From owner-proteins@net.bio.net Wed Sep 01 03:14:00 1999 Path: biosci!newshost.lanl.gov!logbridge.uoregon.edu!newspeer1.nac.net!easynet-tele!easynet.net!tank.news.pipex.net!pipex!server1.netnews.ja.net!hgmp.mrc.ac.uk!tin!jphelan From: "Mr. John P Phelan" Newsgroups: bionet.molbio.proteins Subject: Re: AKTAFPLC Date: Wed, 1 Sep 1999 12:07:25 +0100 Organization: MRC Human Genome Mapping Project Resource Centre Lines: 8 Message-ID: References: <3B675D1AC3E1D111BC1600805FEACC676C331C@lauimls05.qc.dfo.ca> NNTP-Posting-Host: tin.hgmp.mrc.ac.uk Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII X-Trace: niobium.hgmp.mrc.ac.uk 936184049 27316 193.62.192.50 (1 Sep 1999 11:07:29 GMT) X-Complaints-To: news@hgmp.mrc.ac.uk NNTP-Posting-Date: 1 Sep 1999 11:07:29 GMT X-Sender: jphelan@tin In-Reply-To: <3B675D1AC3E1D111BC1600805FEACC676C331C@lauimls05.qc.dfo.ca> Hi there, We have an Akta FPLC and so far it has performed superbly, everything is done via the computer which they supply and is extremely easy to use. As for a bug in the system regarding your own machine I am not strictly sure about his. John From owner-proteins@net.bio.net Wed Sep 01 04:41:00 1999 Path: biosci!newshost.lanl.gov!logbridge.uoregon.edu!newspeer.monmouth.com!newsfeed.mad.ttd.net!news.sarenet.es!not-for-mail From: Carlos Bort Newsgroups: bionet.molbio.proteins Subject: Cen-C protein. Date: Wed, 01 Sep 1999 14:28:39 +0200 Organization: SAREnet SA Lines: 24 Message-ID: <37CD1BF7.CA855B42@teleline.es> Reply-To: carlos_bort@teleline.es NNTP-Posting-Host: 194.30.83.95 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.51 [en] (Win95; I) X-Accept-Language: en X-Priority: 1 (Highest) Hello there, A friend of mine needs some information, for her Ph.D. work, on a protein called Cen-C. It seems it is of vegetable origin, and it plays some role in the digestive system of ruminants. She is specifically working on goat. She would appreciate any information on this protein (sequence, structure, in vivo role, etc.), since she is totally in the dark. I have been unable to find a word on this substance in Web searches. Please send any information to my e-mail address (see below). I do not have access to newsgroups everyday. Please copy your answer also to: belenacho@net.telematic.com.pe If this is not the right news group for the matter, please help me in finding the right one. Thanks. Thanks in advance. Best regards, Carlos Bort - Madrid - Spain carlos_bort@teleline.es Please copy your answer also to: belenacho@net.telematic.com.pe From owner-proteins@net.bio.net Wed Sep 01 10:31:00 1999 From: "RISHIKESH P. BHALERAO" Newsgroups: bionet.molbio.proteins Subject: Post-doctoral position in plant molecular biology Date: Wed, 01 Sep 1999 19:39:37 +0000 Organization: Swedish University of Agricultural Sciences Lines: 39 Message-ID: <37CD80F8.F8@genfys.slu.se> NNTP-Posting-Host: d170.genfys.slu.se Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Macintosh; I; PPC) Path: biosci!news.stanford.edu!newsfeed.stanford.edu!logbridge.uoregon.edu!newsfeed.icl.net!news.algonet.se!algonet!news-peer-europe.sprintlink.net!news.sprintlink.net!newsfeed.sunet.se!news01.sunet.se!news99.sunet.se!newsfeed.uu.se!populus.slu.se!newsmgr We are interested in employing a highly motivated molecular biologist to work in my group to investigate the role of IAA inducible genes in controlling vascular development. We have cloned cDNAs for several IAA inducible genes from the vascular cambium of Hybrid aspen. These IAA inducible genes are differentially regulated in the different cell types of the vascular tissue. The project will focus on: (i) Cloning the promoters of xylem and phloem specific IAA inducible genes from hybrid aspen. (ii) Construction of dominant mutations in selected IAA genes. (iii) Constructing transgenic hybrid aspen plants expressing mutated IAA genes under tissue specific promoter. (iv) Analysis of transgenic plants for alteration in wood quality. The selected individual will work with a team of scientist collaborating closely on studying various aspects of wood formation. Prior knowledge of anatomy will be an advantage. The department of Forest Genetics and Plant Physiology has over 50 scientists and students working on various aspects of wood formation. We have state of the art facilities for performing molecular biology, light and confocal microscopy and analytical chemistry. The position is for 2 years initially and the salary will be SKR 12,000-14,000 depending upon experience. Interested individuals should contact: Rishikesh P. Bhalerao Assistant Professor Department of Forest Genetics and Plant Physiology The Swedish University of Agricultural Sciences S-901 83 Umea Sweden Tel:+46-90-7866282 Fax:+46-90-7865901 Email:rishi.bhalerao@genfys.slu.se From owner-proteins@net.bio.net Wed Sep 01 20:49:00 1999 Path: biosci!GENEMARK.BIOLOGY.GATECH.EDU!john From: john@GENEMARK.BIOLOGY.GATECH.EDU ("John D. Besemer") Newsgroups: bionet.molbio.proteins Subject: UPDATE_Bioinformatics_Conference_in_Atlanta Date: 1 Sep 1999 21:49:43 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 142 Sender: daemon@net.bio.net Distribution: world Message-ID: <199909020121.VAA16931@genemark.biology.gatech.edu> NNTP-Posting-Host: net.bio.net Early registration is now in progress for the Second International Georgia Tech Conference on Bioinformatics. The organizers would like to inform you that the following deadlines are approaching: * September 10 - Deadline for poster abstract submission * October 1 - Deadline for early registration We encourage you to register as soon as possible, as participation will be limited to about 250 people. For more information, please see the conference website at: http://exon.biology.gatech.edu/conference We look forward to seeing you in Atlanta, The Conference Organizers ********************************************************************** SECOND GEORGIA TECH INTERNATIONAL CONFERENCE ON BIOINFORMATICS In silico BIOLOGY: SEQUENCE & STRUCTURE & FUNCION ATLANTA NOVEMBER 11 - 14, 1999 SPONSORS: National Institutes of Health US Department of Energy Alfred P. Sloan Foundation Georgia Tech College of Science Parker H. Petit Institute for Bioengineering & Bioscience SouthEastern Center for Applied Analysis SmithKline Beecham AGENDA: The conference agenda includes keynote lectures, plenary lectures, as well as poster sessions. The list of confirmed speakers is as follows: KEYNOTE SPEAKERS: Russell Doolittle University of California, San Diego, CA Walter Fitch University of California, Irvine, CA PLENARY SPEAKERS: David Baker University of Washington, Seattle, WA Pierre Baldi University of California, Irvine, CA Steven Brenner Stanford University, Stanford, CA Chris Burge MIT, Cambridge, MA Antoine Danchin Institute Pasteur, Paris, France Sean Eddy Washington University School of Medicine, St. Louis, MO Patrick Forterre University Paris-Sud, Paris, France David Haussler University of California, Santa Cruz, CA Samuel Karlin Stanford University, Stanford, CA Jeffrey Lawrence University of Pittsburgh, Pittsburgh, PA Steven Henikoff Fred Hutchinson Cancer Research Center, Seattle, WA Christine Orengo University College, London, UK Burkhard Rost Columbia University, New York, NY Andrej Sali Rockefeller University, New-York, NY William Taylor National Institute for Medical Research, London, UK STEERING/PROGRAM COMMITTEE: Pierre Baldi Net-ID Mark Borodovsky, Co-chair Georgia Tech Soren Brunak Technical University of Denmark Chris Burge MIT Jim Fickett SmithKline Beecham Steven Henikoff Fred Hutchinson Cancer Research Center Eugene Koonin, Co-chair NCBI/NIH Andrej Sali Rockefeller University Chris Sander Millennium Pharmaceuticals Gary Stormo University of Colorado DEADLINES: POSTER SUBMISSION: Deadline for poster abstract submission: September 10, 1999 REGISTRATION: Early registration ends: October 1, 1999 CONFERENCE SCHEDULE: Registration opens at 6:00pm on Thursday, November 11 The program starts 8:00am Friday, November 12 and ends at noon Sunday, November 14. LOCATION: The conference will be held at the Renaissance Atlanta Hotel Downtown located near the center of 1996 Olympic development, close to the Fox Theatre & Georgia Tech. ORGANIZING COMMITTEE: General co-ordination Mark Borodovsky, Georgia Tech mark@amber.gatech.edu "Bioinformatics" magazine publication Chris Burge MIT cburge@mit.edu Poster sessions Scott Sammons Emory University sammons@bimcore.emory.edu Administrative & Public Relations Assistant Amanda Besemer Emory University Amanda_Besemer@bus.emory.edu Publicity John Besemer Georgia Tech john@amber.gatech.edu Registration and general events Michael Moryc Georgia Tech michael.moryc@conted.gatech.edu MORE INFORMATION To obtain more information on poster submission & registration visit the WWW page: http://exon.biology.gatech.edu/conference or contact us by e-mail: register@conted.swann.gatech.edu Phone: (404) 894-2400 Fax: (404) 894-8925 From owner-proteins@net.bio.net Thu Sep 02 04:39:00 1999 Path: biosci!HOTMAIL.COM!pplegal From: pplegal@HOTMAIL.COM Newsgroups: bionet.molbio.proteins Subject: HOT NEW BUSINESS OPPTY. - PERSONAL POSTCARDS! 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Michael Sherrell Grizzly Analytical (USA) 707 887 2919/fax 707 887 9834 www.grizzlyanalytical.com From owner-proteins@net.bio.net Fri Sep 03 04:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.stanford.edu!skynet.be!bignews.mediaways.net!news-fra1.dfn.de!news-lei1.dfn.de!news.uni-weimar.de!news.uni-jena.de!not-for-mail From: "Stefka Stoyanova" Newsgroups: bionet.molbio.proteins Subject: Test Date: Fri, 3 Sep 1999 13:53:35 +1000 Organization: Friedrich-Schiller-University Jena, Germany Lines: 6 Message-ID: <7qocs7$bi4$1@fsuj19.rz.uni-jena.de> NNTP-Posting-Host: wippich.pharmbiol.uni-jena.de X-Priority: 3 X-MSMail-Priority: Normal X-Newsreader: Microsoft Outlook Express 5.00.2014.211 X-MimeOLE: Produced By Microsoft MimeOLE V5.00.2014.211 -- From owner-proteins@net.bio.net Fri Sep 03 12:23:00 1999 Path: biosci!nestgrp.com!SuproTip From: SuproTip@nestgrp.com (SuproTip) Newsgroups: bionet.molbio.proteins Subject: SuproTip, microSPE pipette tips for sample preparation Date: 3 Sep 1999 13:23:31 -0700 Organization: Software Tool & Die - Purveyors to the Trade Lines: 38 Sender: daemon@net.bio.net Distribution: world Message-ID: <37D02C97.F9AEC2BD@nestgrp.com> Reply-To: SuproTip@nestgrp.com NNTP-Posting-Host: net.bio.net To: methods@net.bio.net From: SuproTip@nestgrp.com (The Nest Group, Inc.) 