroups: bionet.molbio.yeast,bionet.molbio.proteins Subject: Re: Codon Bias Effect on Protein Expression Date: Wed, 06 Aug 1997 10:00:27 +0100 Organization: MPIZ-Molecular Genetics Lines: 7 Message-ID: <33E83D2B.CC5@mpiz-koeln.mpg.de> References: <33E6F7FC.4CD3@gmm.gen.emory.edu> Reply-To: email@domain.com NNTP-Posting-Host: mac28.mpiz-koeln.mpg.de Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01Gold (Macintosh; I; 68K) To: burkhold@gmm.gen.emory.edu Xref: biosci bionet.molbio.yeast:7476 bionet.molbio.proteins:11324 Dear Brett, there is a nice review about this: Serrano, R and Villalba, J-M. 1995. Methods in Cell Biology. 50:481-96, and references therein. Indeed there is a strong bias in codon usage and they show a table with th preferred codons. Hope it helps. Marcos From owner-proteins@net.bio.net Tue Aug 05 23:00:00 1997 Path: biosci!bloom-beacon.mit.edu!howland.erols.net!europa.clark.net!128.223.220.30!logbridge.uoregon.edu!news.bc.net!news.sfu.ca!beaufort!rozek From: Annett Rozek Newsgroups: bionet.molbio.proteins Subject: need 15N serine Date: Wed, 6 Aug 1997 10:37:16 -0700 Organization: Simon Fraser University Lines: 29 Message-ID: NNTP-Posting-Host: beaufort.sfu.ca Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII X-Sender: rozek@beaufort Dear news group readers, Is there anybody who can lend us 0.5 g of 15N labeled L-serine-N-t-boc, O-benzyl ether for peptide synthesis? We have this compound on order from Cambridge Isotope Laboratories, but delivery has been delayed for over three months now. Who can help us out? We will replace the serine as soon as we get it from CIL. Sincerely, Annett z \\// z ( --) +-------oOO----(__)--------------+ | Annett Rozek | | Institute of Molecular Biology | | and Biochemistry | | Simon Fraser University | | Burnaby, B.C., V5A 1S6 | | Canada | | ph: (604) 291-5657 | | fax: (604) 291-5583 | +--------------------oOO---------+ |__|__| || || ooO Ooo From owner-proteins@net.bio.net Tue Aug 05 23:00:00 1997 Path: biosci!daresbury!uninett.no!Norway.EU.net!EU.net!howland.erols.net!infeed1.internetmci.com!newsfeed.internetmci.com!portc03.blue.aol.com!newstf02.news.aol.com!audrey02.news.aol.com!not-for-mail From: sford4395@aol.com (SFord4395) Newsgroups: bionet.molbio.proteins Subject: Re: HPLC information? Date: 6 Aug 1997 21:35:09 GMT Lines: 6 Message-ID: <19970806213500.RAA25539@ladder02.news.aol.com> NNTP-Posting-Host: ladder02.news.aol.com X-Admin: news@aol.com Organization: AOL, http://www.aol.co.uk References: <869861390.9807@dejanews.com> Check out any analytical chemistry books from your local collage library. If your more interested in protein and peptide HPLC thing get a bit more complicated. Any good analytical biochemistry book should give you info. Get in touch with any of the HPLC column manufactures (for proteins try Vydac or Biorad), but Waters...any others...usually have Web pages for phone no's etc...and ask for applications notes on peptide/protein HPLC From owner-proteins@net.bio.net Wed Aug 06 23:00:00 1997 Path: biosci!agate!newsgate.duke.edu!solaris.cc.vt.edu!newsrelay.netins.net!usenet.cat.pdx.edu!nntp.reed.edu!amon.reed.edu!jnotis From: John Notis Newsgroups: bionet.molbio.proteins Subject: Re: NP-40 replacement Date: Thu, 7 Aug 1997 15:46:58 -0700 Organization: Reed College, Portland, Oregon Lines: 25 Message-ID: References: <33E1E07B.167EB0E7@virginia.edu> NNTP-Posting-Host: amon.reed.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII To: "John S. Walker" In-Reply-To: <33E1E07B.167EB0E7@virginia.edu> On Fri, 1 Aug 1997, John S. Walker wrote: > G'day > > I'm in a physiology lab that does some 2D-PAGE. Our IEF recipe calls for > NP-40 but our supplier recently told us that this is no longer > available. Is this true or is it our supplier is no longer carrying it? > IF it is true, can anybody suggest a good replacement? > > John Walker > > Sigma chemical told me that NP-40 is no longer made by the original manufacturer, but that a generic form of the same compound is available through them: Igepal CA-630 Sigma Chem# I-3021 So it's supposed to be exactly the same stuff, and it seems to be working fine for us. -John Notis From owner-proteins@net.bio.net Wed Aug 06 23:00:00 1997 Path: biosci!daresbury!lyra.csx.cam.ac.uk!server1.netnews.ja.net!warwick!news.nott.ac.uk!NewsWatcher!user From: kevin.bailey@nottingham.ac.uk () Newsgroups: bionet.molbio.proteins Subject: Re: partial proteolysis in gel Followup-To: bionet.molbio.proteins Date: Thu, 07 Aug 1997 14:42:50 +0100 Organization: Cripps Computing Centre, The University of Nottingham Lines: 20 Message-ID: References: <33E5B24A.38BA@aecom.yu.edu> NNTP-Posting-Host: wpbjwk1.biochem.nottingham.ac.uk In article <33E5B24A.38BA@aecom.yu.edu>, Alan Schoenfeld wrote: > I have been trying to do a partial proteolysis (with V8 and > chymotrypsin). The problem: I cut out the bands for my protein and > digest in the gel. I am comparing it to an in vitro translated product > (NOT cut out from gel). I get digest of the in vitro product but not > the cut out bands. I suspect that there is more digestion in the > liquid > in vitro sample because the gel slices are not accessible (although I > stop the gel for 1 hour while all products are still in the stacking > gel). How can I get equal (and better) digestion? I suspect the only real way is to purify the material you are currently running down the gel in another way (FPLC/HPLC) to get a pure protein in free solution, as you have with the translated product. In-gel digests are often poor, either (as you suggest) due to substrate inaccessibility or possibly some inactivation of the proteolytic enzymes by gel reagents. I've read that a number of proteolytic enzymes are not particularly tolerant to SDS, for example. From owner-proteins@net.bio.net Wed Aug 06 23:00:00 1997 Path: biosci!ucmp.umu.se!Michael.Daws From: Michael.Daws@ucmp.umu.se (Michael Daws) Newsgroups: bionet.molbio.proteins Subject: Tagged active tyrosine kinase Date: 7 Aug 1997 04:11:40 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 19 Sender: daemon@net.bio.net Distribution: world Message-ID: <33E9CA27.2341@ucmp.umu.se> NNTP-Posting-Host: net.bio.net Does anybody know of a commercial source for a tagged (preferably GST or His-Tagged) active tyrosine kinase or kinase domain, or does anybody have such a thing that they could let me have? Thanks in advance for your help. MikeD -- <<<<<<<<<<<<<<<<<<<<>>>>>>>>>>>>>>>>>>>> Michael R. Daws, PhD Umea Center for Molecular Pathogenesis 901 87 Umea, Sweden Phone: 46-90-17-67-93 Fax: 46-90-77-80-07 E-mail: Michael.Daws@ucmp.umu.se <<<<<<<<<<<<<<<<<<<<>>>>>>>>>>>>>>>>>>>> There are two types of people in this world, good and bad. The good sleep better, but the bad seem to enjoy the waking hours much more.....W. Allen From owner-proteins@net.bio.net Wed Aug 06 23:00:00 1997 Path: biosci!daresbury!uninett.no!sn.no!Norway.EU.net!EU.net!howland.erols.net!wnfeed!204.127.130.5!worldnet.att.net!news.u.washington.edu!saul9.u.washington.edu!pearton From: pearton@saul9.u.washington.edu (D. Pearton) Newsgroups: bionet.molbio.proteins Subject: Bipartite NLS prediction Date: 7 Aug 1997 21:14:41 GMT Organization: University of Washington Lines: 25 Message-ID: <5sdds1$t7f@nntp3.u.washington.edu> NNTP-Posting-Host: saul9.u.washington.edu NNTP-Posting-User: pearton X-Newsreader: TIN [version 1.2 PL2] Hi all, I'd like to test a few sequences for the presence of a bipartite Nuclear Loclaisation sequence. Unfortunately the two pages on the web that used to do this (http://www.bio.cam.ac.uk/~cyk10/ and http://cy-mac.welc.cam.ac.uk) are no longer running, it appears. Could anyone point me to a resource that would do this? I'm not particularly interested in other types of NLS. A good reference would also be appreciated. Thanks, -- Dave Pearton pearton@u.washington.edu +++++++++++++++++++++++++++++++++++++++++++++++++++++++++ Down he sank in a chair-ran his hands through his hair- + And chanted in mimsiest tones + Words whose utter inanity proved his insanity, + While he rattled a couple of bones. + + "The Hunting of the Snark" Lewis Carroll + +++++++++++++++++++++++++++++++++++++++++++++++++++++++++ From owner-proteins@net.bio.net Wed Aug 06 23:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!europa.clark.net!205.252.116.205!howland.erols.net!infeed1.internetmci.com!newsfeed.internetmci.com!news.onenet.net!news.ou.edu!not-for-mail From: Bob Steinberg Newsgroups: bionet.molbio.proteins Subject: Re: partial proteolysis in gel Date: Thu, 07 Aug 1997 15:52:03 -0700 Organization: University of Oklahoma Health Sciences Center Lines: 41 Message-ID: <33EA5193.3CBD@etowah.uokhsc.edu> References: <33E5B24A.38BA@aecom.yu.edu> Reply-To: rsteinbe@etowah.uokhsc.edu NNTP-Posting-Host: 157.142.56.147 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Win16; I) kevin.bailey@nottingham.ac.uk wrote: > > In article <33E5B24A.38BA@aecom.yu.edu>, Alan Schoenfeld > wrote: > > > I have been trying to do a partial proteolysis (with V8 and > > chymotrypsin). The problem: I cut out the bands for my protein and > > digest in the gel. I am comparing it to an in vitro translated product > > (NOT cut out from gel). I get digest of the in vitro product but not > > the cut out bands. I suspect that there is more digestion in the > > liquid > > in vitro sample because the gel slices are not accessible (although I > > stop the gel for 1 hour while all products are still in the stacking > > gel). How can I get equal (and better) digestion? > > I suspect the only real way is to purify the material you are currently > running down the gel in another way (FPLC/HPLC) to get a pure protein in > free solution, as you have with the translated product. In-gel digests are > often poor, either (as you suggest) due to substrate inaccessibility or > possibly some inactivation of the proteolytic enzymes by gel reagents. I've > read that a number of proteolytic enzymes are not particularly tolerant to > SDS, for example. I've had good success with in-gel proteolysis using both V8 protease and papain-- I would suggest running all your samples out on a gel first, so they are all equivalent. The procedure I used was to excise bands from a dried gel (I actually used spots from 2-D gels) based on tracings of an autoradiograph or Coomassie-stained pattern (I generally fix, stain, and destain in 7% acetic acid when I want to recover proteins from gels); rehydrate for 30 min at room temperature in SDS Gel Sample Buffer; and place the bands (long axis vertical) directly onto the stacking layer of a 15% SDS-PAGE gel (use a teflon spacer to make a flat surface on the stacking gel about 2-3 mm below the notch of your plates)-- space the bands at about 2.5 to 3 mm intervals. Fix the gel pieces in place with ~0.2 ml of a melted solution of 0.9% electrophoresis-grade agarose in "Cleveland Buffer" (0.125 M Tris, pH 6.8, 0.5% SDS, 0.5% mercaptoethanol, 1 mM EDTA) and, after this has set, overlay with a solution of protease in this same agarose prepared at 42oC to prevent inactivation using about 0.05 ml per centimeter of gel width (using 0.75 mm thick gels). With my proteins, I had good success using 33 micrograms/ml V8 protease or 30 nanograms/ml papain. From owner-proteins@net.bio.net Thu Aug 07 23:00:00 1997 Path: biosci!COMP.UARK.EDU!lpurcell From: lpurcell@COMP.UARK.EDU ("Larry C. Purcell") Newsgroups: bionet.molbio.proteins Subject: Where is Spectra-Physics Date: 8 Aug 1997 12:58:19 -0700 Organization: University of Arkansas Lines: 15 Sender: daemon@net.bio.net Distribution: world Message-ID: <33EB9673.5CB2@comp.uark.edu> NNTP-Posting-Host: net.bio.net Hello, Does anyone know who bought Spectra-Physics or who is now servicing their equipment. I have a SP HPLC that needs service and can not find a contact. Thanks for your help. Larry C. Purcell Department of Agronomy University of Arkansas 276 Altheimer Drive Fayetteville, AR 72704 voice (501)575-3983, fax (501)575-3975 From owner-proteins@net.bio.net Thu Aug 07 23:00:00 1997 Path: biosci!agate!newsgate.cuhk.edu.hk!news.cuhk.edu.hk!usenet From: Kwok-fai Lau Newsgroups: bionet.molbio.proteins Subject: How to characterize a novel protein Date: Sat, 09 Aug 1997 13:51:14 +0800 Organization: The Chinese University of Hong Kong Lines: 6 Message-ID: <33EC0552.6051@mailserv.cuhk.edu.hk> Reply-To: s951874@mailserv.cuhk.edu.hk NNTP-Posting-Host: @137.189.47.153 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Win95; I) Dear everybody, I would like to ask if anybody know there is some methods or strategies to find out the cellular function of a novel protein sequence deduced from a novel cDNA sequence? The cDNA sequence is "fished" out by yeast two-hybrid system. From owner-proteins@net.bio.net Thu Aug 07 23:00:00 1997 Path: biosci!agate!newsgate.cuhk.edu.hk!news.cuhk.edu.hk!usenet From: Kwok-fai Lau Newsgroups: bionet.molbio.proteins Subject: Sorry, correction for the sender of "how to charactrization of novel protein" Date: Sat, 09 Aug 1997 14:00:40 +0800 Organization: The Chinese University of Hong Kong Lines: 8 Message-ID: <33EC0788.6DDD@mailserv.cuhk.edu.hk> Reply-To: s951874@mailserv.cuhk.edu.hk NNTP-Posting-Host: @137.189.47.153 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Win95; I) Dear everybody, Sorry, I have not change the setting of the E-mail address before I sent this article. I would like to ask if anybody know there is some methods or strategies to find out the cellular function of a novel protein sequence deduced from a novel cDNA sequence? The cDNA sequence is "fished" out by yeast two-hybrid system. From owner-proteins@net.bio.net Thu Aug 07 23:00:00 1997 Path: biosci!NBRF.GEORGETOWN.EDU!POSTMASTER From: POSTMASTER@NBRF.GEORGETOWN.EDU Newsgroups: bionet.molbio.proteins Subject: Announcements of the Protein Information Resource Date: 8 Aug 1997 16:11:40 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 176 Sender: daemon@net.bio.net Distribution: world Message-ID: <01IM7HRATAG29BVL8J@NBRF.Georgetown.Edu> NNTP-Posting-Host: net.bio.net Announcements of the Protein Information Resource PIR-International 6 August 1997 Highlights 1. Availability of PIR-International Release 53.00 and Associated Data Sets 2. Unique Features of the PIR-International Protein Sequence Database 3. The NRL_3D Database Updated 4. Format changes anticipated for Release 54.00 5. Ordering the Atlas of Protein and Genomic Sequences CD-ROM 1. Availability of PIR-International Release 53.00 and Associated Data Sets The quarterly (June 30) releases of the PIR-International Protein Sequence Database, the NRL_3D database (corresponding to Brookhaven Protein Data Bank Release 79), and the ALN Database of Protein Sequence Alignments are available. Release Information for PIR-International Data Sets ============================================================================== Data Set Release Entries Residues Description PIR1 53.00 13706 5125282 Section 1. Classified and Annotated Entries PIR2 53.00 77275 24432750 Section 2. Annotated Entries PIR3 53.00 3832 868136 Section 3. Unverified Entries PIR4 53.00 238 43412 Section 4. Unencoded or Untranslated Entries NRL_3D 21.00 10717 1897913 NRL Protein Sequences in Brookhaven PDB ALN 16.00 2896 Database of Protein Sequence Alignments PATCHX 53.00 96102 27691941 Available protein sequences not in PIR ECOLI 4.10 592 3996197 Escherichia coli DNA Database RESID 10.00 236 Residues annotated as features in PIR ------------------------------------------------------------------------------ Availability Information for the Data Sets ============================================================================= Data Set | ATLAS CD-ROM | Magnetic Media | PIR WWW | PIR FTP Site | Online | PIR1 | X | X | X | X | X | PIR2 | X | X | X | X | X | PIR3 | X | X | X | X | X | PIR4 | X | X | X | X | X | NRL_3D | X | X | X | X | X | ALN | X | | X | | | PATCHX | X | | | | X | ECOLI | X | | | | | RESID | X | | | | | ----------------------------------------------------------------------------- The Complex Carbohydrate Structure Database (CCSD) and associated CarbBank program for Windows95/NT are also distributed on the Atlas of Protein and Genomic Sequences CD-ROM. The PIR URL: http://www-nbrf.georgetown.edu/pir/ The PIR anonymous ftp site: nbrf.georgetown.edu 2. Unique Fetaures of the PIR-International Protein Sequence Database The PIR-International Protein Sequence Database is unique among comprehensive public domain protein sequence databases in the following respects: * Beginning with Release 53.00, essentially all sequence entries are classified into families (see below). * The PIR-International Protein Sequence Database contains more citations and more up-to-date data. * Full citations, including the titles of papers cited, are given. * The sequence reported in each citation is represented in a manner that clearly shows any differences from the sequence shown in the entry and allows the reported sequence to be reconstructed automatically. * Cross-references to the nucleotide sequence databases are directly associated with the citation on which they are based. * The most complete and current genetic information is provided, including map position, intron positions, and start codon (if different from ATG), along with pointers to genome databases. * Feature annotations are represented with greater accuracy and consistency because of format and terminology restrictions. The current Guide for PIR Features Annotations is publicly available. * It has consistently adhered to its announced update schedule. The PIR has been updated and publicly released 4 times per year for the last 13 years. * Public access is provided through our Web site and online system to the interim updates normally prepared on a weekly basis (except during holiday periods and during preparation of quarterly releases). Dr. Friedhelm Pfeiffer at MIPS has clustered 93% of the sequences in the PIR database into families whose members have about 50% or more sequence identity. Less than 5% of entries in the database are considered unclassifiable, usually because they are too fragmentary. Only about 2% of entries are not fully analyzed. Over 10,000 alignments of the families that contain at least two sequences are available at the MIPS Web Site http://www.mips.biochem.mpg.de/ Every family classified in this way has been assigned a permanent ID. About half the sequences have been further clustered into superfamilies that have also been assigned permanent IDs. The assignment of permanent IDs to superfamilies allows users to keep track of superfamilies more easily. The permanent family and superfamily numbers will shortly be available on the PIR Web Site by clicking on the "Associated information" when viewing an entry. This information will be available to VAX-VMS users of the Atlas CD-ROM and magnetic tapes. 3. The NRL_3D Database Updated The NRL_3D Database, produced by the PIR since 1989, is a database of protein sequences with determined structures extracted from the Brookhaven Protein Data Bank (PDB) coordinate data files. It provides an interface between the Protein Sequence Database and the PDB and provides access to the PDB data via computerized sequence searching and comparison methods. This release corresponding to the January 1997 release of the Brookhaven Protein Data Bank was produced using the facilities of the National Cancer Institute Biomedical Supercomputer Center, Frederick, MD, and we gratefully acknowledge the cooperation of J.V. Maizel, Jr., S.K. Burt, and G.W. Smythers. 4. Format changes anticipated for Release 54.00 We anticipate that there will be some format changes in Release 54. If you want to receive advance notice of the changes, please E-mail a request for the PIR Developer's Bulletin to PIRMAIL@NBRF.GEORGETOWN.EDU 5. Ordering the Atlas of Protein and Genomic Sequences CD-ROM In addtion to the databases listed previously, the ATLAS CD-ROM also includes an installation guide, an ATLAS User's Guide, the FASTA package, and an Installation Manual and Tutorial for CarbBank. The ATLAS program, which accesses all of the other data sets on the CD-ROM, does not access the Complex Carbohydrate Structure Database. Orders for the ATLAS CD-ROM are accepted, WITHOUT PREPAYMENT on institutional purchase orders, by FAX or E-mail. For further information in the US and the Americas, please contact: Kathryn Sidman, Technical Services Coordinator Protein Information Resource (PIR) National Biomedical Research Foundation (NBRF) 3900 Reservoir Rd., NW Washington DC 20007 FAX: (202) 687-1662 phone: (202) 687-2121 E-mail: PIRMAIL@nbrf.georgetown.edu In Europe contact: Martinsried Institute for Protein Sequences (MIPS) Max-Planck-Institute for Biochemistry 8033 Martinsried, Germany FAX: 49 89 8578 2655 phone: 49 89 8578 2657 E-mail: mewes@ehpmic.mips.biochem.mpg.de In Asia and Oceania contact: Japan International Protein Information Database (JIPID) Science University of Tokyo 2669 Yamazaki, Noda 278 Japan FAX: 81 47 122 1544 phone: 81 48 124 1501 E-mail: Tsugita@JPNSUT31.BITNET For information about CarbBank contact: CarbBank/CCSD 114 W. Magnolia St. Suite 305 Bellingham, WA 98225 Phone: (360) 671-8134 EMail: CarbBank@PacificRim.net PIR is a registered mark of NBRF ------------------------------------------------------------------------ Dr. John S. Garavelli Associate Director Protein Information Resource National Biomedical Research Foundation Washington, DC 20007 PIRMAIL@NBRF.GEORGETOWN.EDU From owner-proteins@net.bio.net Thu Aug 07 23:00:00 1997 Path: biosci!agate!hammer.uoregon.edu!vixen.cso.uiuc.edu!howland.erols.net!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!portc02.blue.aol.com!newsfeed.pitt.edu!dsinc!nntp.upenn.edu!mail.med.upenn.edu!wellsg From: wellsg@mail.med.upenn.edu (Gregg B Wells) Newsgroups: bionet.molbio.proteins Subject: Re: Where is Spectra-Physics Date: 8 Aug 1997 22:13:43 GMT Organization: University of Pennsylvania Lines: 27 Distribution: world Message-ID: <5sg5mn$rt$1@netnews.upenn.edu> References: <33EB9673.5CB2@comp.uark.edu> NNTP-Posting-Host: mail.med.upenn.edu X-Newsreader: TIN [version 1.2 PL2-upenn1.1] Larry C. Purcell (lpurcell@COMP.UARK.EDU) wrote: : Hello, : Does anyone know who bought Spectra-Physics or who is now servicing their : equipment. I have a SP HPLC that needs service and can not find a : contact. : Thanks for your help. : Larry C. Purcell You might try calling Spectra-Physics Lasers, Inc. at 800-SPL-LASER (also 1-415-966-5628 for non-USA callers) with your question. The web site for SPL (www.splasers.com) suggests that it is a descendant of the original Spectra-Physics. They might know who currently is taking care of the rest of the original divisions of Spectra-Physics. This answer is a little indirect, but the telephone call might be worthwhile if you run out of other options. Gregg Wells Department of Pathology University of Pennsylvania Philadelphia, Pennsylvania USA email: wellsg@mail.med.upenn.edu From owner-proteins@net.bio.net Thu Aug 07 23:00:00 1997 Path: biosci!agate!newsgate.cuhk.edu.hk!news.cuhk.edu.hk!usenet From: Chan Siu Hong Newsgroups: bionet.molbio.proteins Subject: Sorry, correction for the sender of "how to charactrization of novel protein" Date: Sat, 09 Aug 1997 14:03:21 +0800 Organization: The Chinese University of Hong Kong Lines: 6 Message-ID: <33EC0829.544@mailserv.cuhk.edu.hk> Reply-To: s960637@mailserv.cuhk.edu.hk NNTP-Posting-Host: @137.189.47.153 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Win95; I) Dear everybody, I would like to ask if anybody know there is some methods or strategies to find out the cellular function of a novel protein sequence deduced from a novel cDNA sequence? The cDNA sequence is "fished" out by yeast two-hybrid system. From owner-proteins@net.bio.net Fri Aug 08 23:00:00 1997 Path: biosci!daresbury!uninett.no!Norway.EU.net!EU.