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SuproTip microSPE tips are available in eleven different chemistries and two different sample capacity ranges to desalt or capture a few microliters of femtomole-level molecules in under 60 seconds. Custom chemistry tips are also available. For more product specs, user guides, tech notes and ordering information please see: http://world.std.com/~nestgrp/protocols/protocol.html or http://world.std.com/~nestgrp/protocols/AmiKa/SuproTip.pdf From owner-proteins@net.bio.net Fri Sep 03 17:43:00 1999 Path: biosci!N2.COM!topbizmill From: topbizmill@N2.COM Newsgroups: bionet.molbio.proteins Subject: Voted #1 e-Business Date: 3 Sep 1999 18:43:51 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 31 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Dear Online Marketer, We have found that you are doing business on the internet and hope everything is going well for you. 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From owner-proteins@net.bio.net Sun Sep 05 02:21:00 1999 Path: biosci!news.stanford.edu!newsfeed.stanford.edu!logbridge.uoregon.edu!dispose.news.demon.net!demon!newsfeed.tli.de!newscore.gigabell.net!newscore.ipf.de!news0.de.colt.net!news-fra1.dfn.de!news-lei1.dfn.de!news.uni-leipzig.de!news.uni-halle.de!not-for-mail From: moqch@mlucom6.replace.uni-halle.de (Georg Wille) Newsgroups: bionet.molbio.proteins Subject: Re: protein crystals? Date: 5 Sep 1999 10:01:48 GMT Organization: University Halle, Germany Message-ID: <7qtf2c$nv3$1@mlucom4.urz.uni-halle.de> References: <37cc0011.401045@news.uni-hamburg.de> NNTP-Posting-Host: dial-045.urz.uni-halle.de Mime-Version: 1.0 Content-Type: Text/Plain; charset=US-ASCII X-Newsreader: WinVN 0.99.9 (Released Version) (x86 32bit) Lines: 37 >purification step. During the elution with >a buffer containing 50mM Hepes pH 7.4, NaCl >450 mM, DTT 5mM, Pefabloc 1 mM (Merck, PMSF >like stuff) we have noticed brownish stuff at >the tip of the elution tube. We have taken >some stuff from the tip onto a glass slid >and under the microscope >there were apparent big NaCl crystals >and much smaller yellow - brownish crystals. I wonder whether this could really be NaCl crystals, 450mM is perfectly soluble. In fact even 2M is soluble. I doubt that DTT or DNA would form crystals under these conditions either. >On the other hand could the brown yellowish color >be compatible with a hemoprotein? And maybe the >protein under investigation just crystallizes like >hell? The color may be consistent, but this is just guessing. Why don't you just take the pellet of these crystals and run a SDS-PAGE, preferably with a sample of the supernatant as well. Sincerely Georg -------------- To reply replace "replace" in the E-mail address with "urz". From owner-proteins@net.bio.net Sun Sep 05 02:48:00 1999 Path: biosci!GMX.DE!aavatar From: aavatar@GMX.DE ("aavatar") Newsgroups: bionet.molbio.proteins Subject: Men-Learn to Inrease Your Sexual Pleasure-Book Date: 5 Sep 1999 03:48:38 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 50 Sender: daemon@net.bio.net Distribution: world Message-ID: <199909051041.TAA23008@dri.co.jp> NNTP-Posting-Host: net.bio.net Now any man, regardless of age, can easily learn: * To be multi-orgasmic * To greatly increase the intensity of his orgasm * Triple the length of his orgasm * The secrets to penis enlargement * Discover the male G-spot * To greatly increase semen volume * The facts about Viagra, plus new drugs * To eliminate premature ejaculation * The secrets of getting his partner to want more sex * To eliminate impotence at any age * To have up to a three hour erection New, Easy to Read Book on Men's Sexual Secrets Male Sexual Secret's Written by Robert Winter and Jeff Rutgard, M.D. Most Men Only Get A Fraction of The Pleasure They Can From Sex-After Only A Few Pages You Can Easily Triple That Amount Of Pleasure. This is a fantastic new book covering so many little known and unknown sexual secret's you'll be amazed. If you don't learn something new in the first few pages that greatly increases your sex life we'll return your money. The total cost of this book is only $12.95 plus $3.95 shipping and handling. To order " Men's Secrets" Call 800-335-9977 24hours a day or you can send your check or money order for $16.90 to Avatar Publishing 168 second Ave #PBM 285 New York, NY. 10003 This message is sent in compliance of the new e-mail bill: SECTION 301. Per Section 301, Paragraph(a)(2)(c) of S.1618 To be removed from our e-mail list please call 888-248-2594 or you can e-mail us at aavatar99@hotmail.com From owner-proteins@net.bio.net Tue Sep 07 05:58:00 1999 Path: biosci!news.stanford.edu!newsfeed.stanford.edu!nntp.cs.ubc.ca!newsflash.concordia.ca!pitt.edu!not-for-mail From: pxpst2@vms.cis.pitt.edu (Peter) Newsgroups: bionet.molbio.proteins Subject: Re: protein crystals? Date: Tue, 07 Sep 1999 09:50:02 -0400 Organization: University Of Pittsburgh Lines: 21 Message-ID: References: <37cc0011.401045@news.uni-hamburg.de> <7qtf2c$nv3$1@mlucom4.urz.uni-halle.de> NNTP-Posting-Host: pelli.pathology.pitt.edu Mail-Copies-To: pxpst2@vms.cis.pitt.edu X-Newsreader: MT-NewsWatcher 2.4.4 In article <7qtf2c$nv3$1@mlucom4.urz.uni-halle.de>, moqch@mlucom6.replace.uni-halle.de (Georg Wille) wrote: > >purification step. During the elution with > >a buffer containing 50mM Hepes pH 7.4, NaCl > >450 mM, DTT 5mM, Pefabloc 1 mM (Merck, PMSF > >like stuff) we have noticed brownish stuff at > >the tip of the elution tube. We have taken > >some stuff from the tip onto a glass slid > >and under the microscope > >there were apparent big NaCl crystals > >and much smaller yellow - brownish crystals. > > > I wonder whether this could really be NaCl crystals, 450mM is perfectly > soluble. In fact even 2M is soluble. I doubt that DTT or DNA would form > crystals under these conditions either. I have noticed from time to time that PMSF will crystallize. Peter From owner-proteins@net.bio.net Wed Sep 08 00:06:00 1999 Path: biosci!newsfeed.stanford.edu!logbridge.uoregon.edu!mozo.cc.purdue.edu!not-for-mail From: dokland@deft.cc.purdue.edu (Terje Dokland) Newsgroups: bionet.molbio.proteins Subject: deglycosylation of proteins Date: 8 Sep 1999 02:59:05 -0500 Organization: Purdue University Computing Center Lines: 9 Message-ID: <7r5509$jo6@deft.cc.purdue.edu> NNTP-Posting-Host: deft.cc.purdue.edu X-Trace: mozo.cc.purdue.edu 936777545 24498 128.210.250.52 (8 Sep 1999 07:59:05 GMT) X-Complaints-To: usenet@mozo.cc.purdue.edu NNTP-Posting-Date: 8 Sep 1999 07:59:05 GMT Does anybody know of a non-destructive chemical method to deglycosylate proteins? Enzymatic methods would work, but the enzymes are forbiddingly expensive in the amounts that I would most likely require. The chemical methods that I have seen in the literature (anhydrous hydrogen fluoride or TFMS) seem rather harsh and would destroy the structure I am interested in, namely a virus. Any suggestions would be appreciated. Thanks, terje From owner-proteins@net.bio.net Wed Sep 08 08:04:00 1999 Path: biosci!AOL.COM!DOCTORHIM From: DOCTORHIM@AOL.COM Newsgroups: bionet.molbio.proteins Subject: Re: deglycosylation of proteins Date: 8 Sep 1999 09:04:13 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 3 Sender: daemon@net.bio.net Distribution: world Message-ID: <30513a56.2507e139@aol.com> NNTP-Posting-Host: net.bio.net Why don't you contact the company that makes deglycosylation kits   http://www.prozyme.com/glycopro and ask for the cost of bulk deglycosylation enzymes From owner-proteins@net.bio.net Wed Sep 08 08:27:00 1999 Path: biosci!newsfeed.stanford.edu!cyclone.bc.net!rover.ucs.ualberta.ca!karlster From: karlster@excite.com (karlster) Newsgroups: bionet.molbio.proteins Subject: Re: deglycosylation of proteins Date: Wed, 08 Sep 1999 10:24:00 -0600 Organization: A land far, far away... Lines: 22 Message-ID: References: <7r5509$jo6@deft.cc.purdue.edu> NNTP-Posting-Host: andromeda.glaxo.med.ualberta.ca X-Newsreader: MT-NewsWatcher 2.4.4 In article <7r5509$jo6@deft.cc.purdue.edu>, dokland@deft.cc.purdue.edu (Terje Dokland) wrote: > Does anybody know of a non-destructive chemical method to > deglycosylate proteins? Enzymatic methods would work, but the enzymes > are forbiddingly expensive in the amounts that I would most likely > require. The chemical methods that I have seen in the literature > (anhydrous hydrogen fluoride or TFMS) seem rather harsh and would > destroy the structure I am interested in, namely a virus. Any > suggestions would be appreciated. Thanks, > terje Terje, This suggestion might be coming from left-field but is it possible to change the amino acid target(s) for glycosylation (N-linked/O-linked) through site-directed mutagenesis possible for your virus without terminally impairing replication? Just an thought karlster From owner-proteins@net.bio.net Thu Sep 09 04:29:00 1999 Path: biosci!newsfeed.stanford.edu!newsfeed.berkeley.edu!news.maxwell.syr.edu!newsfeed.nacamar.de!fu-berlin.de!jussieu.fr!news.infobiogen.fr!lovelace.infobiogen.fr!besmond From: Claude Besmond Newsgroups: bionet.molbio.proteins Subject: raw to GCG converter Date: Thu, 9 Sep 1999 14:17:46 +0200 Organization: "GIS INFOBIOGEN, 7 rue Guy Moquet BP8, 94801 VILLEJUIF, France" Lines: 12 Message-ID: Reply-To: Claude Besmond NNTP-Posting-Host: lovelace.infobiogen.fr Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII X-Trace: caroll.infobiogen.fr 936879499 7869 193.52.226.4 (9 Sep 1999 12:18:19 GMT) X-Complaints-To: newsmaster@infobiogen.fr NNTP-Posting-Date: 9 Sep 1999 12:18:19 GMT Hello, I am looking for a utility program (W3 somewhere or downloadable for Mac or Unix) that translate raw 1-letter protein sequence (no number, no annotations, no signs) into GCG format. Readseq doesn't seem to do it, or does it?? Thanks Claude From owner-proteins@net.bio.net Thu Sep 09 04:46:00 1999 Path: biosci!pravda.ucr.edu!ihnp4.ucsd.edu!sdd.hp.com!usc!howland.erols.net!portc02.blue.aol.com!pitt.edu!not-for-mail From: Rich Dudley Newsgroups: bionet.molbio.proteins Subject: Re: raw to GCG converter Date: Thu, 09 Sep 1999 08:44:47 -0400 Organization: University of Pittsburgh, Dept. of Cell Biology and Physiology Lines: 33 Message-ID: <37D7ABBE.59FD388F@pitt.edu> References: Reply-To: rdudley+@pitt.edu NNTP-Posting-Host: whill.cbp.pitt.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.61 [en] (Win95; I) X-Accept-Language: en Are you using GCG? If you are, you can put the sequence in something of a FASTA format, and use the GCG command "reformat". The file should look looke like this: >seqence_name .. sequence It's important to include the two dots after the sequence name. Good luck! rich Claude Besmond wrote: > Hello, > > I am looking for a utility program (W3 somewhere or downloadable for Mac > or Unix) that translate raw 1-letter protein sequence (no number, no > annotations, no signs) into GCG format. > Readseq doesn't seem to do it, or does it?? > > Thanks > Claude -- --- --- --- -- -- -- --- --- --- Richard J. Dudley (rdudley+@pitt.edu) Research Specialist V Dept. of Cell Biology and Physiology University of Pittsburgh http://www.cbp.pitt.edu ---> search BIONET archives at http://www.bio.net <--- From owner-proteins@net.bio.net Thu Sep 09 08:40:00 1999 Path: biosci!newsfeed.stanford.edu!nntp.cs.ubc.ca!news-spur1.maxwell.syr.edu!news.maxwell.syr.edu!newsfeed.nacamar.de!fu-berlin.de!watson.bio.nat.tu-bs.DE!not-for-mail From: Andrea Hansen Newsgroups: bionet.molbio.proteins Subject: Re: raw to GCG converter Date: 09 Sep 1999 18:33:51 +0200 Lines: 19 Sender: andi@watson.bio.nat.tu-bs.de Message-ID: References: NNTP-Posting-Host: watson.bio.nat.tu-bs.de (134.169.70.93) Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Access: 16 [4974] X-Trace: fu-berlin.de 936894852 15756 (none) 134.169.70.93 X-Newsreader: Gnus v5.5/XEmacs 20.4 - "Emerald" Claude Besmond writes: Hi Claude, > I am looking for a utility program (W3 somewhere or downloadable for Mac > or Unix) that translate raw 1-letter protein sequence (no number, no > annotations, no signs) into GCG format. Have you tried REFORMAT? It is a program in the GCG package: REFORMAT rewrites sequence files so that they can be read by GCG programs. Andrea -- Andrea Hansen Institut fuer Genetik Tel : ++49-531-391-5783 TU Braunschweig Fax : ++49-531-391-5765 Spielmannstr. 7 email : webmaster@bioinformatik.de 38106 Braunschweig http://www.bioinformatik.de From owner-proteins@net.bio.net Fri Sep 10 07:16:00 1999 Path: biosci!newsfeed.stanford.edu!arclight.uoregon.edu!hammer.uoregon.edu!newsfeed.direct.ca!fu-berlin.de!jussieu.fr!news.infobiogen.fr!lovelace.infobiogen.fr!besmond From: Claude Besmond Newsgroups: bionet.molbio.proteins Subject: Re: raw to GCG converter (Thanks) Date: Fri, 10 Sep 1999 17:07:44 +0200 Organization: "GIS INFOBIOGEN, 7 rue Guy Moquet BP8, 94801 VILLEJUIF, France" Lines: 14 Message-ID: References: <37D7ABBE.59FD388F@pitt.edu> NNTP-Posting-Host: lovelace.infobiogen.fr Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII X-Trace: caroll.infobiogen.fr 936976084 22492 193.52.226.4 (10 Sep 1999 15:08:04 GMT) X-Complaints-To: newsmaster@infobiogen.fr NNTP-Posting-Date: 10 Sep 1999 15:08:04 GMT In-Reply-To: <37D7ABBE.59FD388F@pitt.edu> Rich and Andrea, thank you for your help about Reformat. It works well. Claude --------------------------------------- Claude Besmond Hopital Robert Debre, Inserm Unite 458 48 boulevard Serurier, 75019 Paris, France tel: 33(0)1 4003 1927 fax: 33(0)1 4003 1903 e-mail: besmond@infobiogen.fr From owner-proteins@net.bio.net Sun Sep 12 05:34:00 1999 Path: biosci!BIOLS.SUSX.AC.UK!barbarac From: barbarac@BIOLS.SUSX.AC.UK (Barbara Ciani) Newsgroups: bionet.molbio.proteins Subject: free energy estimation from CD Date: 12 Sep 1999 06:34:25 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 50 Sender: daemon@net.bio.net Distribution: world Message-ID: <37DBAA4E.DEE5E548@biols.susx.ac.uk> NNTP-Posting-Host: net.bio.net --------------C971CA15BB094AC02087EA9E Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit Hi there! Does anyone knows about software or algorithms to calculate free energy and other thermodynamic quantities from CD thermal denaturation curves? Barbara -- Barbara Ciani Protein Design Group School of Biological Sciences University of Sussex Falmer Brighton BN1 9QG phone:01273-678683 email:barbarac@biols.susx.ac.uk http://bionix.biols.susx.ac.uk/biols/Biochem/Woolfson/html/group.html --------------C971CA15BB094AC02087EA9E Content-Type: text/html; charset=us-ascii Content-Transfer-Encoding: 7bit Hi  there!
Does anyone knows about software or algorithms to calculate free energy and other thermodynamic quantities from CD thermal denaturation curves?

Barbara 

-- 
Barbara Ciani
Protein Design Group 
School of Biological Sciences
University of Sussex
Falmer
Brighton BN1 9QG

phone:01273-678683
email:barbarac@biols.susx.ac.uk
http://bionix.biols.susx.ac.uk/biols/Biochem/Woolfson/html/group.html
  --------------C971CA15BB094AC02087EA9E-- From owner-proteins@net.bio.net Mon Sep 13 01:01:00 1999 Path: biosci!internet!biosci!not-for-mail From: biohelp (BIOSCI Administrator) Newsgroups: bionet.molbio.proteins Subject: BIOSCI/bionet miniFAQ & Fundraiser Date: 13 Sep 1999 02:00:28 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 239 Sender: daemon@net.bio.net Distribution: world Message-ID: <199909130900.CAA04379@net.bio.net> NNTP-Posting-Host: net.bio.net (LAST REVISION: 14-AUG-99) 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. -------------------------------------------------------- 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 -------------------------------------------------------------------- the UK-HGMP-Resource Centre (known as hgmp.mrc.ac.uk): ----------------------------------------------------- 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@hgmp.mrc.ac.uk. 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 majordomo@hgmp.mrc.ac.uk. 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 Please ask for help at biosci@hgmp.mrc.ac.uk 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. 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 Mon Sep 13 01:53:00 1999 Path: biosci!newsfeed.stanford.edu!news-feed.inet.tele.dk!bofh.vszbr.cz!unlisys!news.snafu.de!cs.tu-berlin.de!news.uni-hamburg.de!not-for-mail From: behrends@plexus.uke.uni-hamburg.de (soenke behrends) Newsgroups: bionet.molbio.proteins Subject: test for bacterial expression? Date: Mon, 13 Sep 1999 09:42:51 GMT Organization: UKE Lines: 17 Message-ID: <7rih43$mpg$2@rzsun02.rrz.uni-hamburg.de> Reply-To: behrends@plexus.uke.uni-hamburg.de NNTP-Posting-Host: mendel.uke.uni-hamburg.de X-Newsreader: Forte Free Agent v0.55 Dear netters, when you express a protein in E. coli and you get the impression that you have inclusion bodies. Is there a quick method for diagnosis? How do you test whether the protein just sticks to the membrane or whether you have inclusion bodies? Thanks for your time and help Soenke From owner-proteins@net.bio.net Mon Sep 13 09:08:00 1999 Path: biosci!newsfeed.stanford.edu!logbridge.uoregon.edu!newsfeed.esat.net!diablo.theplanet.net!colt.net!news.belnet.be!news.rediris.es!News.cica.es!""!not-for-mail From: Antonio J. =?iso-8859-1?Q?P=E9rez?= Newsgroups: bionet.molbio.proteins Subject: SwissProt with non-homologs protein sequences Date: Mon, 13 Sep 1999 18:58:07 +0200 Organization: C.I.C.A. Lines: 12 Message-ID: <37DD2D1F.7D6CE0A9@uma.es> NNTP-Posting-Host: puma.genetica.uma.es Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Trace: mercurio.cica.es 937241816 5421 150.214.50.212 (13 Sep 1999 16:56:56 GMT) X-Complaints-To: news@cica.es NNTP-Posting-Date: 13 Sep 1999 16:56:56 GMT X-Mailer: Mozilla 4.61 [en] (Win98; I) X-Accept-Language: en Where could I find a SwissProt with non-homologs sequences above of a determinate identity level? -- Antonio J. Pérez Pulido Universidad of Málaga-Facultad de Ciencias Dpto. Biología Celular y Genética (Unidad de Genética) Campus Universitario de Teatinos, 29071 Málaga (Spain) Tfno. 952131957 / Fax 952285897 e-mail aperezp@uma.es From owner-proteins@net.bio.net Thu Sep 16 03:08:00 1999 Path: biosci!fcs280s.ncifcrf.gov!fcrdcnews.NCIFCRF.GOV!washdc3-snf1!washdc3-snh1.gtei.net!nyc-news-feed1.bbnplanet.com!news.gtei.net!easynet-tele!easynet.net!newsfeed.icl.net!newsfeed.nacamar.de!news.maxwell.syr.edu!nntp2.deja.com!nnrp1.deja.com!not-for-mail From: malgorzatawilczynska@my-deja.com Newsgroups: bionet.molbio.proteins Subject: free sulfhydryl detection kit??? Date: Thu, 16 Sep 1999 10:42:27 GMT Organization: Deja.com - Share what you know. Learn what you don't. Lines: 9 Message-ID: <7rqhii$39g$1@nnrp1.deja.com> NNTP-Posting-Host: 130.239.34.75 X-Article-Creation-Date: Thu Sep 16 10:42:27 1999 GMT X-Http-User-Agent: Mozilla/4.5 [en] (Win95; I) X-Http-Proxy: 1.0 x38.deja.com:80 (Squid/1.1.22) for client 130.239.34.75 X-MyDeja-Info: XMYDJUIDmalgorzatawilczynska Hi, does anybody know a kit or a reliable method for free sulfhydryl groups quantification in proteins? Thanks Malgorzata Sent via Deja.com http://www.deja.com/ Share what you know. Learn what you don't. From owner-proteins@net.bio.net Thu Sep 16 07:21:00 1999 Path: biosci!newsfeed.stanford.edu!logbridge.uoregon.edu!newsfeed.icl.net!newsfeed.nacamar.de!fu-berlin.de!pc207-60.biochem.uni-potsdam.DE!not-for-mail From: Frank =?iso-8859-1?Q?F=FCrst?