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!howland.erols.net!news.starnet.net!news.starnet.net!news.cc.ukans.edu!usenet From: PGegen@UKans.edu (Dr. Peter Gegenheimer) Newsgroups: bionet.molbio.proteins Subject: Re: Sorry, correction for the sender of "how to charactrization of novel protein" Date: Sat, 09 Aug 1997 11:38:36 Organization: Univ. Kansas (Biochemistry) Lines: 31 Message-ID: <5si6hj$rc8@raven.cc.ukans.edu> Reply-To: PGegen@UKans.edu NNTP-Posting-Host: rnaworld.bio.ukans.edu X-Newsreader: ProNews/2 Version 1.00eb2B On Sat, 9 Aug 1997 15:37:53, Chan Siu Hong wrote: > Dear everybody, > > I would like to ask if anybody know there is some methods or strategies > to find out the cellular function of a novel protein sequence deduced > from a novel cDNA sequence? The cDNA sequence is "fished" out by yeast > two-hybrid system. Wasn't there a time when "science" meant "experimental"??? o---------------------------------------------------------------------o | Dr. Peter Gegenheimer | Vox: 785-864-3939 FAX: 785-864-5321 | | Departments of Biochemistry | PGegen@UKans.edu | | and of Botany | http://RNAworld.Bio.UKans.edu/ | | | | | University of Kansas | | | 2045 Haworth Hall | "The sleep of reason produces | | Lawrence KS 66045-2106 | monsters." Goya | o_____________________________|_______________________________________o From owner-proteins@net.bio.net Fri Aug 08 23:00:00 1997 Path: biosci!agate!hammer.uoregon.edu!vixen.cso.uiuc.edu!howland.erols.net!rill.news.pipex.net!pipex!join.news.pipex.net!pipex!iafrica.com!not-for-mail From: hoboes@iafrica.com (S. Rutherford) Newsgroups: bionet.molbio.proteins Subject: bacteriolytic peptide/lysozyme? Date: Sun, 10 Aug 1997 04:49:14 GMT Organization: UUNET Internet Africa Lines: 3 Message-ID: <33ed4820.3396926@dbn-news.iafrica.com> NNTP-Posting-Host: 196-31-98-147.iafrica.com X-Newsreader: Forte Free Agent 1.1/16.230 does anyone know of a bacteriolytic peptide/lysozyme which has a pH optimum in the region of 5 - 6? From owner-proteins@net.bio.net Sat Aug 09 23:00:00 1997 Path: biosci!bloom-beacon.mit.edu!eru.mt.luth.se!feed1.news.erols.com!cpk-news-hub1.bbnplanet.com!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!news.bbnplanet.com!news.ums.edu!gamera.cbl.umces.edu!cbl.umces.edu!reno From: "Reno T. Nguyen" Newsgroups: bionet.molbio.proteins Subject: TCA precipitation Date: Sun, 10 Aug 1997 10:39:18 -0400 Organization: University of Maryland, Chesapeake Biological Lab. Lines: 26 Message-ID: NNTP-Posting-Host: cbl.umces.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII Is anyone aware of a study which evaluated the minimum molecular weight of polypeptides/proteins that are precipitated on addition of TCA? I have heard that polypeptides > ~2000 Da are precipitated. This approximate MW cutoff is supposedly based on the study of ampholyte mixtures. I would like to obtain the literature on this information, but have been unsuccessful at finding it. Thanks for any clues. Reno ************************************************************************* Reno Nguyen Chesapeake Biological Laboratory /----\ /-----\ / Univ. MD Center for Environmental Science / / / / P.O. Box 38 / /______/ / Solomons, MD 20688 / / \ / / / // Tel: (410) 326-7261 \_______/ \_______/ \_______/ (410) 326-7409 Fax: (410) 326-7341 E-mail: reno@cbl.umces.edu ************************************************************************ From owner-proteins@net.bio.net Sun Aug 10 23:00:00 1997 Path: biosci!agate!hammer.uoregon.edu!csulb.edu!news-ext.gatech.edu!gatech!nntp-xfer.ncsu.edu!calvert From: calvert@eos.ncsu.edu (Phil Calvert) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins,bionet.cellbiol Subject: Dissolving Amino Acids in Scintillation Fluid Date: Tue, 12 Aug 1997 01:49:37 -0500 Organization: North Carolina State University Lines: 17 Distribution: world Message-ID: NNTP-Posting-Host: mac-27rd.ce.ncsu.edu Mime-Version: 1.0 Content-Type: text/plain; charset=ISO-8859-1 Content-Transfer-Encoding: 8bit X-no-archive: yes X-Newsreader: Yet Another NewsWatcher 2.4.0 Xref: biosci bionet.molbio.methds-reagnts:60346 bionet.molbio.proteins:11349 bionet.cellbiol:7914 I hydrolyzed some protein with HCl and passed the hydrolyzate through a small ion-exchange column. The amino acids were then eluted with 4M ammonium hydroxide. When I tried mixing this with scintillation fluid (Ultima Gold) it became very cloudy. I figured it must be the ammonia, so I blew it down to dryness with nitrogen. Well, I still couldn't get it to dissolve properly. This makes sense because amino acids don't dissolve very well in organic solvents, generally speaking. Is there a trick to this? I was thinking that maybe using a co-solvent like DMSO, DMF, pyridine, etc. might work, but this is just a hunch. Any suggestions? -- ------------------------------------ !-----------Phil Calvert-----------! !---calvert.NoSpam@eos.ncsu.edu----! ------------------------------------ NOTE: If present, the phrase ".NoSpam" must be removed from my return address before sending your reply. From owner-proteins@net.bio.net Sun Aug 10 23:00:00 1997 Path: biosci!em.uni-frankfurt.de!beschmann From: beschmann@em.uni-frankfurt.de ("Heike A. Beschmann") Newsgroups: bionet.molbio.proteins Subject: oxidized proteins Date: 11 Aug 1997 03:51:28 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 24 Sender: daemon@net.bio.net Distribution: world Message-ID: <3.0.2.32.19970811125102.0069121c@popmail.server.uni-frankfurt.de> NNTP-Posting-Host: net.bio.net As I am new to the field of protein research there are questions I hope someone can help me with. Purpose of our study is to determine if there are differences between the oxidation status of the cellular proteins of sperm cells of patients with unexplained infertility. The analysis will content different steps as there are: 1. isolating the proteins from the cells (making whole cell protein extract) 2. SDS-PAGE electrophoresis 3. ECL (luminescence) detection of protein carbonyl groups concerning step 1: Protein extraction is done by cell disrupture by detergent in the presence of several protease inhibitors. Unfortunately not only proteins are relased from the cells but also DNA (more or less) which interferes with the carbonyl group detection. Therefore I am looking for an easy and rapid method to remove the DNA out of the protein extract without loss of proteins as material is very limited. Some days ago somebody has mentioned Streptomycin precipitation. What is it and how does it work? Why does Streptomycin precipitate DNA? Thanks a lot for every input. Heike From owner-proteins@net.bio.net Sun Aug 10 23:00:00 1997 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!infeed1.internetmci.com!newsfeed.internetmci.com!portc03.blue.aol.com!newstf02.news.aol.com!audrey01.news.aol.com!not-for-mail From: prorenin@aol.com (Prorenin) Newsgroups: bionet.molbio.proteins Subject: Bio-Rad IEF mini tube gels Date: 11 Aug 1997 12:56:15 GMT Lines: 14 Message-ID: <19970811125601.IAA10456@ladder01.news.aol.com> NNTP-Posting-Host: ladder01.news.aol.com X-Admin: news@aol.com Organization: AOL http://www.aol.com Hi, Our lab currently has a high school student doing a school research project on protein iso-forms. She has been experimenting with the Bio-Rad mini IEF gels. She is having problems with Bio-Rads 2-D molecular weight markers. The only protien that develops when she silver stains the gel is BSA. Has anyone else had problems with this standard? How long do you run the tube gels? Jane has been running them at 750 V for close to 3 hours. Any help will be greatly appreciated. Tina Pitarresi CVC Cornell University Medical College From owner-proteins@net.bio.net Sun Aug 10 23:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!europa.clark.net!4.1.16.34!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news-ext.gatech.edu!gatech!nntp-xfer.ncsu.edu!calvert.NoSpam From: calvert.NoSpam@eos.ncsu.edu (Phil Calvert) Newsgroups: bionet.molbio.proteins Subject: Re: Where is Spectra-Physics Date: Tue, 12 Aug 1997 01:26:29 -0500 Organization: North Carolina State University Lines: 18 Distribution: world Message-ID: References: <33EB9673.5CB2@comp.uark.edu> NNTP-Posting-Host: mac-27rd.ce.ncsu.edu Mime-Version: 1.0 Content-Type: text/plain; charset=ISO-8859-1 Content-Transfer-Encoding: 8bit X-no-archive: yes X-Newsreader: Yet Another NewsWatcher 2.4.0 In article <33EB9673.5CB2@comp.uark.edu>, lpurcell@COMP.UARK.EDU ("Larry C. Purcell") wrote: > Does anyone know who bought Spectra-Physics or who is now servicing their > equipment. I have a SP HPLC that needs service and can not find a > contact. Larry, Spectra-Physics merged with some other company to form a new company called Thermo Separation Products. You can reach them at (800) 456-4552. -- ------------------------------------ !-----------Phil Calvert-----------! !---calvert.NoSpam@eos.ncsu.edu----! ------------------------------------ NOTE: If present, the phrase ".NoSpam" must be removed from my return address before sending your reply. From owner-proteins@net.bio.net Mon Aug 11 23:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!news-spur1.maxwell.syr.edu!news.maxwell.syr.edu!infeed2.internetmci.com!newsfeed.internetmci.com!jump.net!grunt.dejanews.com!not-for-mail Date: Tue, 12 Aug 1997 12:52:33 -0600 From: s953393@umslvma.umsl.edu Subject: Standard melting agarose Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Message-ID: <871407615.9508@dejanews.com> Reply-To: s953393@umslvma.umsl.edu Organization: UMSL To: s953395@umslvma.umsl.edu Electrophoresis: Agarose X-Article-Creation-Date: Tue Aug 12 17:40:16 1997 GMT X-Originating-IP-Addr: 205.139.231.175 (pm5x15.dialip.mo.net) X-Http-User-Agent: Mozilla/3.01 (Win95; I) X-Authenticated-Sender: s953393@umslvma.umsl.edu Lines: 10 Xref: biosci bionet.molbio.methds-reagnts:60359 bionet.molbio.proteins:11351 I am currently using FMC Seakem standard gelling agarose, but I am wondering if anyone has some suggestions on some other less expensive sources of standard melting agarose. I need to find a cheaper all purpose agarose. I would appreciate any help you could give. Thanking you in advance. Mike S. -------------------==== Posted via Deja News ====----------------------- http://www.dejanews.com/ Search, Read, Post to Usenet From owner-proteins@net.bio.net Mon Aug 11 23:00:00 1997 Path: biosci!daresbury!lyra.csx.cam.ac.uk!drm21 From: drm21@mole.bio.cam.ac.uk (David Micklem) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: Standard melting agarose Date: Tue, 12 Aug 1997 21:35:49 +0000 Organization: Wellcome/CRC Institute Lines: 33 Message-ID: References: <871407615.9508@dejanews.com> NNTP-Posting-Host: drm-mac1.welc.cam.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=ISO-8859-1 Content-Transfer-Encoding: 8bit X-Newsreader: Yet Another NewsWatcher 2.3.0 Xref: biosci bionet.molbio.methds-reagnts:60367 bionet.molbio.proteins:11352 In article <871407615.9508@dejanews.com>, s953393@umslvma.umsl.edu wrote: >I am currently using FMC Seakem standard gelling agarose, but I am >wondering if anyone has some suggestions on some other less expensive >sources of standard melting agarose. I need to find a cheaper all purpose >agarose. I would appreciate any help you could give. Thanking you in >advance. > >Mike S. You could save money by re-using it - assuming that it is being used mostly for analytic gels.. I used to pour gels with a comb inserted at both ends. The first comb was loaded and the gel run as normal (with EtBr in the gel and buffer). After photographing, the gel is returned to the tank, and run backwards for a few minutes. Next time a gel is needed, use the second set of wells. Run the gel far enough for the first set of DNA to run back to the first set of wells, past them, and off the gel. Repeat indefinitely (or until you trash the wells), using fresh buffer quite frequently and rinsing the wells with a pasteur before each use. David -- D.R.Micklem, Time flies like an arrow... Wellcome/CRC Institute, Fruit flies like a banana. Cambridge CB2 1QR, UK Email:drm21@mole.bio.cam.ac.uk Unsolicited mail will incur a US$100 processing charge. From owner-proteins@net.bio.net Mon Aug 11 23:00:00 1997 Path: biosci!agate!newsfeed.kornet.nm.kr!news.maxwell.syr.edu!news.apfel.de!newscore.univie.ac.at!03-newsfeed.univie.ac.at!news.univie.ac.at!news-admin@univie.ac.at From: harald@mol.univie.ac.at Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins,bionet.cellbiol Subject: Re: Dissolving Amino Acids in Scintillation Fluid Date: Tue, 12 Aug 97 15:11:25 PST Organization: Institute of Molecular Biology Lines: 21 Distribution: world Message-ID: <5spnga$ffo@www.univie.ac.at> References: Reply-To: harald@mol.univie.ac.at NNTP-Posting-Host: p2522.mol.univie.ac.at Mime-Version: 1.0 X-Newsreader: WinVN 0.93.0 Xref: biosci bionet.molbio.methds-reagnts:60352 bionet.molbio.proteins:11350 bionet.cellbiol:7918 In article , calvert@eos.ncsu.edu says... > >I hydrolyzed some protein with HCl and passed the hydrolyzate through a >small ion-exchange column. The amino acids were then eluted with 4M >ammonium hydroxide. When I tried mixing this with scintillation fluid >(Ultima Gold) it became very cloudy. I figured it must be the ammonia, so >I blew it down to dryness with nitrogen. Well, I still couldn't get it to >dissolve properly. This makes sense because amino acids don't dissolve >very well in organic solvents, generally speaking. Is there a trick to >this? I was thinking that maybe using a co-solvent like DMSO, DMF, >pyridine, etc. might work, but this is just a hunch. Any suggestions? Sure. I suggest using Dioxan or a commercially available Szintillation cocktail (I don t recall the name) which allows mixing with water. Also, you could try to bring the AA in the salt form by adding a small amount of e.g. NaOH in order to make them dissolve better in water, since amino acids sometime just refuse to dissolve, but the salts should not. DMSO is a common solvent for both inorganic and organic salts and is definitely worth a try. hope this helps, Harald From owner-proteins@net.bio.net Mon Aug 11 23:00:00 1997 Path: biosci!news.Stanford.EDU!nntp.Stanford.EDU!not-for-mail From: punk@leleand.stanford.edu Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: Standard melting agarose Date: Tue, 12 Aug 1997 13:55:57 -0700 Organization: Stanford University Lines: 1 Message-ID: <33F0CDDC.12F9@leleand.stanford.edu> References: <871407615.9508@dejanews.com> NNTP-Posting-Host: d322-powermac8500.stanford.