= Newsgroups: bionet.molbio.proteins Subject: Re: free sulfhydryl detection kit??? Date: Thu, 16 Sep 1999 17:17:00 +0200 Lines: 45 Message-ID: <7rr1g9$ks8$1@fu-berlin.de> References: <7rqhii$39g$1@nnrp1.deja.com> NNTP-Posting-Host: pc207-60.biochem.uni-potsdam.de (141.89.207.60) Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Access: 16 49 955 X-Trace: fu-berlin.de 937494857 21384 (none) 141.89.207.60 X-Mailer: Mozilla 4.51 [de]C-CCK-MCD QXW03200 (Win95; I) X-Accept-Language: de-DE,en malgorzatawilczynska@my-deja.com wrote: > > Hi, > does anybody know a kit or a reliable method for free sulfhydryl groups > quantification in proteins? > Thanks > Malgorzata > Ellmans test works well. The principle is the reaction of DTNB, 5,5'-Dithio-2,2'-dinitro-dibenzoic acid with the free -SH, which forms a yellow dye (2-Nitro benzoic acid-5-thiolate or something like that should be the name). Then you can quantitate the free sulfhydryls by measuring the absorbance at 412 nm, epsilon(TNB-)=1415 M-1 cm-1 I learned the method from a colleague and got the numbers from her, so perhaps better check the literature (see below). I used it to measure exact concentrations of DTT at pH 9, but I know that it also works with proteins, so I think pH is not important. Only at acidic pH I'd expect the reaction to slow down (and at very low pH you'd get the TNB- protonated of course). The reaction isn't too fast anyway, and one has to take into account that the DTNB-solution absorbs a little, too. And I had the impression that DTNB-solution is unstable under aerobic conditions, so I didn't keep it longer than some hours. DTNB is poorly soluble in water, but well in buffer like 50 mM Tris. Now the literature, taken from the catalogue of Merck KG [the german company, not the american one]: 1. Ellman, G.L., Arch. Biochem. Biophys. 74, 443 (1958) 2. Riddles, PlW., et al., Anal. Biochem. 94, 75 (1979) 3. Wieker, H.J., et al., Z. Physiol. Chem. 353, 771 (1972) bye, Frank -- Frank Fuerst, Institute of Biochemistry of Potsdam University Im Biotechnologiepark, D-14943 Luckenwalde Tel.: +49-3371-681334; Fax: +49-3371-681339 I'm SignatureVirus 99! Copy me into your signature and join the fun! From owner-proteins@net.bio.net Fri Sep 17 02:33:00 1999 Path: biosci!cnn.nas.nasa.gov!newsfeed.berkeley.edu!dispose.news.demon.net!demon!insnet.net!peernews.cix.co.uk!news.cix.co.uk!not-for-mail From: "Andrew M Smith" Newsgroups: bionet.molbio.proteins Subject: Find conference information here Date: Fri, 17 Sep 1999 11:09:28 +0100 Organization: www.selectscience.net Message-ID: <7rt4al$ao1$15@plutonium.compulink.co.uk> NNTP-Posting-Host: asmithi.compulink.co.uk X-Newsreader: Microsoft Outlook Express 4.72.3110.5 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3110.3 Lines: 20 At www.selectscience.net we have details on nearly 350 conferences, symposia, exhibitions, short courses and lectures taking place from today through to 2002. Earlier this year, we have featured information on over 350 more. If you want to find out about upcoming events in your discipline, come to www.selectscience.net If you are organising a meeting, please drop me a line (andrew@selectscience.net) so I can add your event to our database. I'm looking forward to hearing from you, Andrew Smith (andrew@selectscience.net) Editor, www.selectscience.net From owner-proteins@net.bio.net Sun Sep 19 05:24:00 1999 Path: biosci!interstroom.nl!nieuwenhuizen From: nieuwenhuizen@interstroom.nl ("R. vd Nieuwenhuizen") Newsgroups: bionet.molbio.proteins Subject: Protein quantitation Date: 19 Sep 1999 06:24:51 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 57 Sender: daemon@net.bio.net Distribution: world Message-ID: <001201bf02ec$eba8aa20$a87debc2@nieuwenhuizen> NNTP-Posting-Host: net.bio.net This is a multi-part message in MIME format. ------=_NextPart_000_000F_01BF02B2.3EE51CE0 Content-Type: text/plain; charset="iso-8859-1" Content-Transfer-Encoding: quoted-printable Dear scientists, I'm a 2nd grade student chemistry, I was wondering how one can (quickly) = analyse=20 the total-protein concentration in a solid sample (powder). And what methods can be used tot extract all proteins from a solid = sample. I was thinking about analysing on a spectrophotometer. Can anyone provide me an awnser ??? Or send me some links on the=20 subject ?? Thanks, ------=_NextPart_000_000F_01BF02B2.3EE51CE0 Content-Type: text/html; charset="iso-8859-1" Content-Transfer-Encoding: quoted-printable
Dear scientists,
 
I'm a 2nd grade student chemistry, I = was wondering=20 how one can (quickly) analyse
the total-protein concentration in a = solid sample=20 (powder).
And what methods can be used tot = extract all=20 proteins from a solid sample.
I was thinking about analysing on a=20 spectrophotometer.
Can anyone provide me an awnser ??? Or = send me some=20 links on the
subject ??
 
Thanks,
 
------=_NextPart_000_000F_01BF02B2.3EE51CE0-- From owner-proteins@net.bio.net Sun Sep 19 05:28:00 1999 Path: biosci!interstroom.nl!nieuwenhuizen From: nieuwenhuizen@interstroom.nl ("R. vd Nieuwenhuizen") Newsgroups: bionet.molbio.proteins Subject: quantitating protein in solid sample Date: 19 Sep 1999 06:28:04 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 19 Sender: daemon@net.bio.net Distribution: world Message-ID: <000501bf02ed$60c39b80$a87debc2@nieuwenhuizen> NNTP-Posting-Host: net.bio.net Dear scientists, I'm a 2nd grade student chemistry, I was wondering how one can (quickly) = analyse=20 the total-protein concentration in a solid sample (powder). And what methods can be used tot extract all proteins from a solid = sample. I was thinking about analysing on a spectrophotometer. Can anyone provide me an awnser ??? Or send me some links on the=20 subject ?? Thanks, rembrand@mail.com From owner-proteins@net.bio.net Sun Sep 19 14:44:00 1999 Path: biosci!newsfeed.stanford.edu!uchinews!newsswitch.lcs.mit.edu!howland.erols.net!sunqbc.risq.qc.ca!newsflash.concordia.ca!pitt.edu!not-for-mail From: pxpst2@vms.cis.pitt.edu (Peter) Newsgroups: bionet.molbio.proteins Subject: Re: Protein quantitation Date: Sun, 19 Sep 1999 18:35:06 -0400 Organization: University Of Pittsburgh Lines: 29 Distribution: world Message-ID: References: <001201bf02ec$eba8aa20$a87debc2@nieuwenhuizen> NNTP-Posting-Host: pelli.pathology.pitt.edu Mail-Copies-To: pxpst2@vms.cis.pitt.edu X-Newsreader: MT-NewsWatcher 2.4.4 First, please do not post in MIME format on usenet. It makes reading difficult. In article <001201bf02ec$eba8aa20$a87debc2@nieuwenhuizen>, nieuwenhuizen@interstroom.nl ("R. vd Nieuwenhuizen") wrote: > > I'm a 2nd grade student chemistry, I was wondering how one can (quickly) = > analyse=20 > the total-protein concentration in a solid sample (powder). Do you want to keep it a powder for the analysis? If you answer "no" then weigh out some and dissolve it into a fix volume of water. Now you can do a bradford, Lowry or BCA protein assay. These are all relatively easy and quick to perform. Only equipment needed is a spectrophotometer, preferably a plate spectrophotometer. If you do not want to dissolve it, then you will have to use some very exotic techniques and your sensitivity is going to suffer. Techniques I am thinking of are photoaccustic IR. But it may not work well if you do not know the specific protein. > And what methods can be used totally extract all proteins from a solid > sample. Phenol ether extraction. Good for nanagrams of protein to grams. email me if you want the protocol or look it up through the bionet archives. regards, Peter Pediaditakis From owner-proteins@net.bio.net Sun Sep 19 18:45:00 1999 Path: biosci!sprint.ca!suehan From: suehan@sprint.ca ("SUE HAN") Newsgroups: bionet.molbio.proteins Subject: PROTEIN EXPRESSION Date: 19 Sep 1999 19:45:02 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 92 Sender: daemon@net.bio.net Distribution: world Message-ID: <028501bd7fc3$d2cc5a40$039294d1@hqian> NNTP-Posting-Host: net.bio.net This is a multi-part message in MIME format. ------=_NextPart_000_0282_01BD7F89.2551B200 Content-Type: text/plain; charset="gb2312" Content-Transfer-Encoding: quoted-printable Do you have too many projects and limited time? If high level protein = expression is one of your goals, C&P Biotech is ready to help you with = sub-cloning and protein purification in E. coli. 1.. Sub-cloning, site-directed mutagenesis, large deletion and = insertion mutation. PRICE: US$ 500.00 per sample. 2.. Protein expression and purification based on GST or = histidine-tag fusion. PRICE: US$ 900.00, 5.0 mg electrophoresis pure protein is guaranteed. Give yourself the chances to concentrate on other important projects = with knowing that professionals are handling your expression and = purification work.=20 NO CHARGE IF WE FAILED. CONTACT ADDRESS: Sue Han C&P Biotech 3 Hampton Way Thornhill, Ontario Canada, L3T 5C8 Email: suehan@sprint.ca Tel: 416 992 3089 Fax: 905 886 1898 ------=_NextPart_000_0282_01BD7F89.2551B200 Content-Type: text/html; charset="gb2312" Content-Transfer-Encoding: quoted-printable

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------=_NextPart_000_0282_01BD7F89.2551B200-- From owner-proteins@net.bio.net Mon Sep 20 03:17:00 1999 Path: biosci!newsfeed.stanford.edu!newsfeed.berkeley.edu!hermes.visi.com!news-out.visi.com!newsfeed.enteract.com!news.maxwell.syr.edu!nntp2.deja.com!nnrp1.deja.com!not-for-mail From: Kresten Newsgroups: bionet.molbio.proteins Subject: Re: free sulfhydryl detection kit??? Date: Mon, 20 Sep 1999 11:02:30 GMT Organization: Deja.com - Share what you know. Learn what you don't. Lines: 86 Message-ID: <7s5482$8rh$1@nnrp1.deja.com> References: <7rqhii$39g$1@nnrp1.deja.com> <7rr1g9$ks8$1@fu-berlin.de> NNTP-Posting-Host: 130.226.183.6 X-Article-Creation-Date: Mon Sep 20 11:02:30 1999 GMT X-Http-User-Agent: Mozilla/4.5 [en] (Win95; I) X-Http-Proxy: 1.0 www2.crc.dk:3128 (Squid/2.1.RELEASE), 1.0 x37.deja.com:80 (Squid/1.1.22) for client 130.226.182.217, 130.226.183.6 X-MyDeja-Info: XMYDJUIDkresten In article <7rr1g9$ks8$1@fu-berlin.de>, Frank =?iso-8859-1?Q?F=FCrst?= wrote: > malgorzatawilczynska@my-deja.com wrote: > > > > Hi, > > does anybody know a kit or a reliable method for free sulfhydryl > > groups > > quantification in proteins? > > Thanks > > Malgorzata > > > Ellmans test works well. Yep > > The principle is the reaction of DTNB, > 5,5'-Dithio-2,2'-dinitro-dibenzoic acid with the free -SH, which forms > a yellow dye (2-Nitro benzoic acid-5-thiolate or something like that > should be the name). Then you can quantitate the free sulfhydryls by > measuring the absorbance at 412 nm, epsilon(TNB-)=1415 M-1 cm-1 I think the number is about ten-fold higher. > > I learned the method from a colleague and got the numbers from her, so > perhaps better check the literature (see below). > > I used it to measure exact concentrations of DTT at pH 9, but I know > that it also works with proteins, so I think pH is not important. Only > at acidic pH I'd expect the reaction to slow down (and at very low pH > you'd get the TNB- protonated of course). pH is important for the abovementioned reasons. As the reactive group is the thiolate in the protein the reaction will run much faster at the higher pH. But be aware that many unwanted sidereactions also run faster at the higher pH - e.g. oxidation of thiols by air. Can be a problem. > The reaction isn't too fast anyway, and one has to take into account > that the DTNB-solution absorbs a little, too. And I had the impression > that DTNB-solution is unstable under aerobic conditions, so I didn't > keep it longer than some hours. I keep mine as 10mM in 0.5M KPi at pH 7.0, 4 degrees C. I have had no problems wih stability. Usually one uses DTNB at high excess so that some instability can be tolerated if appropriate background measurements are made. Also one thing to remember is that some free thiols in the protein may not react with Ellman's reagent due to inaccesability. Therefore it can be a good idea to make two measurements, one in water and one in high concentration of denaturant (eg 6M GuaHCl). Epsilon is a bit different in 6M GuaHCl but the value is somewhere in the litterature - and can be measured easily if the library is more than 5 min away. HTH Kresten > > DTNB is poorly soluble in water, but well in buffer like 50 mM Tris. > > Now the literature, taken from the catalogue of Merck KG [the german > company, not the american one]: > > 1. Ellman, G.L., Arch. Biochem. Biophys. 74, 443 (1958) > 2. Riddles, PlW., et al., Anal. Biochem. 94, 75 (1979) > 3. Wieker, H.J., et al., Z. Physiol. Chem. 353, 771 (1972) > > bye, Frank > > -- > Frank Fuerst, Institute of Biochemistry of Potsdam University > Im Biotechnologiepark, D-14943 Luckenwalde > Tel.: +49-3371-681334; Fax: +49-3371-681339 > I'm SignatureVirus 99! Copy me into your signature and join the fun! > -- The address kresten@my-dejanews.com is for spambots only. Please mail me at LysLeuLeu@crc.dk transforming the pre@-part into my initials. Kresten Lindorff Larsen, Dept. Yeast Genetics Sent via Deja.com http://www.deja.com/ Share what you know. Learn what you don't. From owner-proteins@net.bio.net Mon Sep 20 03:17:00 1999 Path: biosci!newsfeed.stanford.edu!arclight.uoregon.edu!wn4feed!worldnet.att.net!207.172.3.37!feed1.news.rcn.net!rcn!news.maxwell.syr.edu!nntp2.deja.com!nnrp1.deja.com!not-for-mail From: Kresten Newsgroups: bionet.molbio.proteins Subject: Re: free sulfhydryl detection kit??? Date: Mon, 20 Sep 1999 11:01:54 GMT Organization: Deja.com - Share what you know. Learn what you don't. Lines: 86 Message-ID: <7s546u$8r8$1@nnrp1.deja.com> References: <7rqhii$39g$1@nnrp1.deja.com> <7rr1g9$ks8$1@fu-berlin.de> NNTP-Posting-Host: 130.226.183.6 X-Article-Creation-Date: Mon Sep 20 11:01:54 1999 GMT X-Http-User-Agent: Mozilla/4.5 [en] (Win95; I) X-Http-Proxy: 1.0 www2.crc.dk:3128 (Squid/2.1.RELEASE), 1.0 x35.deja.com:80 (Squid/1.1.22) for client 130.226.182.217, 130.226.183.6 X-MyDeja-Info: XMYDJUIDkresten In article <7rr1g9$ks8$1@fu-berlin.de>, Frank =?iso-8859-1?Q?F=FCrst?= wrote: > malgorzatawilczynska@my-deja.com wrote: > > > > Hi, > > does anybody know a kit or a reliable method for free sulfhydryl > > groups > > quantification in proteins? > > Thanks > > Malgorzata > > > Ellmans test works well. Yep > > The principle is the reaction of DTNB, > 5,5'-Dithio-2,2'-dinitro-dibenzoic acid with the free -SH, which forms > a yellow dye (2-Nitro benzoic acid-5-thiolate or something like that > should be the name). Then you can quantitate the free sulfhydryls by > measuring the absorbance at 412 nm, epsilon(TNB-)=1415 M-1 cm-1 I think the number is about ten-fold higher. > > I learned the method from a colleague and got the numbers from her, so > perhaps better check the literature (see below). > > I used it to measure exact concentrations of DTT at pH 9, but I know > that it also works with proteins, so I think pH is not important. Only > at acidic pH I'd expect the reaction to slow down (and at very low pH > you'd get the TNB- protonated of course). pH is important for the abovementioned reasons. As the reactive group is the thiolate in the protein the reaction will run much faster at the higher pH. But be aware that many unwanted sidereactions also run faster at the higher pH - e.g. oxidation of thiols by air. Can be a problem. > The reaction isn't too fast anyway, and one has to take into account > that the DTNB-solution absorbs a little, too. And I had the impression > that DTNB-solution is unstable under aerobic conditions, so I didn't > keep it longer than some hours. I keep mine as 10mM in 0.5M KPi at pH 7.0, 4 degrees C. I have had no problems wih stability. Usually one uses DTNB at high excess so that some instability can be tolerated if appropriate background measurements are made. Also one thing to remember is that some free thiols in the protein may not react with Ellman's reagent due to inaccesability. Therefore it can be a good idea to make two measurements, one in water and one in high concentration of denaturant (eg 6M GuaHCl). Epsilon is a bit different in 6M GuaHCl but the value is somewhere in the litterature - and can be measured easily if the library is more than 5 min away. HTH Kresten > > DTNB is poorly soluble in water, but well in buffer like 50 mM Tris. > > Now the literature, taken from the catalogue of Merck KG [the german > company, not the american one]: > > 1. Ellman, G.L., Arch. Biochem. Biophys. 74, 443 (1958) > 2. Riddles, PlW., et al., Anal. Biochem. 94, 75 (1979) > 3. Wieker, H.J., et al., Z. Physiol. Chem. 353, 771 (1972) > > bye, Frank > > -- > Frank Fuerst, Institute of Biochemistry of Potsdam University > Im Biotechnologiepark, D-14943 Luckenwalde > Tel.: +49-3371-681334; Fax: +49-3371-681339 > I'm SignatureVirus 99! Copy me into your signature and join the fun! > -- The address kresten@my-dejanews.com is for spambots only. Please mail me at LysLeuLeu@crc.dk transforming the pre@-part into my initials. Kresten Lindorff Larsen, Dept. Yeast Genetics Sent via Deja.com http://www.deja.com/ Share what you know. Learn what you don't. From owner-proteins@net.bio.net Mon Sep 20 03:54:00 1999 From: "Andrew M Smith" Newsgroups: bionet.molbio.proteins Subject: Last week's news headlines Date: Mon, 20 Sep 1999 12:00:49 +0100 Organization: www.selectscience.net Message-ID: <7s56s9$oeu$15@plutonium.compulink.co.uk> NNTP-Posting-Host: asmithi.compulink.co.uk Mime-Version: 1.0 Content-Type: text/plain; charset="iso-8859-1" Content-Transfer-Encoding: quoted-printable X-Newsreader: Microsoft Outlook Express 4.72.3110.5 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3110.3 Path: biosci!rutgers!nntp.upenn.edu!logbridge.uoregon.edu!dispose.news.demon.net!demon!newsfeed.icl.net!news.netkonect.net!news.intensive.net!peernews.cix.co.uk!news.cix.co.uk!not-for-mail Lines: 60 In the last seven days, www.selectscience.net has reported on 24 news stories the day they happened, so if you need to keep up to speed on the latest business developments in chemistry and life science, make the news ticker on www.selectscience.net part of your daily routine. Andrew Smith (andrew@selectscience.net) Editor, www.selectscience.net The last week's headlines were: deCODE genetics of Iceland Maps a Gene Linked to pre-eclampsia Peptide Therapeutics Group plc: Interim Results for the Six Months Ended = 30 June 1999 & Proposed Spin-Out of Drug Discovery Activities Affymetrix, Inc. Announces Exercise of Share Purchase Option Affymetrix, Inc. Raises $125 Million in Convertible Subordinated Debt = Placement Bacterial Virus And Human Adenovirus Found to be Similar Biotech entrepreneurs need assistance, say workshop participants Oxford GlycoSciences and Cellular Genomics, Inc., Establish Joint = Proteome Program PE Corporation to Enter Alliance With New Venture Led By Dr. Noubar = Afeyan Celera Genomics Completes Sequencing Phase of Drosophila Genome Project Aurora Biosciences Announces Director Resignation Aurora Biosciences Announces Drug Discovery Program Funded By The Cystic = Fibrosis Foundation Affymetrix to Acquire Genetic Mictosystems DuPont and MIT Form Biotechnology Alliance Judgement in Favor of Affymetrix in Patent Interferences Oxford GlycoSciences Reports 1999 Interim Results Abbott Laboratories Declares 303rd Consecutive Quarterly Dividend Hughes Institute Scientists Announce a New Paradigm for the Treatment Of = Asthma and Allergy New Chemical Entities, Inc. and Thetagen, Inc. Merge Myriad Genetics Forms Strategic Alliance With Galileo Genomics Cellomics Announces Major Expansion Into Europe From owner-proteins@net.bio.net Fri Sep 24 02:18:00 1999 Path: biosci!newshost.lanl.gov!logbridge.uoregon.edu!remarQ73!supernews.com!remarQ.com!remarQ69!WReNclone!WReNphoon3.POSTED!WReN!not-for-mail From: AHRickard Subject: Biotin maleimide Newsgroups: bionet.molbio.proteins Message-ID: <1e2537bb.5f810fe0@usw-ex0108-059.remarq.com> Lines: 14 Bytes: 524 X-Originating-Host: 194.83.240.20 Organization: http://www.remarq.com: The World's Usenet/Discussions Start Here X-Wren-Trace: eICljYyV0pjTzZuFhseAm8Czg5GQgI6bhILBgorajMvP15nVwJjGwN7V1MvckA== Date: Thu, 23 Sep 1999 07:16:08 -0700 NNTP-Posting-Host: 10.0.2.59 X-Complaints-To: wrenabuse@remarq.com X-Trace: WReNphoon3 938167753 10.0.2.59 (Fri, 24 Sep 1999 03:09:13 PDT) NNTP-Posting-Date: Fri, 24 Sep 1999 03:09:13 PDT Hi, Biotin maleidmide is a sulfhydryl specific biotinylating reagent. Does anyone know what is the strength of the interaction between cysteine (present in long chains of polypeptides and proteins)and biotin maleimide. More to the point will SDS or heat cause dissassociation of BM from cysteine.Thanks for your time, Alex Rickard PME Manchester University * Sent from RemarQ http://www.remarq.com The Internet's Discussion Network * The fastest and easiest way to search and participate in Usenet - Free! From owner-proteins@net.bio.net Fri Sep 24 08:57:00 1999 Path: biosci!candseek.com!915608 From: 915608@candseek.com Newsgroups: bionet.molbio.proteins Subject: JOBOP LABORATORY TECHNICIAN CELL CULTURE Date: 24 Sep 1999 09:57:00 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 76 Sender: daemon@net.bio.net Distribution: world Message-ID: <199909241656.JAA05075@net.bio.net> NNTP-Posting-Host: net.bio.net Since your email address was listed on a related web site page or database, I thought you might help. I am seeking an individual within the following conditions: A laboratory technician who will work for a biotech company that is developing novel therapeutic products including interventional therapeutics, cancer vaccines and blood cell growth factors for the treatment of cancer and cancer-related disorders. The candidate should have experience in mammalian cell culture and manipulation. This person will be responsible for culture and maintenance of adherent and suspension mammalian cells, media and reagent preparation, DNA transfection of eukaryotic cells, gene expression assays, and good record keeping. The candidate should possess a B.S.,M.S.or MD degree in the biological sciences. Our client is a leading biotech firm with research facilities in New York and can provide excellent benefits (health insurance, dental, and vision plan, paid vacation and more). A high impact, high profile position with excellent opportunity for advancement. Geographic Location of Position: US-NY If you know anyone that might be interested, please forward this to them or contact: Sam Sanders DMC Fax: 609-584-9575 Voice: 609-584-8733 Ext. 218 Email: 915608@candseek.com To permanently discontinue receiving employment opportunity notices from any and all help wanted advertisers using the Candidate Seeker system, click your "Reply" button and type the word "re- move" without spaces between the letters into the SUBJECT field then click the "Send" button. Your email address will be permanently filtered from ALL future job opportunity notifications sent via the Candidate Seeker system. To temporarily filter employment opportunity notices sent via the Candidate Seeker system, type the acronym "JOBOP" into your subject filter. All employment opportunity notices sent via the Candidate Seeker system contain the acronym "JOBOP" in the subject so they may be easily filtered or blocked if so desired. Other email addresses may be permanently deleted from future contact by emailing a single blank message from the desired address to nomail@candseek.com. Enter additional addresses into the body of the message and they will also be added to the "nomail" list Please feel free to contact the candidateseeker.com feedback line at 609-584-5499. Do not use this number for job related questions. All job related questions should be directed to the employer by replying to contact addresses or phone numbers indicated at the end of the job description message. Mailed By: candseek.com 510 Horizon Center Robbinsville, NJ 08691 From owner-proteins@net.bio.net Sun Sep 26 13:36:00 1999 Path: biosci!newsfeed.stanford.edu!newsfeed.berkeley.edu!nntp2.deja.com!nnrp1.deja.com!not-for-mail From: Antonia125 Newsgroups: bionet.molbio.proteins Subject: beta barrel volume calc. Date: Sun, 26 Sep 1999 21:16:34 GMT Organization: Deja.com - Before you buy. Lines: 28 Message-ID: <7sm2fc$4te$1@nnrp1.deja.com> NNTP-Posting-Host: 144.92.44.76 X-Article-Creation-Date: Sun Sep 26 21:16:34 1999 GMT X-Http-User-Agent: Mozilla/4.06 [en] (Win95; U) X-Http-Proxy: 1.0 x41.deja.com:80 (Squid/1.1.22) for client 128.104.48.249, 144.92.44.76 X-MyDeja-Info: XMYDJUIDantonia125 Hello, I was wondering if anyone could help me with this What dimensions should I be using to calculate these answers? The question states: The tertiary structure of Ig VI domain is a beta-barrel of antiparallel strands. It can be described as a cylinder ofvdimensions 25 angstroms and 41 angstroms in length. (a). How many amino acid residues would you expect to find in this domain? (b). How many beta strands would you expect to find? (c). Given the number of beta strands, estimate the maximum number of residues that could be considered to adopt the conformation expected for a beta-sheet. I know the total volume would be pi times radius squared, and that it is 4.4 angstroms between alpha carbons, but I can't find a way to incorporate that and solve the questions. Thanks in advance, Antonia Sent via Deja.com http://www.deja.com/ Before you buy. From owner-proteins@net.bio.net Sun Sep 26 13:36:00 1999 Path: biosci!newsfeed.stanford.edu!newsfeed.berkeley.edu!nntp2.deja.com!nnrp1.deja.com!not-for-mail From: Antonia125 Newsgroups: bionet.molbio.proteins Subject: beta barrel volume calc. Date: Sun, 26 Sep 1999 21:18:02 GMT Organization: Deja.com - Before you buy. Lines: 28 Message-ID: <7sm2i4$4to$1@nnrp1.deja.com> NNTP-Posting-Host: 144.92.44.76 X-Article-Creation-Date: Sun Sep 26 21:18:02 1999 GMT X-Http-User-Agent: Mozilla/4.06 [en] (Win95; U) X-Http-Proxy: 1.0 x41.deja.com:80 (Squid/1.1.22) for client 128.104.48.249, 144.92.44.76 X-MyDeja-Info: XMYDJUIDantonia125 Hello, I was wondering if anyone could help me with this What dimensions should I be using to calculate these answers? The question states: The tertiary structure of Ig VI domain is a beta-barrel of antiparallel strands. It can be described as a cylinder of dimensions 25 angstroms and 41 angstroms in length. (a). How many amino acid residues would you expect to find in this domain? (b). How many beta strands would you expect to find? (c). Given the number of beta strands, estimate the maximum number of residues that could be considered to adopt the conformation expected for a beta-sheet. I know the total volume would be pi times radius squared, and that it is 4.4 angstroms between alpha carbons, but I can't find a way to incorporate that and solve the questions. Thanks in advance, Antonia Sent via Deja.com http://www.deja.com/ Before you buy. From owner-proteins@net.bio.net Sun Sep 26 13:37:00 1999 Path: biosci!newsfeed.stanford.edu!logbridge.uoregon.edu!newsfeed2.news.nl.uu.net!sun4nl!xs4all!jacqg From: jacqg@zpam.dds.nl (jw) Newsgroups: bionet.molbio.proteins Subject: Re: Biotin maleimide Date: Sun, 26 Sep 1999 23:31:56 +0200 Organization: reddeloos,redeloos&radeloos Lines: 26 Sender: wimerik@dc2-modem334.dial.xs4all.nl Message-ID: <1dyqim5.18qb0sf9xjajiN@dc2-modem334.dial.xs4all.nl> References: <1e2537bb.5f810fe0@usw-ex0108-059.remarq.com> NNTP-Posting-Host: dc2-modem334.dial.xs4all.nl X-Trace: news1.xs4all.nl 938381415 27166 194.109.129.78 (26 Sep 1999 21:30:15 GMT) X-Complaints-To: abuse@xs4all.nl NNTP-Posting-Date: 26 Sep 1999 21:30:15 GMT X-Newsreader: MacSOUP 2.3 AHRickard wrote: > Hi, > Biotin maleidmide is a sulfhydryl specific biotinylating > reagent. Does anyone know what is the strength of the > interaction between cysteine (present in long chains of > polypeptides and proteins)and biotin maleimide. More to the > point will SDS or heat cause dissassociation of BM from > cysteine.Thanks for your time, maleimide and its derivatives will covalently attach to free sulfhydryl groups, so you will need harsh and specific agents to break the thioether linkage formed. neither SDS nor heat will destroy the bonds. note that only free sulfhydryl moieties will react with maleimide, cysteine present in disulfide bridges will not become labeled. moreover, if the protein is folded not all free cysteines may be accessible. if you want to make sure to modify all cysteines, you should perform the labeling experiment under denaturing conditions (eg SDS). first reduce the cysteines with DTT or b-mercaptoethanol, then add molar excess (eg 10 fold) label. as a last note of warning, at higher pH (>8) the reaction with amines (lysine) will increase, maleimide specificity for sulfhydryl is at neutral or acidic pH. good luck, jw From owner-proteins@net.bio.net Mon Sep 27 00:58:00 1999 Path: biosci!newsfeed.stanford.edu!logbridge.uoregon.edu!dispose.news.demon.net!demon!newsfeed.nacamar.de!fu-berlin.de!pc207-60.biochem.uni-potsdam.DE!not-for-mail From: Frank =?iso-8859-1?Q?F=FCrst?