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0 (Macintosh; I; PPC) Xref: biosci bionet.molbio.methds-reagnts:60369 bionet.molbio.proteins:11353 We use gibco agarose I don't know if it is cheaper . works fine From owner-proteins@net.bio.net Tue Aug 12 23:00:00 1997 Path: biosci!vt.edu!akarabay From: akarabay@vt.edu (Arzu Karabay) Newsgroups: bionet.molbio.proteins Subject: Extinction coefficient Date: 13 Aug 1997 13:00:13 -0700 Organization: student Lines: 16 Sender: daemon@net.bio.net Distribution: world Message-ID: <33F221F5.58AC@vt.edu> Reply-To: akarabay@vt.edu NNTP-Posting-Host: net.bio.net I have been trying to measure the extinction coefficients of the recombinant fusion proteins that I purified. I have used Guanidium hydrochloride. The proteins are purified with denaturation method and are in high grade protein running buffer (0.025 M Tris, pH8.3, 0.192 M glycine,and 0.1 % SDS). I used 6 M Guanidium hydrochloride (150 ul of 8 M Guanidium hydrocholoride and 50 ul of the protein in 200 ul volume). every time I did the measurement with the same protein I got different results.. I have this reference: Analytical Biochemistry 182, 319-326 (1989), but it does not have a clear protocol. I do not know if there is a certain incubation period with GndHCl. Then I decided to do a time course measurement, and I got different measurements at different time points. If anybody have any explanation for this and a clearer protocol on measurement of the extinction coefficient, and exactly how to calculate it this will help me a lot. I really am looking forward to your great ideas. Thanks for your help. From owner-proteins@net.bio.net Tue Aug 12 23:00:00 1997 Date: Wed, 13 Aug 1997 11:18:08 +0200 From: fof1@chclu.chemie.uni-konstanz.de (Frank O. Fackelmayer) Newsgroups: bionet.molbio.proteins Subject: Re: Sorry, correction for the sender of "how to charactrization of novel protein" Message-ID: References: <5si6hj$rc8@raven.cc.ukans.edu> NNTP-Posting-Host: zellkultur.biologie.uni-konstanz.de Organization: University of Constance, Germany Lines: 66 Path: biosci!agate!hammer.uoregon.edu!vixen.cso.uiuc.edu!howland.erols.net!newsfeed.nacamar.de!fu-berlin.de!news.belwue.de!news.uni-konstanz.de!zellkultur.biologie.uni-konstanz.de!user In article <5si6hj$rc8@raven.cc.ukans.edu>, PGegen@UKans.edu wrote: > On Sat, 9 Aug 1997 15:37:53, Chan Siu Hong > wrote: > > > Dear everybody, > > > > I would like to ask if anybody know there is some methods or strategies > > to find out the cellular function of a novel protein sequence deduced > > from a novel cDNA sequence? The cDNA sequence is "fished" out by yeast > > two-hybrid system. > > Wasn't there a time when "science" meant "experimental"??? > > o---------------------------------------------------------------------o > | Dr. Peter Gegenheimer | Vox: 785-864-3939 FAX: > 785-864-5321 | > | Departments of Biochemistry | PGegen@UKans.edu > | > | and of Botany | http://RNAworld.Bio.UKans.edu/ > | > | | > | > | University of Kansas | > | > | 2045 Haworth Hall | "The sleep of reason produces > | > | Lawrence KS 66045-2106 | monsters." Goya > | > o_____________________________|_______________________________________o Hi Peter, Your comment is not very polite. You´re on the wrong track if you assume that "some methods or strategies to find out the cellular function of a novel protein" refers to anything other than experiments. What´s a "method" for you? As to the original question: Yeah, that´s a problem many people have to face these days. I would suggest the following procedure: * database comparison: is there any protein homologous to yours, and what function does it have -> positive: try whether your protein has comparable activity -> negative: * look for conserved protein motifs, e.g. with the PROSITE database -> positive: do experiments in that direction (e.g. activity tests) -> negative: * guess: what could be the activity of your protein when it interacts with a protein you know (it was picked by two hybrid screen)? possible: activate, inhibit, regulate.... -> idea: test it -> no idea: * make sure the interaction is not a two-hybrid artifact * produce antibodies against the protein or epitope-tag your protein to see where it is in the cell * what could it do there: (eg nuclear protein: DNA or RNA binding) If you don´t come up with anything this way, you will have no choice but to biochemically characterize the protein, hoping you get a hint as to the function. Hope this helps, Frank From owner-proteins@net.bio.net Tue Aug 12 23:00:00 1997 Path: biosci!bloom-beacon.mit.edu!panix!howland.erols.net!csir.uni.net.za!uct.uni.net.za!iafrica.com!not-for-mail From: hoboes@iafrica.com (S. Rutherford) Newsgroups: bionet.molbio.proteins Subject: bacteriolytic peptides? Date: Thu, 14 Aug 1997 03:34:03 GMT Organization: UUNET Internet Africa Lines: 3 Message-ID: <33f27c91.1410589@dbn-news.iafrica.com> NNTP-Posting-Host: 196-31-98-33.iafrica.com X-Newsreader: Forte Free Agent 1.1/16.230 does anyone know of a bacteriolytic peptide/lysozyme which has a pH optimum in the region of 5 - 6? From owner-proteins@net.bio.net Tue Aug 12 23:00:00 1997 Path: biosci!agate!newsfeed.kornet.nm.kr!howland.erols.net!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!sunqbc.risq.net!athena.ulaval.ca!poste43-156.fsaa.ulaval.ca!user From: aai140@agora.ulaval.ca (Savita Visal) Newsgroups: bionet.molbio.proteins Subject: induction of jasmonic acid Date: Wed, 13 Aug 1997 11:48:05 -0500 Organization: CRH, Pav. Envirotron, Univ.Laval, Quebec, Canada Lines: 14 Message-ID: NNTP-Posting-Host: poste43-156.fsaa.ulaval.ca Keywords: JA, gamma-linolenic acid, defence pathway Hello Netters, I have a question: Is gamma-linolenic acid involved in the defence pathway?.. I have'nt got a reference stating the involvement of this molecule...in contrast there are lot many papers about alfa linolenic acid as a precursor molecule in the synthesis of JA.... Does anyone have info/ref about gamma LA being an intermediate molecule in the JA biosynthesis pathway? Thanks Savita email: aai140@agora.ulaval.ca PH: 1 418 656 2131 x 8874 Fax: 1 418 656 7871 From owner-proteins@net.bio.net Tue Aug 12 23:00:00 1997 Path: biosci!agate!howland.erols.net!infeed1.internetmci.com!newsfeed.internetmci.com!192.48.96.125!in3.uu.net!138.133.17.7!amgen!news From: John Philo Newsgroups: bionet.molbio.proteins Subject: Re: Extinction coefficient Date: Wed, 13 Aug 1997 14:20:46 -0700 Organization: Amgen Lines: 92 Message-ID: <33F2252E.7956@amgen.com> References: <33F221F5.58AC@vt.edu> Reply-To: jphilo@amgen.com NNTP-Posting-Host: worf.amgen.com Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01GoldC-CIS0597c (Win95; U) To: akarabay@vt.edu Arzu Karabay wrote: > > I have been trying to measure the extinction coefficients of the > recombinant fusion proteins that I purified. I have used Guanidium > hydrochloride. The proteins are purified with denaturation method and > are in high grade protein running buffer (0.025 M Tris, pH8.3, 0.192 M > glycine,and 0.1 % SDS). I used 6 M Guanidium hydrochloride (150 ul of > 8 M Guanidium hydrocholoride and 50 ul of the protein in 200 ul > volume). every time I did the measurement with the same protein I got > different results.. I have this reference: Analytical Biochemistry > 182, 319-326 (1989), but it does not have a clear protocol. I do not > know if there is a certain incubation period with GndHCl. Then I > decided to do a time course measurement, and I got different > measurements at different time points. If anybody have any explanation > for this and a clearer protocol on measurement of the extinction > coefficient, and exactly how to calculate it this will help me a lot. > I really am looking forward to your great ideas. > Thanks for your help. Arzu, I agree the Gill & von Hippel paper is not very clear about the purpose of the GuHCl measurement or how to do it. Much of this confusion arises because what many people describe as a "measurement" of an extinction coefficient is not truly (in my opinion) a measurement, but merely an experimental correction to a theoretical extinction coefficient calculated from the amino acid composition of the protein (commonly using the method described in that paper). Fundamentally, the basis of the Gill & von Hippel method relies on data from H. Edelhoch, Biochemistry 6, pages 1948-1954, 1956 for the extinction coefficients of Trp, Tyr, and cystine in 6 M guanidineHCl at 280 nm (a wavelength where other amino acids do not contribute). The underlying assumption, then, is that since proteins are unfolded in 6M GuHCl (at least usually), their extinction coefficients in that solvent should be independent of sequence and can be calculated simply by adding (on a molar basis) values the appropriate numbers of Trp, Tyr, and cystines. Basically what Gill & von Hippel argue is that since, in practice, the difference in extinction at 280 between unfolded and native proteins is usually only a few percent, one can get a reasonable accurate calculated extinction coefficient for the NATIVE protein using the number calculated for 6M GuHCl. The purpose of actually measuring a particular protein in GuHCl is to experimentally determine the difference between the folded and unfolded protein, in order to correct the number calculated for GuHCl and get a more accurate number for the folded protein. (While some people call the result of this correction an "experimental" extinction coefficient, it really is just a corrected theoretical one.) Thus simply putting your protein into GuHCl and measuring the spectrum actually tells you nothing, since you still have no independent measurement of the protein concentration from which to calculate a true experimental extinction coefficient. What you may want to do, however, is to do a side-by-side comparison of your protein in GuHCl and under native conditions in order to correct the theoretical number for the changes induced by folding. To do this you need to measure the ratio (extinction native)/(extinction in GuHCl). This is usually done by careful, side-by-side dilutions of a high concentration stock of your native protein, diluting either with native buffer or 8M GuHCl such that you end up with equal protein concentrations in 6 M GuHCl and native buffer. (Obviously how equal the concentrations are depends on the accuracy of the dilutions). The dilutions can either be done into a pair of matched cuvettes, or sequentially into the same cuvette, and the ratio of the absorbance at 280 of these two solutions gives you the ratio of folded versus unfolded extinction coefficients. With regard to calculating the number in GuHCl, this calculation is done by most sequence analysis packages such as GCG (although they usually don't actually say what numbers they are using). One thing to be aware of is that such packages generally assume that all cysteines in the sequence become oxidized to disulfides (cystines), EVEN WHEN THE NUMBER IS ODD!!! Your protein may be intracellular, and therefore unlikely to contain disulfides. If you want to do the calculation manually, if x, y, & z are the number of Trp, Tyr, and disulfides, respectively, and the total molecular weight is M, then the extinction coefficient (absorbance at 280 nm for 1 mg/ml protein and 1 cm pathlength) in GuHCl is: (5690*x + 1280*y + 120*z)/M Lastly, you may want to look at Pace et al. (1995) Protein Science 4, 2411-2423 where there is a slightly different set of numbers which is supposed to directly apply to the folded protein. 'Hope this helps, John Philo, Protein Chemistry, Amgen From owner-proteins@net.bio.net Tue Aug 12 23:00:00 1997 Path: biosci!internet!biosci!not-for-mail From: biohelp (BIOSCI Administrator) Newsgroups: bionet.molbio.proteins Subject: BIOSCI/bionet miniFAQ & Fundraiser Date: 13 Aug 1997 02:00:06 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 233 Sender: daemon@net.bio.net Distribution: world Message-ID: <199708130900.CAA16024@net.bio.net> NNTP-Posting-Host: net.bio.net (LAST REVISION: 30-JUL-95) This BIOSCI "miniFAQ" is designed to answer the questions that come up the *most frequently*. The main BIOSCI FAQ (Frequently Asked Questions) is accessible on the World Wide Web at URL http://www.bio.net/. If you can not find an answer to your question in this or other documentation, the BIOSCI technical support staff answers e-mail queries sent to biosci-help@net.bio.net We can only answer questions about the use of the newsgroups and mailing lists. We unfortunately do not have the staff to do Internet information searches or answer scientific questions. Please post those to the appropriate BIOSCI/bionet newsgroups. Contents: -------- 0) BIOSCI NEEDS YOUR SUPPORT!! 1) Using the WWW to access the BIOSCI/bionet newsgroups. 2) What to do about "spams," i.e., junk mail, ads, etc. 3) Examples of subscribing and unsubscribing to the mailing lists. 4) The BIOSCI user address and research interest directory. 0) BIOSCI NEEDS YOUR SUPPORT!! ------------------------------ BIOSCI's government funding has been expended, and we are now operating solely from advertising revenue that we have raised from our Web site at http://www.bio.net/. We need just a few minutes of your time to help us serve you. You can do two important things which will take very little time for you individually and will immensely help us continue to help you. First, please use our WWW system at http://www.bio.net/ to access the archives. You can post or reply to messages via your Web browser as described in item #1 below. Your usage helps attract sponsors. If you contact any of our sponsors, please be sure to thank them for supporting BIOSCI. It is critical for them to get this feedback if they are to continue their sponsorship for the long term. Second, if you work for a company or organization that provides products or services of interest to the biology community, please pass this message on to your marketing or marketing communications department or other appropriate group. Please ask them to help support BIOSCI by sponsoring our Web site and explain the uses and benefits of the system to the biology community. If they are interested, they can then contact us for further information at our tech support address, biosci-help@net.bio.net. 1) Using the WWW to access the BIOSCI/bionet newsgroups. -------------------------------------------------------- As of 10 December 1995, all BIOSCI/bionet full newsgroups are accessible through the World Wide Web (WWW) at URL http://www.bio.net. One can read and reply publicly or privately to both recent postings and archived messages through one's Web browser if it is configured properly to send e-mail. Each newsgroup is equipped with its own WAIS index. The main BIOSCI home page also has access to the BIO-JOURNALS Table of Contents database WAIS index and the BIOSCI user address database described in another item further below. 2) What to do about "spams," i.e., junk mail, ads, etc. ------------------------------------------------------- BIOSCI is a set of parallel USENET newsgroups (the "bionet" groups), mailing lists, and a hypermail archive at URL http://www.bio.net/. The same postings are distributed on all media (except for a small number of mailing-list-only groups at net.bio.net). Unfortunately it is becoming a despicable practice on the Internet (by a few people out to make a fast buck) to do automated mass postings to thousands of newsgroups and mailing lists. These attempts to grab free advertising are refered to as "spams" in the usual, somewhat boneheaded, net terminology. USENET is more susceptible to this practice, and many spams originate on the USENET groups and then are passed on to the mailing lists. However, spammers also get lists of mailing addresses and hit these too, so neither medium is immune. What should you do personally if you get junk mail? --------------------------------------------------- Just delete it and move on without reading it further. Filing a protest is becoming increasingly useless because spammers are often disguising the addresses where the messages are sent from. Unless you really understand Internet mail systems, your attempt at protest by sending replies to the message will often end up being sent to the address of an innocent person that the spammer is victimizing. What can BIOSCI/bionet do to protect its newsgroups? ---------------------------------------------------- The only solution currently available is to moderate the newsgroup. If this newsgroup is already moderated, then you are in good shape. Moderation protects the USENET distribution from about 95% of the spams that are being sent to date and protects the mailing lists completely. Moderation means, however, that someone has to take the time to review each message before it goes out. We have set up software here that simply allows the moderator to forward to an address at net.bio.net messages that (s)he wishes to have distributed. This takes no more time than that needed to read the message and pass it on, say about 1 min. per message. Most newsgroups currently have a discussion leader who is responsible for their newsgroup. The discussions leaders and their e-mail addresses are listed in the BIOSCI Information Sheet which is available on the Web at http://www.bio.net/. If a newsgroup is being hit with too many junk postings, please contact the discussion leader for that group and see if there is interest in moderating the group. Please do not assume that by simply posting a complaint to the newsgroup itself, anyone on the BIOSCI staff will act on your complaint. With close to 100 newsgroups to run, the BIOSCI staff has to rely on the discussion leaders of each newsgroup to report problems directly to us at biosci-help@net.bio.net. We will moderate any of our newsgroups if the discussion leader tells us that the readership of the group wishes to do so and if a moderator is willing to do the work. For most BIOSCI/bionet groups, this entails only a few minutes of work each day. Moderating a newsgroup will resolve probably 95% of the junk postings on the USENET distribution. Unfortunately there are easy ways for determined spammers to override the moderation mechanism on USENET, but we can protect our e-mail subscribers from unwanted postings if the newsgroup is moderated. You can also access our newsgroups over the WWW at URL http://www.bio.net. While this Web interface will not stop spammers from trying to post to the groups, this will give you yet another way, besides using USENET news, to keep the junk out of your personal mail files. For those of you with local USENET news systems, the Web interface will also give you faster access to new newsgroups and recent postings. 3) Examples of subscribing and unsubscribing to the mailing lists. ------------------------------------------------------------------ PLEASE NOTE: The BIOSCI management does NOT act on subscription/unsubscription requests that are posted improperly to the newsgroups and mailing lists. People who do this only bother everyone on the lists to no avail. Please be sure to follow the proper procedures below. Gory details are in the BIOSCI Information sheets on the Web at http://www.bio.net. Below we give an example utilizing the METHODS-AND-REAGENTS list at both of our two BIOSCI sites: Users in the Americas and Pacific Rim countries who use the BIOSCI ------------------------------------------------------------------ node at computer net.bio.net: ---------------------------- A) Determine the "listname" which is the <=8 character mail address ^^^^^^^^^^^^^ for the group. These can be found in the BIOSCI Info. Sheet. For the METHODS-AND-REAGENTS group the mailing address is methods@net.bio.net. The listname is the portion of the address to the left of the @ sign, i.e., "methods". The listname is used with the "subscribe" and "unsubscribe" commands illustrated below. B) Mail all commands in the body of a mail message addressed to biosci-server@net.bio.net. Do NOT send commands to the newsgroup posting addresses! Leave the Subject: line blank, any text on it will be ignored. C) In the body of your message put one or more of the following commands with an "end" command on the last line, e.g., subscribe methods unsubscribe methods end Do NOT put your e-mail address or other text on these lines. The server only allows you to cancel your subscription if the address on your mail header matches the address on our mailing list. Please ask for help at biosci-help@net.bio.net if your address has changed, e.g., if you know you are on the list but the server tells you that you are not a member. Users in Europe, Africa, and Central Asia who use the BIOSCI node at -------------------------------------------------------------------- computer daresbury.ac.uk (also known as dl.ac.uk): ------------------------------------------------- To subscribe and unsubscribe to/from the BIOSCI lists, you need to specify the full USENET newsgroup name with "bionet-news." prepended. The USENET newsgroup names are listed in the BIOSCI Information sheet on the Web at http://www.bio.net/. For the METHODS-AND-REAGENTS list the USENET newsgroup name is bionet.molbio.methds-reagnts, thus the appropriate commands are sub bionet-news.bionet.molbio.methds-reagnts unsub bionet-news.bionet.molbio.methds-reagnts These commands are included in a message addressed to mxt@dl.ac.uk, NOT to the newsgroup mailing addresses. As usual, include the text in the body of the message as text on the Subject: line is ignored. To unsubscribe from all the lists at the UK node, use unsub bionet-news Please note that if the address in the list is different than the one in your mail message header, you will not be able to unsubscribe by this method. If you have problems, please mail biosci@daresbury.ac.uk. 4) The BIOSCI user address and research interest directory. ----------------------------------------------------------- Please take this opportunity to add your name, address, and research interest information to the BIOSCI User Address Database if you have not already done so. You can fill out the address form directly through our Web page at URL http://www.bio.net/adrform.html. The address database is reindexed nightly for WWW access (the URL is http://www.bio.net/). If you are not directly on the Internet but can reach it by e-mail, please use our waismail server to access the user directory. waismail use is described above. You can also request a user address form by e-mail from biosci-help@net.bio.net. Please check your database entry from time-to-time to see if your address information is still up-to-date. Because of our limited personnel resources, we ask that you resubmit a *complete* form to revise your entry; we only replace complete entries and do not have resources to edit old forms. From owner-proteins@net.bio.net Tue Aug 12 23:00:00 1997 Path: biosci!agate!howland.erols.net!rill.news.pipex.net!pipex!join.news.pipex.net!pipex!server1.netnews.ja.net!server5.netnews.ja.net!daresbury!not-for-mail From: Newsgroups: bionet.molbio.proteins Subject: Is Your Web Site A Secret? Date: 14 Aug 1997 04:58:23 +0100 Lines: 131 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <5stvov$mhc@mserv1.dl.ac.uk> Reply-To: owl@owlsnest.com Original-To: proteins@dl.ac.uk Is your web site the best kept secret on the Internet? We'll promote it to 50 search engines and indexes for $85 and complete the job in 2 business days. Satisfaction is guaranteed! If you have a great product, but are not getting many