= Newsgroups: bionet.molbio.proteins Subject: Re: beta barrel volume calc. Date: Mon, 27 Sep 1999 10:54:40 +0200 Message-ID: <7snb60$2pa$1@fu-berlin.de> References: <7sm2fc$4te$1@nnrp1.deja.com> NNTP-Posting-Host: pc207-60.biochem.uni-potsdam.de (141.89.207.60) Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Access: 16 49 955 X-Trace: fu-berlin.de 938422272 2858 (none) 141.89.207.60 X-Mailer: Mozilla 4.51 [de]C-CCK-MCD QXW03200 (Win95; I) X-Accept-Language: de-DE,en Lines: 61 Antonia125 wrote: (I don't think this type of questions is very sensible, but well...) > Hello, I was wondering if anyone could help me with this > > What dimensions should I be using to calculate these answers? > > The question states: The tertiary structure of Ig VI domain is a > beta-barrel of antiparallel strands. It can be described as a cylinder > ofvdimensions 25 angstroms and 41 angstroms in length. the 25 angstroms is the diameter? > (a). How many amino acid residues would you expect to find in this > domain? > > (b). How many beta strands would you expect to find? You should consider b) first. To do this, first calculate the circumference of the cylinder (Pi x diameter) and then divide it by the average width of a beta strand (You must look up this, don't know it). Thus you get the number of beta-strands. Now you need the average "length" of an amino acid in a beta-strand backbone, or the number of amino acids in a given length of beta strand. You would have to look this up (or start rasmol and measure some representative structures...). Oh, I just read that you know it yet: > 4.4 angstroms between alpha carbons, but I can't find a way to But I would be aware of this number beeing different in alpha-helices, beta-strands and loops. Now calculate the number of amino acids in 41 angstroms and multiply by the number of strands: answer to question a (except that I would expect to find loops, which complicates the calculation. Or indeed it shows that this kind of calculation is not very sensible) > I know the total volume would be pi times radius squared, and that it is Be aware that the volume is not important for this question. (But indeed IMHO the only _sensible_ figure to remember would be the average volume of an amino acid in a folded protein, or the number of aa in, say, 100 square angstroms). > (c). Given the number of beta strands, estimate the maximum number of > residues that could be considered to adopt the conformation expected for > a beta-sheet. I must admit that I do not understand this question. From only the diameter and length of the barrel, how can one estimate how much is in strands and how much in loops? Or is the question how big a protein of that structure can get? -- Frank Fuerst, Institute of Biochemistry of Potsdam University Im Biotechnologiepark, D-14943 Luckenwalde Tel.: +49-3371-681334; Fax: +49-3371-681339 I'm SignatureVirus 99! Copy me into your signature and join the fun! From owner-proteins@net.bio.net Mon Sep 27 01:36:00 1999 Path: biosci!newsfeed.stanford.edu!arclight.uoregon.edu!newsfeed.worldnet.att.net.MISMATCH!wn4feed!worldnet.att.net!128.230.129.106!news.maxwell.syr.edu!newspeer.monmouth.com!nntp2.deja.com!nnrp1.deja.com!not-for-mail From: Kresten Newsgroups: bionet.molbio.proteins Subject: Re: Biotin maleimide Date: Mon, 27 Sep 1999 09:24:38 GMT Organization: Deja.com - Before you buy. Lines: 38 Message-ID: <7snd4h$c3$1@nnrp1.deja.com> References: <1e2537bb.5f810fe0@usw-ex0108-059.remarq.com> NNTP-Posting-Host: 130.226.183.6 X-Article-Creation-Date: Mon Sep 27 09:24:38 1999 GMT X-Http-User-Agent: Mozilla/4.5 [en] (Win95; I) X-Http-Proxy: 1.0 www2.crc.dk:3128 (Squid/2.1.RELEASE), 1.0 x32.deja.com:80 (Squid/1.1.22) for client 130.226.182.217, 130.226.183.6 X-MyDeja-Info: XMYDJUIDkresten Thiols react covalently with maleimides, thus the biotin should not dissociate from the protein under SDS-treatment. If you expose your mixture to too much heat/acid/base/etc. you could of course get bond cleavages. HTH Kresten In article <1e2537bb.5f810fe0@usw-ex0108-059.remarq.com>, AHRickard wrote: > Hi, > Biotin maleidmide is a sulfhydryl specific biotinylating > reagent. Does anyone know what is the strength of the > interaction between cysteine (present in long chains of > polypeptides and proteins)and biotin maleimide. More to the > point will SDS or heat cause dissassociation of BM from > cysteine.Thanks for your time, > > Alex Rickard > PME > Manchester University > > * Sent from RemarQ http://www.remarq.com The Internet's Discussion Network * > The fastest and easiest way to search and participate in Usenet - Free! > > -- The address kresten@my-dejanews.com is for spambots only. Please mail me at LysLeuLeu@crc.dk transforming the pre@-part into my initials. Kresten Lindorff Larsen, Dept. Yeast Genetics Sent via Deja.com http://www.deja.com/ Before you buy. From owner-proteins@net.bio.net Mon Sep 27 04:36:00 1999 Path: biosci!newsfeed.stanford.edu!cyclone.bc.net!news.maxwell.syr.edu!news-was.dfn.de!news-han1.dfn.de!news.gwdg.de!not-for-mail From: Marc Saric Newsgroups: bionet.molbio.proteins Subject: Re: beta barrel volume calc. Date: Mon, 27 Sep 1999 13:51:12 +0200 Organization: Max Planck Institut =?iso-8859-1?Q?f=FCr?= molekulare Physiologie Lines: 30 Message-ID: <37EF5A2F.38526FC5@mpi-dortmund.mpg.de> References: <7sm2fc$4te$1@nnrp1.deja.com> <7snb60$2pa$1@fu-berlin.de> NNTP-Posting-Host: m99106.mpi-dortmund.mpg.de Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Trace: gwdu67.gwdg.de 938433071 2784 141.5.193.151 (27 Sep 1999 11:51:11 GMT) X-Complaints-To: news@gwdg.de NNTP-Posting-Date: 27 Sep 1999 11:51:11 GMT X-Mailer: Mozilla 4.6 [de] (WinNT; I) X-Accept-Language: de Frank Fürst schrieb: > > (c). Given the number of beta strands, estimate the maximum number of > > residues that could be considered to adopt the conformation expected for > > a beta-sheet. > > I must admit that I do not understand this question. From only > the diameter and length of the barrel, how can one estimate how > much is in strands and how much in loops? Or is the question how > big a protein of that structure can get? One could try to estimate the minimum turn radius between two adjactent beta-sheets (don´t know that number) and the minimum number of amino-acids needed for such a minimum-turn. Then one would be able to calculate the total number of residues for an "ideal" beta-barrel and calculate the ratio between the turn-AA and the sheet AA or so. But I also really don´t understand that question. -- Bye, Marc Saric Max-Planck-Institut für molekulare Physiologie Otto-Hahn-Strasse 11 44227 Dortmund phone: +49 231 133 2168 From owner-proteins@net.bio.net Mon Sep 27 08:07:00 1999 Path: biosci!washburn.edu!zzbart From: zzbart@washburn.edu (Janice Barton) Newsgroups: bionet.molbio.proteins Subject: Re: beta barrel volume calc. Date: 27 Sep 1999 09:07:35 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 79 Sender: daemon@net.bio.net Distribution: world Message-ID: References: <7snb60$2pa$1@fu-berlin.de> NNTP-Posting-Host: net.bio.net You man not like the question, but it appears to be one posed to a class of students. And, you just suggested to the student how to attack the problem, defeating the purpose of the exercise. Janice S. Barton, Ph.D. Professor and Chair of Chemistry Department of Chemistry Washburn University, Topeka, KS 66621 zzbart@washburn.edu http://www.washburn.edu/cas/chemistry/jbarton 785-231-1010-x1269 On Mon, 27 Sep 1999, Frank [iso-8859-1] Fürst wrote: > Antonia125 wrote: > > (I don't think this type of questions is very sensible, but > well...) > > > Hello, I was wondering if anyone could help me with this > > > > What dimensions should I be using to calculate these answers? > > > > The question states: The tertiary structure of Ig VI domain is a > > beta-barrel of antiparallel strands. It can be described as a cylinder > > ofvdimensions 25 angstroms and 41 angstroms in length. > > the 25 angstroms is the diameter? > > > (a). How many amino acid residues would you expect to find in this > > domain? > > > > (b). How many beta strands would you expect to find? > > You should consider b) first. To do this, first calculate the > circumference of the cylinder (Pi x diameter) and then divide it > by the average width of a beta strand (You must look up this, > don't know it). Thus you get the number of beta-strands. > Now you need the average "length" of an amino acid in a > beta-strand backbone, or the number of amino acids in a given > length of beta strand. You would have to look this up (or start > rasmol and measure some representative structures...). Oh, I just > read that you know it yet: > > > 4.4 angstroms between alpha carbons, but I can't find a way to > > But I would be aware of this number beeing different in > alpha-helices, beta-strands and loops. Now calculate the number > of amino acids in 41 angstroms and multiply by the number of > strands: answer to question a (except that I would expect to find > loops, which complicates the calculation. Or indeed it shows that > this kind of calculation is not very sensible) > > > I know the total volume would be pi times radius squared, and that it is > > Be aware that the volume is not important for this question. > (But indeed IMHO the only _sensible_ figure to remember would be > the average volume of an amino acid in a folded protein, or the > number of aa in, say, 100 square angstroms). > > > (c). Given the number of beta strands, estimate the maximum number of > > residues that could be considered to adopt the conformation expected for > > a beta-sheet. > > I must admit that I do not understand this question. From only > the diameter and length of the barrel, how can one estimate how > much is in strands and how much in loops? Or is the question how > big a protein of that structure can get? > > -- > Frank Fuerst, Institute of Biochemistry of Potsdam University > Im Biotechnologiepark, D-14943 Luckenwalde > Tel.: +49-3371-681334; Fax: +49-3371-681339 > I'm SignatureVirus 99! Copy me into your signature and join the > fun! > > From owner-proteins@net.bio.net Mon Sep 27 09:19:00 1999 Path: biosci!newshost.lanl.gov!awabi.library.ucla.edu!128.230.129.106!news.maxwell.syr.edu!tank.news.pipex.net!pipex!server1.netnews.ja.net!leicester!usenet From: "A.F. Simpson" Newsgroups: bionet.molbio.proteins Subject: Re: beta barrel volume calc. Date: Mon, 27 Sep 1999 18:08:41 -0700 Organization: University of Leicester Lines: 15 Message-ID: <37F01519.26B@le.ac.uk> References: <7snb60$2pa$1@fu-berlin.de> NNTP-Posting-Host: pc42.cmht.le.