inquiries from your Web site, you may not be adequately listed on the Web's search engines and indexes. Millions of viewers daily use these facilities to find the products and services they are looking for. But if your site is not listed, no one will see it. Listings on most of these services are free. However, locating and filling out the forms required to get a listing can take several days, and most people just don't have the time to do it. That is why we offer a web site promotion service. WHAT'S THE DEAL? We will submit your site to 50 indexes and search engines for $85. We will accept the return of this E-mail, with the form below filled out, as an order. We will bill you upon completion of the promotion. Our terms are net 15 days from date of invoice. Satisfaction guaranteed! HOW LONG WILL IT TAKE? Generally, we complete the submissions within 48 hours of receiving your order. It can take any individual search engine or index up to three weeks to process your submission, although most are much faster. WHAT SEARCH ENGINES AND INDEXES ARE INCLUDED IN THE PROMOTION? The list changes from time to time. This is our current list: Abaweb!, Alta Vista, Been There, BizWeb, Central Source Yellow Pages, Enterpreneurs on the Web, Excite, Four11, Galaxy, I-Network I-Systems Spiral Business Directory, I-World Web Pointer, Infoseek, Inktomi, Innovator's Network Yellow Pages, Internet Mall, Jayde Online Directory, Jumpcity, Jumper Hot Links, Linkmaster, Lycos, Magellan, Mega Mall, Net-Happenings, Net Navigator, Net Mall, NTG's List, NYNEX Big Yellow, One World Plaza, OnLine's WWWeb Index, Rex, Starting Point, Truenorth, URL Tree, Virtual Lynx, Web Point, WebCentral, Web Venture Hotlist, Webcrawler, Websurf, Win Mag/NetGuide Hotspots, WhatUSeek, Worldwide Announce Archive, WWW Business Yellow Pages, World Wide Yellow Pages, WWW Worm, YelloWWWeb. HOW WILL I KNOW THAT YOU HAVE PROMOTED MY SITE? When we have completed the promotion, we will send you an HTML file as an attachment to your E-mail bill. Save this file to your disk, and view it through your Web browser. It provides links to the search engine we submitted your site to, plus any comments we received from them when we did it. ARE THERE ANY GUARANTEES? We do not require prepayment. Your satisfaction is guaranteed or you don't pay the bill. WHO IS OWL'S EYE PRODUCTIONS? We are a web site promotion company located at: Owl's Eye Productions, Inc. 260 E. Main Street Brewster, NY 10509 Phone: (914) 278-4933 Fax: (914) 278-4507 Email: owl@owlsnest.com HOW DO I ORDER? The easiest way to order is by e-mail. Just hit the REPLY button on your e-mail program and fill out the following information. (This information will be posted to the search engines/indexes): Your name: Company Name: Address: City: State/Prov: Zip/Postal Code: Telephone: Fax: Email address: URL: http:// Site Title: Description (about 25 words): Key words (maximum of 25, in descending order of importance): Proofs (Where shall we e-mail proofs): If billing a different address, please complete the following: Addressee: Company Name: Address: City: State/Prov: Zip/Postal Code: Telephone: Fax: Email address: We will bill via Email. (7519) Terms: By returning this document via Email, you agree as follows: You have the authority to purchase this service on behalf of your company. Terms are net 15 days. Accounts sent to collections will be liable for collection costs. You agree to protect and indemnify Owl's Eye Productions, Inc. in any claim for libel, copyright violations, plagiarism, or privacy and other suits or claims based on the content or subject matter of your site. WHAT HAPPENS NEXT? When we receive your order, we will input the information into our system, and send you a proof. After we process any corrections, we will run your promotion, capturing any comments from search engines as we go. We will incorporate these into an HTML-formatted report to you, which we will attach to your bill. ===Web Promotions=====Press Releases=====Link Exchanges========= Owl's Eye Productions, Inc. 260 E. Main Street Brewster, NY 10509 Ph: 914-278-4933 Fx: 914-278-4507 E-mail: owlseye@owlsnest.com From owner-proteins@net.bio.net Wed Aug 13 23:00:00 1997 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!baron.netcom.net.uk!netcom.net.uk!news-peer!btnet!newsfeed.cableol.net!server4.netnews.ja.net!news.cc.ic.ac.uk!not-for-mail From: "Dr. Said Dermime" Newsgroups: bionet.molbio.proteins Subject: Re: Tris/Tricine gels Date: Thu, 14 Aug 1997 10:30:08 +0100 Organization: Dept. Immunology, RPMS, DuCane Road, London W12 0NN, UK Lines: 22 Message-ID: <33F2D01D.7547@rpms.ac.uk> References: <54nnfc$7uc_001@leeds.ac.uk> NNTP-Posting-Host: imm5cwb2.rpms.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01Gold (Macintosh; I; PPC) > Dear Netters, > How does the Tris/Tricine PAGE method of Schaegger and von Jagow succeed over > that of Laemmli in resolving low molecular weight polypeptides? > A (quick) look over the literature hasn't given me any clear description of > the theory behind the methods. > An explanation or reference giving a good explanation would be most helpful. > Thank you > Rob > Dept. of Biochemistry and Molecular Biology > University of Leeds > W. Yorks., England, U. K. Dear Rob, In my experience, yes. With Laemmli system the lower limit is somewhere around 15kD, with tricine (which migrates faster than glycine) I can reach down to about 3kDa in 13.5% gel prepared from 40% premix obtained from BioRad (other companies' solutions work as well). The protein standard I use is from NEB broad-range which covers everything from 2 kD to 200 kD. Armin a.sepp@rpms.ac.uk From owner-proteins@net.bio.net Wed Aug 13 23:00:00 1997 Path: biosci!agate!ihnp4.ucsd.edu!gondor!newshub.sdsu.edu!newshub.csu.net!csulb.edu!news-ext.gatech.edu!gatech!newsfeed.pitt.edu!pitt.edu!dsinc!nntp.upenn.edu!taurus.fccc.edu!sauder From: sauder@polaris.fccc.edu (John Michael Sauder) Newsgroups: bionet.molbio.proteins Subject: Re: Extinction coefficient Date: 14 Aug 1997 18:27:29 GMT Organization: Fox Chase Cancer Center, Philadelphia, PA Lines: 13 Message-ID: <5svimh$4bb$1@taurus.fccc.edu> References: <33F221F5.58AC@vt.edu> NNTP-Posting-Host: polaris.fccc.edu In article <33F221F5.58AC@vt.edu> akarabay@vt.edu writes: > I have been trying to measure the extinction coefficients of the > recombinant fusion proteins that I purified. I have used Guanidium > hydrochloride. > I do not know if there is a certain incubation period with GndHCl. Then > I decided to do a time course measurement, and I got different measurements > at different time points. Sounds like you're watching unfolding kinetics (or maybe photo- bleaching). Keep monitoring it; hopefully you will "soon" get a baseline signal. Many proteins unfold within seconds in concentrated GndHCl, although I'm sure there are exceptions. Make sure your final signal is relatively unchanging. From owner-proteins@net.bio.net Thu Aug 14 23:00:00 1997 Path: biosci!agate!howland.erols.net!newsfeed.nacamar.de!news-kar1.dfn.de!news-fra1.dfn.de!news-koe1.dfn.de!uni-muenster.de!news From: "Ralf Spenneberg" Newsgroups: bionet.molbio.proteins Subject: FIRST ANNOUNCEMENT: FIFTH EUROPEAN SYMPOSIUM ON CALCIUM BINDING PROTEINS IN NORMAL AND TRANSFORMED CELLS Date: Fri, 15 Aug 1997 15:00:30 GMT Organization: Westfaelische Wilhelms-Universitaet Muenster, Germany Lines: 50 Sender: "Ralf Spenneberg" Message-ID: <5t0r9d$ssm@majestix.uni-muenster.de> NNTP-Posting-Host: ifmbpc01.uni-muenster.de X-Newsreader: Forte Free Agent 1.0.82 FIRST ANNOUNCEMENT FIFTH EUROPEAN SYMPOSIUM ON CALCIUM BINDING PROTEINS IN NORMAL AND TRANSFORMED CELLS MUENSTER, GERMANY, JULY 30 - AUGUST 2, 1998 Topics: Ca2+ sensing and signalling Ca2+ fluxes and channels Ca2+ regulated membrane/protein transport Ca2+ binding proteins (EF hand proteins, annexins, C2 domain proteins) Speakers and chairpersons include: R. Barraclough (Liverpool, UK), C.E. Creutz (Charlottesville, VA, USA), T.N. Davis (Seattle, WA, USA), J. Dedman (Cincinnati, OH, USA), R.R. Frants (Leiden, Netherlands), D.J. Gawler (Leeds, UK), H. Haigler (Irvine, CA, USA), R. Huber (Martinsried, Germany), C.B. Klee (Bethesda, MD, USA), A. Konnerth (Homburg, Germany), R.H. Kretsinger (Charrlottesville, VA,USA), T.F.J. Martin (Madison, WI, USA), S.E. Moss (London, UK), E. Neher (Goettingen, Germany), A. Polans (Madison, WI, USA), J. Putney (Res. Triangle Pk., NC, USA), B.W. Schäfer (Zuerich, Switzerland), E. Schiebel (Martinsried, Germany), C. Sorg (Muenster, Germany), R.V. Thakker (London, UK), R.J.P. Williams (Oxford, UK). More Information: http://www-ifi.uni-muenster.de/Dokumente/ZMBE/institute/ifmb/CaBP_Meeting/start..html Ralf Spenneberg Institut für medizinische Biochemie Tel. +49 251 8356723 ZMBE-Center for molecular biology of inflammation Fax. +49 251 8356748 Von Esmarchstraße 56 D-48149 Muenster Germany From owner-proteins@net.bio.net Thu Aug 14 23:00:00 1997 Path: biosci!daresbury!uninett.no!Norway.EU.net!EU.net!iagnet.net!128.255.40.11!news.uiowa.edu!news.physics.uiowa.edu!newsrelay.iastate.edu!news.iastate.edu!gbh From: gbh@iastate.edu (Joel Nott) Newsgroups: bionet.molbio.proteins Subject: Protein Workshop Date: 14 Aug 1997 21:23:39 GMT Organization: Iowa State University, Ames, Iowa, USA Lines: 20 Message-ID: <5svt0r$151$1@news.iastate.edu> NNTP-Posting-Host: pv0a25.vincent.iastate.edu X-Newsreader: NN version 6.5.1 (NOV) We are pleased to announce our third annual protein workshop "Protein/Peptide Structure, Function and Characterization" to be held in the Molecular Biology Building of Iowa State University in Ames, IA on August 21 and August 22. The workshop will consist of talks, posters, information and hands-on workshops dealing with protein/peptide structure, function and characterization. Registration is free and everyone is w elcome to attend. If you would like more information or would like to register, feel free to call us at 515-294-3267, FAX at 515-294-9968 or e-mail at: protein@iastate.edu. More information is also available online at http://www.biotech.iastate.edu/facilities/protein/ Joel Nott Iowa State University Protein Facility 1182 Molecular Biology Building Ames, IA 50011 -- Joel Nott From owner-proteins@net.bio.net Thu Aug 14 23:00:00 1997 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!howland.erols.net!newsfeed.nacamar.de!blackbush.xlink.net!rz.uni-karlsruhe.de!news.urz.uni-heidelberg.de!news.dkfz-heidelberg.de!news From: U.Deligezer@DKFZ-Heidelberg.de Newsgroups: bionet.molbio.proteins Subject: aggregate formation Date: 15 Aug 1997 07:40:40 GMT Organization: DKFZ Lines: 4 Message-ID: <5t115o$h90@news.inet.dkfz-heidelberg.de> Reply-To: U.Deligezer@DKFZ-Heidelberg.de NNTP-Posting-Host: atv-alt.inet.dkfz-heidelberg.de X-Newsreader: WinVN 0.92.6+ Hallo, I work with hepatitis B virus x protein and its variants. Have you an idea how to prevent aggregate formation of protein at purification. From owner-proteins@net.bio.net Thu Aug 14 23:00:00 1997 Path: biosci!OWLSNEST.COM!owl From: owl@OWLSNEST.COM Newsgroups: bionet.molbio.proteins Subject: Is Your Web Site A Secret? Date: 15 Aug 1997 18:04:51 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 131 Sender: daemon@net.bio.net Distribution: world Message-ID: <199708160059.UAA21509@owlsnest.com> Reply-To: owl@owlsnest.com NNTP-Posting-Host: net.bio.net Is your web site the best kept secret on the Internet? We'll promote it to 50 search engines and indexes for $85 and complete the job in 2 business days. Satisfaction is guaranteed! If you have a great product, but are not getting many inquiries from your Web site, you may not be adequately listed on the Web's search engines and indexes. Millions of viewers daily use these facilities to find the products and services they are looking for. But if your site is not listed, no one will see it. Listings on most of these services are free. However, locating and filling out the forms required to get a listing can take several days, and most people just don't have the time to do it. That is why we offer a web site promotion service. WHAT'S THE DEAL? We will submit your site to 50 indexes and search engines for $85. We will accept the return of this E-mail, with the form below filled out, as an order. We will bill you upon completion of the promotion. Our terms are net 15 days from date of invoice. Satisfaction guaranteed! HOW LONG WILL IT TAKE? Generally, we complete the submissions within 48 hours of receiving your order. It can take any individual search engine or index up to three weeks to process your submission, although most are much faster. WHAT SEARCH ENGINES AND INDEXES ARE INCLUDED IN THE PROMOTION? The list changes from time to time. This is our current list: Abaweb!, Alta Vista, Been There, BizWeb, Central Source Yellow Pages, Enterpreneurs on the Web, Excite, Four11, Galaxy, I-Network I-Systems Spiral Business Directory, I-World Web Pointer, Infoseek, Inktomi, Innovator's Network Yellow Pages, Internet Mall, Jayde Online Directory, Jumpcity, Jumper Hot Links, Linkmaster, Lycos, Magellan, Mega Mall, Net-Happenings, Net Navigator, Net Mall, NTG's List, NYNEX Big Yellow, One World Plaza, OnLine's WWWeb Index, Rex, Starting Point, Truenorth, URL Tree, Virtual Lynx, Web Point, WebCentral, Web Venture Hotlist, Webcrawler, Websurf, Win Mag/NetGuide Hotspots, WhatUSeek, Worldwide Announce Archive, WWW Business Yellow Pages, World Wide Yellow Pages, WWW Worm, YelloWWWeb. HOW WILL I KNOW THAT YOU HAVE PROMOTED MY SITE? When we have completed the promotion, we will send you an HTML file as an attachment to your E-mail bill. Save this file to your disk, and view it through your Web browser. It provides links to the search engine we submitted your site to, plus any comments we received from them when we did it. ARE THERE ANY GUARANTEES? We do not require prepayment. Your satisfaction is guaranteed or you don't pay the bill. WHO IS OWL'S EYE PRODUCTIONS? We are a web site promotion company located at: Owl's Eye Productions, Inc. 260 E. Main Street Brewster, NY 10509 Phone: (914) 278-4933 Fax: (914) 278-4507 Email: owl@owlsnest.com HOW DO I ORDER? The easiest way to order is by e-mail. Just hit the REPLY button on your e-mail program and fill out the following information. (This information will be posted to the search engines/indexes): Your name: Company Name: Address: City: State/Prov: Zip/Postal Code: Telephone: Fax: Email address: URL: http:// Site Title: Description (about 25 words): Key words (maximum of 25, in descending order of importance): Proofs (Where shall we e-mail proofs): If billing a different address, please complete the following: Addressee: Company Name: Address: City: State/Prov: Zip/Postal Code: Telephone: Fax: Email address: We will bill via Email. (7519) Terms: By returning this document via Email, you agree as follows: You have the authority to purchase this service on behalf of your company. Terms are net 15 days. Accounts sent to collections will be liable for collection costs. You agree to protect and indemnify Owl's Eye Productions, Inc. in any claim for libel, copyright violations, plagiarism, or privacy and other suits or claims based on the content or subject matter of your site. WHAT HAPPENS NEXT? When we receive your order, we will input the information into our system, and send you a proof. After we process any corrections, we will run your promotion, capturing any comments from search engines as we go. We will incorporate these into an HTML-formatted report to you, which we will attach to your bill. ===Web Promotions=====Press Releases=====Link Exchanges========= Owl's Eye Productions, Inc. 260 E. Main Street Brewster, NY 10509 Ph: 914-278-4933 Fx: 914-278-4507 E-mail: owlseye@owlsnest.com From owner-proteins@net.bio.net Thu Aug 14 23:00:00 1997 Path: biosci!botany.uq.edu.au!J.Marcus From: J.Marcus@botany.uq.edu.au ("Marcus, Dr J.") Newsgroups: bionet.molbio.proteins Subject: Re: aggregate formation Date: 15 Aug 1997 15:01:59 -0700 Organization: Dept of Botany, Univ of Queensland Lines: 25 Sender: daemon@net.bio.net Distribution: world Message-ID: <199708152202.IAA19552@bunyip.cc.uq.edu.au> References: <5t115o$h90@news.inet.dkfz-heidelberg.de> Reply-To: Marcus@tpp.uq.edu.au NNTP-Posting-Host: net.bio.net Betaine or Taurine are sometimes used as additives to prevent aggregation due to charged residues. John Marcus > Hallo, > I work with hepatitis B virus x protein and its variants. > Have you an idea how to prevent aggregate formation of protein > at purification. > > _________________________________________________________ John Marcus Marcus@tpp.uq.edu.au (Dr J.Marcus) Cooperative Research Centre for Tropical Plant Pathology 5th Level John Hines Building University of Queensland St. Lucia, QLD 4072 AUSTRALIA Fax: 61-7-3365-4771 Phone: 61-7-3365-4764 From owner-proteins@net.bio.net Thu Aug 14 23:00:00 1997 Path: biosci!vt.edu!kkniel From: kkniel@vt.edu (Kali Phelps) Newsgroups: bionet.molbio.proteins Subject: Malachite Green Assay Date: 15 Aug 1997 13:59:02 -0700 Organization: student Lines: 7 Sender: daemon@net.bio.net Distribution: world Message-ID: <33F4C1E9.555A@vt.edu> Reply-To: kkniel@vt.edu NNTP-Posting-Host: net.bio.net Hello, I am about to use the malachite green ATPase assay on motor proteins expressed in E.coli. I have had some success with the coupled pyruvate-kinase reaction. I would like to know if anyone has used this malachie green assay and found success or hardships. Also, any tips or hints you can provide would be greatly appreciated! Thanks, Kali From owner-proteins@net.bio.net Thu Aug 14 23:00:00 1997 Path: biosci!daresbury!uninett.no!Norway.EU.net!EU.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!newsfeed.nacamar.de!fu-berlin.de!derma0001.ukbf.fu-berlin.DE!not-for-mail From: Oliver Politz Newsgroups: bionet.molbio.proteins Subject: protein splicing Date: Fri, 15 Aug 1997 13:45:48 +0100 Organization: Freie Universitaet Berlin Lines: 16 Message-ID: <33F44F7C.CA1D34D4@medizin.fu-berlin.de> NNTP-Posting-Host: derma0001.ukbf.fu-berlin.de (160.45.173.203) Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Access: 16 17 19 X-Mailer: Mozilla 4.01 [en] (WinNT; I) X-Priority: 3 (Normal) Hello netters, I have cloned a bacterial protein which shows a "protein splicing" motive and therefore I would like to get some more information about this : - good reviews to read - what is the mechanism ? - examples of known spliced proteins - how to analyse My problem is that this protein has two domains just separated by this "intein" and when I take this away I have high similarities to other proteins from the databank which don't have such an intein. So the question is if my protein gets functional by a splicing mechanism or not. Thanks and please simply email to politz@medizin.fu-berlin.de From owner-proteins@net.bio.net Thu Aug 14 23:00:00 1997 Path: biosci!agate!ihnp4.ucsd.edu!munnari.OZ.AU!bunyip.cc.uq.edu.au!not-for-mail From: M.Mason@mailbox.uq.edu.au (Mike) Newsgroups: bionet.molbio.proteins Subject: knock a protein off a membrane Date: 15 Aug 1997 10:34:18 GMT Organization: Nothing Lines: 13 Message-ID: <5t1bbb$4m6$1@nargun.cc.uq.edu.au> Reply-To: M.Mason@Botany.uq.edu.au NNTP-Posting-Host: btmmason.slip.cc.uq.edu.au Mime-Version: 1.0 Content-Type: Text/Plain; charset=US-ASCII X-Newsreader: WinVN 0.99.8 (16bit) Fellow scientists, I am trying to isolate a protein which is bound to the membrane through a lipid modification. I wish to isolate the protein without denaturing it. Does anyone have some sugguestions on the matter? I am currently trying different detergent percentages, although I am worried they might be denaturing the protein. Thanks in advance, Mike Mason Botany Department, University of Queensland Australia M.Mason@Botany.uq.edu.au From owner-proteins@net.bio.net Fri Aug 15 23:00:00 1997 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.direct.ca!news.he.net!hermes.louisville.edu!not-for-mail From: "Robert D. Gray" Newsgroups: bionet.molbio.proteins Subject: Re: Extinction coefficient Date: Sat, 16 Aug 1997 11:41:36 -0400 Organization: University of Louisville Lines: 27 Message-ID: <33F5CA30.177@ulkyvm.louisville.edu> References: <33F221F5.58AC@vt.edu> <33F2252E.7956@amgen.com> Reply-To: rdgray01@ulkyvm.louisville.edu NNTP-Posting-Host: rgray3.biochemistry.louisville.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01Gold (Win95; I) > Arzu Karabay wrote: > > > > I have been trying to measure the extinction coefficients of the > > recombinant fusion proteins that I purified. I have used Guanidium > > hydrochloride. The proteins are purified with denaturation method and > > are in high grade protein running buffer (0.025 M Tris, pH8.3, 0.192 M > > glycine,and 0.1 % SDS). I used 6 M Guanidium hydrochloride (150 ul of > > 8 M Guanidium hydrocholoride and 50 ul of the protein in 200 ul > > volume). every time I did the measurement with the same protein I got > > different results.. I have this reference: Analytical Biochemistry > > 182, 319-326 (1989), but it does not have a clear protocol. I do not > > know if there is a certain incubation period with GndHCl. Then I > > decided to do a time course measurement, and I got different > > measurements at different time points. If anybody have any explanation > > for this and a clearer protocol on measurement of the extinction > > coefficient, and exactly how to calculate it this will help me a lot. > > I really am looking forward to your great ideas. > > Thanks for your help. You might also wish to consult the following paper: "Statistical Determination of the Average Values of the Extinction Coefficients of Tryptophan and Tyrosine in Native Proteins," by Mach, Middaugh & Lewis, Anal. Biochem. 200, 74-80 (1992). -- Robert D. Gray Department of Biochemistry University of Louisville, Louisville, KY 40292 From owner-proteins@net.bio.net Fri Aug 15 23:00:00 1997 Path: biosci!agate!hammer.uoregon.edu!news.oru.edu!news-spur1.maxwell.syr.edu!news.maxwell.syr.edu!infeed1.internetmci.com!newsfeed.internetmci.com!152.163.199.19!portc03.blue.aol.com!newstf02.news.aol.com!audrey02.news.aol.com!not-for-mail From: huntpharm@aol.com (Huntpharm) Newsgroups: bionet.molbio.proteins Subject: US-NC-HEAD OF STRUCTURAL CHEMISTRY Date: 16 Aug 1997 19:48:22 GMT Lines: 13 Message-ID: <19970816194800.PAA17917@ladder02.news.aol.com> NNTP-Posting-Host: ladder02.news.aol.com X-Admin: news@aol.com Organization: AOL http://www.aol.com I am looking for a PhD Scientist to be the Head of the Department of Structural Chemistry. The candidate will have a strong background in either X-Ray Crystallography or Computational Chemistry . As the person in this visionary role, you will be responsible for leading a group of sixteen scientists and growing. You should possess a PhD with 5+ years experience and strong publication record. We are a leading pharmaceutical company with research facilities in North Carolina and can provide excellent benefits (health insurance, dental, and vision plan, bonus program, paid vactation and more). Excellent compensation package. A high impact, high profile position with excellent opportunity for advancement. Please contact Scott Shanes by phone at 609-584-8733 Ext. 218, fax CV and cover letter to 609-584-9575 or E-Mail to sis@diedremoire.com From owner-proteins@net.bio.net Sat Aug 16 23:00:00 1997 Path: biosci!biosci!not-for-mail From: Pier Carlo Montecucchi <100143.765@CompuServe.COM> Newsgroups: bionet.molbio.proteins,sci.bio.technology Subject: Peptide synthetizer Date: 17 Aug 1997 14:57:55 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 8 Sender: daemon@net.bio.net Distribution: world Message-ID: <5t7s53$fpc@net.bio.net> NNTP-Posting-Host: net.bio.net I am looking for some reviews describing the commercial available apparatus for peptide synthesis (advantages / disandvantages). Sincerely Pier Carlo Montecucchi Email: pcmontecucchi@compuserve.com fax: 212-208-2648 From owner-proteins@net.bio.net Sun Aug 17 23:00:00 1997 Path: biosci!agate!hammer.uoregon.edu!vixen.cso.uiuc.edu!howland.erols.net!infeed1.internetmci.com!newsfeed.internetmci.com!195.99.66.215!news-feed1.eu.concert.net!news-peer.bt.net!btnet!baron.netcom.net.uk!netcom.net.uk!server3.netnews.ja.net!news.ox.ac.uk!andypmac.zoo.ox.ac.uk!user From: enquiries@vei.co.uk Newsgroups: bionet.molbio.proteins Subject: Bioinformatics Conference Date: Mon, 18 Aug 1997 09:44:50 +0100 Organization: Virtual Environments International Lines: 207 Message-ID: NNTP-Posting-Host: andypmac.zoo.ox.ac.uk Bioinformatics IEC-1 - FIRST INTERNET-EXTENDED BIOINFORMATICS CONFERENCE ************************************************************************ The First Internet-Extended Bioinformatics conference will be held from Monday Nov 17 -> Friday Nov 28 1997 and will combine the use of a virtual conference and workshop (http://www.vei.co.uk/bioinfiec1/)followed by a physical conference and workshop in the Paragon hotel in London, UK. The virtual conference and workshop will run from Nov 17, the physical workshop will take place on Nov 26 and the physical conference on Nov 27-28. The focus of the conference is on the methods of bioinformatics and their application to current topical areas of research including genomics, datamining, lead drug discovery, sequence retrieval, alignment and modelling, molecular evolution, phylogenetic analysis, genetic variation, functional analysis and visualisation. A special feature of the event will be the use of Internet technology to enhance and extend the activities of the event both in the use and demonstration of methods and software and in the extension of the physical event to include virtual activities before and during the physical conference. Bioinformatics has unique and significant activities in both the private and public sectors and the target audience for the physical conference will be senior researchers and managers in both. The virtual facilities will also extend the event to a broader international audience both to corporate intranets and to academic researchers in remote locations. The virtual conference will allow the presentation of virtual lectures, papers and posters via the WWW, their discussion in a real-time virtual conference centre and the demonstration of methods, software and tools. The virtual workshop will allow registrants to participate in tutorials and applications over the Internet in the ten days proceeding the physical conference which will host an all-day hands-on physical workshop at an on-site bank of computers with an Internet conection. The physical conference will consist of lectures, panel discussions, an electronic poster session, and exhibitor stands. The lectures will be broadcast live over the Internet using audio, live cameras and a virtual slide facility with questions and comments accepted live from virtual participants. Presentations for the virtual conference must be prepared in Hypertext Markup Language (HTML) with figures in GIF or other Web-compatible formats so that participants can view the papers via the World Wide Web (The presentations may also include enhancements such as 3D structures, VRML, Java, RealAudio, Quicktime movies etc.) Powerpoint presentations will be used for the virtual presentation of conference lectures. Aid and consultation is provided to participants who may address their queries to the conference hot-line at bioinforg@vei.co.uk. Further details will be given in the authors' guide accessible via http://www.vei.co.uk/bioinfiec1/. Conference sections by topic are: Molecular Evolution, Virology, Datamining; Lead Drug Discovery; Sequence retrieval, Alignment and Modelling; Phylogenetic analysis; Genetic variation; Functional Analysis; Visualisation; Population Analysis; Internet Databases & Information Retrieval; Structural Biology; HIV Modelling; Prions; and Applications in Cancer Research. During the virtual conference interaction, presentations and discussions will take place via the Internet using a Java-based virtual conference centre, WWW-based discussion forums and an electronic mailing list. Before the conference, a timetable for lectures and discussion sessions for each section will be posted. The virtual and physical workshops will cover topics including search engines, biological resources on the WWW, bibliographic databases, sequence retrieval and alignment, structure generation and advanced modelling and visualization techniques. The Conference will feature a Virtual Exhibition where sponsors and exhibitors will be able to describe the activities of their organization, or display their products and services and interact with registrants. Potential sponsors and exhibitors should contact the conference organisers at bioinforg@vei.co.uk. DEADLINES AND DATES 1) DO NOW - The Bioinformatics Conference mailing list Conference-related news and announcements will be posted regularly to the mailing list (bioinf@vei.co.uk). If you are receiving this message from the bioinf list you are already subscribed. If you wish to subscribe to the bioinf list send the following one line message to bioinf-request@vei.co.uk: subscribe bioinf@vei.co.uk your_email@address To unsubscribe send the following message: unsubscribe bioinf@vei.co.uk your_email@address 2) Registration If you intend to participate in Bioinformatics IEC-1 please use the special registration form accessible via http://www.vei.co.uk/bioinfiec1/. The electronic registration will be used to construct a registrant database for the conference which will generate the conference mailing list and handle assignment of userids and passwords. Virtual Conference & Virtual Workshop: 75 pounds (125 dollars) (88 pounds inc. VAT) "Combined" Physical & Virtual Conference: 540 pounds (900 dollars) (635 pounds inc. VAT) "Combined" Academic fee (limited availability): 180 pounds (300 dollars) (212 pounds inc. VAT) Additional Fee for Physical Workshop Attendance: 200 pounds (335 dollars) (235 pounds inc. VAT) In addition it is necessary to pay for registration via ordinary means: Registration payments can be accepted by credit card, direct bank transfer, cheque or bank draft. Payment Instructions: a) Credit Card We can currently accept Visa, Mastercard, Switch or JCB. Please send your credit card type, number and expiration date via a) regular mail to: Bioinformatics IEC-1 Registration, VEI, Oxford Centre for Innovation, Mill St, Oxford, OX2 0JX, UK. b) fax to Bioinformatics IEC-1 Registration at +44 1865 793165 c) phone Bioinformatics IEC-1 Registration at +44 1865 793644 b) Bank Transfer Your payment can be made in sterling by direct bank transfer into the following account: Account Number: 20-6518 40787523 Account Name: Virtual Environments International Ltd Bank: Barclays Bank, Oxford City Centre Branch, P.O. Box 333, Oxford, OX1 3HS, UK --Please ensure your full name is listed with the transfer details. --You are responsible for any bank charges associated with the transfer from your bank. --Please notify us directly of your transfer payment at bioinforg@vei.co.uk. c) Cheque/Bank Draft Cheques or bank orders (in pounds or dollars) should be made out to Virtual Environments International Ltd and mailed to: Bioinformatics IEC-1 Registration, VEI, Oxford Centre for Innovation, Mill St, Oxford, OX2 0JX, UK. Acknowledgement of Registration and Payment: When you first register at the conference site you should receive an acknowledgement email with a password and userid. After receipt of your registration fee you will receive a further acknowledgement and your userid and password will be validated to allow access to the conference site (when it opens). 3) DEADLINE for receipt of ABSTRACT. The deadline for receipt of presentation abstracts is Oct 1. Email your abstract directly to bioinfabstracts@vei.co.uk. Fuller details of the scope of each section is given in the authors' guide accessible via http://www.vei.co.uk/bioinfiec1/ Your abstract should be no longer than 300 words. And remember to state which forms of presentation you will use (virtual poster presentation, virtual lecture, physical poster presentation) you wish and which subject section the presentation is being placed in. For example you could present a WWW poster on Visualization in the Virtual Conference and accompany it with a physical poster at the physical conference. Computers will be available at the physical conference for the presentation and discussion of the virtual posters and the demonstration of software and methods. 4) DEADLINE for receipt of PRESENTATION The deadline for receipt of presentations is November 1. You must deposit your text and graphics files at the conference ftp site following the instructions available at http://www.vei.co.uk/bioinfiec1/. Any general e-mails (such as registration queries, maillist queries, HTML queries, password queries, timetable queries, general technical advice on browsers and graphics, etc) should be sent to: bioinforg@vei.co.uk From owner-proteins@net.bio.net Sun Aug 17 23:00:00 1997 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!server3.netnews.ja.net!news.cc.ic.ac.uk!not-for-mail From: Koen De Smet Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Q: what are sepabeads? Date: Mon, 18 Aug 1997 16:56:59 +0100 Organization: Department of Medical Microbiology Lines: 33 Message-ID: <33F870CB.7589@nospam.ic.ac.uk> Reply-To: k.desmet@nospam.ic.ac.uk NNTP-Posting-Host: cb-22.sm.med.ic.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.02 (Macintosh; I; PPC) Xref: biosci bionet.molbio.methds-reagnts:60505 bionet.molbio.proteins:11376 Hi, I am just reading an article on the purification of an enzyme. It includes a step involving Sepabeads FP-DA13 gel (Mitsubishi Chemical Industries ltd). Can anybody give me more information on this resin? Is it anion exchange, cation exchange, gelfiltration or affinity chromatography? I don't know this company (I presume it is Japanese, and the paper is from a japanese group) so we don't have their catalogue. Thanks for any advice! Koen -- _______________________________________________________________ To reply by email, remove "nospam." from the above address _______________________________________________________________ Dr Koen A.L. De Smet Research Fellow Department of Medical Microbiology Imperial College Medical School at St Mary's Norfolk Place London W2 1PG Great Britain Tel: (+44)-(0)171-594 3946 Fax: (+44)-(0)171-262-6299 http://www.sm.ic.ac.uk/medmicro/home.htm _______________________________________________________________ From owner-proteins@net.bio.net Sun Aug 17 23:00:00 1997 Path: biosci!daresbury!uninett.no!nntp.uio.no!news-spur1.maxwell.syr.edu!news.maxwell.syr.edu!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsxfer3.itd.umich.edu!oleane!jussieu.fr!univ-lyon1.fr!pcb-briolay.univ-lyon1.fr!user From: nosjean@univ-lyon1.fr (Olivier Nosjean) Newsgroups: bionet.molbio.proteins Subject: Re: knock a protein off a membrane Date: 18 Aug 1997 15:03:22 GMT Organization: Univ. C. Bernard - Lyon 1 . France Lines: 52 Message-ID: References: <5t1bbb$4m6$1@nargun.cc.uq.edu.au> NNTP-Posting-Host: pcb-briolay.univ-lyon1.fr In article (Dans l'article) <5t1bbb$4m6$1@nargun.cc.uq.edu.au>, M.Mason@Botany.uq.edu.au wrote (écrivait) : > Fellow scientists, > I am trying to isolate a protein which is bound to the membrane through a lipid > modification. I wish to isolate the protein without denaturing it. Does anyone > have some sugguestions on the matter? I am currently trying different detergent > percentages, although I am worried they might be denaturing the protein. > > Thanks in advance, Hello, Concerning the possible denaturation of your protein, you should be safe by employing a non-ionic detergent, such as Triton X-100, Triton X-114, Octylglucoside, Nonidet P40. Besides, some proteins can stand low concentrations of anionic (SDS) or zwiterionic (CHAPS) detergents, or even organic solvents (n-butanol). Then, parameters which will influence your choice are a) the rate of solubilization of your protein of interest, and also b) the rate of solubilization of miscellaneous hydrophobic of amphipatic contaminating membrane components. Unfortunately, there is no other way to know than to try each of them ! Other parameters you should take care of are the CMC of the detergent (the higher, the easier to dialize), the lipids/detergent ratio, the salt concentration, the temperature, the duration of incubation, the pH. And here again, the try-and-see approach may be the most appropriate for a specific protein. You may find some help in : Hjelmeland, L. (1984) Meth. Enzymol. 104, pp305-318 Furth, A. (1984) Meth Enzymol. 104, pp318-328 Furth A. (1980) Anal. Biochem. 109, 207-215 and good luck ! Olivier Nosjean _ _ _ \/ \/ {O} Olivier Nosjean |_| Laboratoire de Physico-Chime Biologique ||| Univ. C. Bernard - Lyon 1 | France From owner-proteins@net.bio.net Mon Aug 18 23:00:00 1997 Path: biosci!daresbury!uninett.no!news-feed.inet.tele.dk!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news.maxwell.syr.edu!news-out.communique.net!communique!news2.epix.net!inet.guthrie.org!zlchang!zlchang From: zlchang@inet.guthrie.org (zlchang