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: rook.le.ac.uk 938451803 1379186 143.210.176.54 (27 Sep 1999 17:03:23 GMT) X-Complaints-To: usenet@le.ac.uk NNTP-Posting-Date: 27 Sep 1999 17:03:23 GMT X-Mailer: Mozilla 3.01 (Win16; I) Janice Barton wrote: > > You man not like the question, but it appears to be one posed to a > class of students. And, you just suggested to the student how to attack > the problem, defeating the purpose of the exercise. What's wrong with asking for help with a problem you're having trouble with? It's not as if the questioner either a) tried to pretend it wasn't that sort of question or b) just said 'tell me the answer'. How are people supposed to learn if they don't ask questions? > Janice S. Barton, Ph.D. love Anna From owner-proteins@net.bio.net Mon Sep 27 13:05:00 1999 Path: biosci!newsfeed.stanford.edu!logbridge.uoregon.edu!news-out.uswest.net!dca1-feed1.news.digex.net!intermedia!news.ums.edu!gamera.cbl.umces.edu!news.umces.edu!cbl.umces.edu!reno From: "Reno T. Nguyen" Newsgroups: bionet.molbio.proteins Subject: Very High MW proteins Date: Mon, 27 Sep 1999 16:36:53 -0400 Organization: University of Maryland Chesapeake Biological Laboratory Lines: 25 Message-ID: NNTP-Posting-Host: cbl.umces.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII X-Trace: gamera.cbl.umces.edu 938464615 2750 131.118.224.6 (27 Sep 1999 20:36:55 GMT) X-Complaints-To: feedback@cbl.umces.edu NNTP-Posting-Date: 27 Sep 1999 20:36:55 GMT Hi, Does anyone know the relative contribution of very high MW proteins, let's say > 700 kDa, to the total protein pool in a bacterial cell, or some animal cell ? Reno Nguyen ************************************************************************* Reno T. 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 Mon Sep 27 16:39:00 1999 Path: biosci!BIGFOOT.COM!getamoreasales1 From: getamoreasales1@BIGFOOT.COM Newsgroups: bionet.molbio.proteins Subject: Don't Get Left Behind! Date: 27 Sep 1999 17:39:43 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 35 Sender: daemon@net.bio.net Distribution: world Message-ID: <7727BBF21663.AAA5C0E@bw86zhb.bluewin.ch> NNTP-Posting-Host: net.bio.net Increase Your Revenues Up To 1500% Accepting Credit Cards & Electronic Checks! ACCEPT CREDIT CARDS OVER THE INTERNET *** NO SETUP FEES Good Credit / Bad Credit / No Credit **** NO PROBLEM ****!!! It Just Doesn't Matter - Everyone Gets Approved Limited Offer So Take Advantage Of It!! 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From owner-proteins@net.bio.net Tue Sep 28 09:21:00 1999 Path: biosci!newsfeed.stanford.edu!logbridge.uoregon.edu!newsfeed.icl.net!newsfeed.nacamar.de!fu-berlin.de!server1.netnews.ja.net!pegasus.csx.cam.ac.uk!hgmp.mrc.ac.uk!tin!dswan From: dan Newsgroups: bionet.molbio.proteins Subject: same old homology questions... Date: Tue, 28 Sep 1999 18:14:07 +0100 Organization: MRC Human Genome Mapping Project Resource Centre Lines: 38 Message-ID: NNTP-Posting-Host: tin.hgmp.mrc.ac.uk Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII X-Trace: niobium.hgmp.mrc.ac.uk 938538851 20743 193.62.192.50 (28 Sep 1999 17:14:11 GMT) X-Complaints-To: news@hgmp.mrc.ac.uk NNTP-Posting-Date: 28 Sep 1999 17:14:11 GMT X-Sender: dswan@tin X-No-Archive: Yes I have seen a number of debates on various bionet.* groups about sequence identity, similarity and homology. I have a couple of brief questions I haven't been able to answer though. I have clones of a novel gene from human, xenopus, chick and mouse. The sequences however are not full length (the mouse sequence is), ie the other species have truncations which removes part of the putative N-terminal sequence. If I want to calculate the sequence identity at the DNA level, or sequence similarity at the protein level, do I need some method of factoring for the fact that the mouse always has a larger number of bases/amino acids as it is a full length clone? Second, although *I* am sure that the gene is the homolog of the murine gene in each species (ie no other family members, high level of visual similarity on alignment), there must be some convention saying this gene is definitely the homolog in this species.. What is this? Is it necessary to work from an evolutionary basis and establish some kind of phylogenetic tree? If so how? Hmm that turned into more questions than I anticipated :-/ thanks in advance for your time! Dan --------------------------------------------------------- |Dan Swan, |Lab : +44 (0)131 6505862 | |Centre for Genome Research, |Office : +44 (0)131 6505865 | |The University of Edinburgh,|Fax: +44 (0)131 6670164 | |King's Buildings, |Mobile: +44 (0)973 873 181 | |West Mains Road, ---------------------------- |Edinburgh, |If an experiment works | |EH9 3JQ |something has gone wrong.. | --------------------------------------------------------- From owner-proteins@net.bio.net Tue Sep 28 11:20:00 1999 Path: biosci!IMTECH.ERNET.IN!bhupesh From: bhupesh@IMTECH.ERNET.IN (ewald) Newsgroups: bionet.molbio.proteins Subject: Non-specific disulphides! Date: 28 Sep 1999 12:20:12 -0700 Organization: Institute of Microbial Technology, Sector 39-A, Chandigarh-160036 Lines: 34 Sender: daemon@net.bio.net Distribution: world Message-ID: <37F1C043.41C3@lion.imtech.ernet.in> Reply-To: bhupesh@imtech.ernet.in NNTP-Posting-Host: net.bio.net Hi all! This is a pretty naive question and must have been posted on the newsgroup many times in some way or the other! I am sorry to be posting it again but I couldn't find a satisfactory answer in the archives. My protein has three cysteines. I use 10mM bME during early stages of purification. During the last step,I see a very intersting (and intriguing) observation during purification over a Superdex 75 column. The protein separates cleanly as a dimer (column buffer containing 3mM bME as more would affect column stability). However, on storage of less than 24 hrs (in buffer+3mM bME), or on concentration, the protein shows multimeric forms: 2-mer (native), 4-mer, 6-mer, 8-mer, 10-mer etc on the Sup-75 column. Also, some or more of the protein precipates while using centricon concentrators with the "purified" dimer. Can anybody guess what's wrong? Is 3mM bME not enough as a reducing agent? Can I resolubilize the precipitate again(unsuccessful with upto 50 mM bME in buffer!)? Also, would 10mM bME affect the Superdex-75 Column (altho' I should probably be posting this question to Pharmacia)? Our lab has little experience with proteins with disulhides, hence the long mail. For the same reason, all suggestions would be exteremely helpful. If I inadvertently left out some details that might be required, I shall be happy to post another mail. Hope to get help soon! Radha. 29-9-99. **************************** Radha Chauhan, Graduate student, Institute of Microbial Technology, Sector 39-A, Chandigarh - 160 036. INDIA. e-mail : radha@lion.imtech.ernet.in radha75@mailcity.com ***************************** From owner-proteins@net.bio.net Tue Sep 28 18:32:00 1999 Path: biosci!newsfeed.stanford.edu!logbridge.uoregon.edu!nntp.upenn.edu!dialin0716.upenn.edu!user From: dalby@mail.med.upenn.edu (Pablo) Newsgroups: bionet.molbio.proteins Subject: Re: Non-specific disulphides! Date: Tue, 28 Sep 1999 22:32:06 -0500 Organization: home Lines: 28 Distribution: world Message-ID: References: <37F1C043.41C3@lion.imtech.ernet.in> NNTP-Posting-Host: dialin0716.upenn.edu In article <37F1C043.41C3@lion.imtech.ernet.in>, bhupesh@imtech.ernet.in wrote: > bME as more would affect column stability). However, on storage of less > than 24 hrs (in buffer+3mM bME), or on concentration, the protein shows > multimeric forms: 2-mer (native), 4-mer, 6-mer, 8-mer, 10-mer etc on the > Sup-75 column. Also, some or more of the protein precipates while using > centricon concentrators with the "purified" dimer. This sounds more like an aggregation problem which is unrelated to the cysteines. At higher concentrations of proteins the reaction rates of association increase, and higher orders of aggregation occur. To reverse the problem I would suggest rediluting the purified protein into GdmCl (not urea), followed by slow dialysis back into water and then keeping the protein stored in aliquots at the lower concentration. This is a very common problem which hampers the study of many proteins, especially for techniques requiring high (>100uM) concentrations of protein (eg NMR, CD, DSC etc). You may have to perform your studies in low concentrations of denaturant to reduce aggregation. -- brothers gonna work it out From owner-proteins@net.bio.net Wed Sep 29 03:52:00 1999 Path: biosci!IMTECH.ERNET.IN!bhupesh From: bhupesh@IMTECH.ERNET.IN (ewald) Newsgroups: bionet.molbio.proteins Subject: Disulphide or aggregation? Date: 29 Sep 1999 04:52:10 -0700 Organization: Institute of Microbial Technology, Sector 39-A, Chandigarh-160036 Lines: 22 Sender: daemon@net.bio.net Distribution: world Message-ID: <37F2AAFC.3C58@lion.imtech.ernet.in> Reply-To: bhupesh@imtech.ernet.in NNTP-Posting-Host: net.bio.net Dear Dalby (Pablo)! Thank you for your reply to my query posted on the the bionet newsgroup. Here's a query as a followup to your mail which I hope you would help me clarify. During overexpression the protein is perfectly soluble and no inclusion bodies occur. Although the anomaly occurs only after further concentrating the protein, I feel it is due to non-specific disulphide formation as I clearly see 2-mer, 4-mer, 6-mer and so on, whereas I thought that in non-specific aggregations, the majority population would be higher aggregates. Although I can probably check this by modification of cys, I want to avoid it as at least two cys are thought to be involved in the active site. Also, we would have liked to use higher concentrations of the protein to try crystallizations. Are my arguments good enough? A naive protein chemist ************************** Radha Chauhan Graduate student email: radha@lion.imtech.ernet.in radha75@mailcity.com ************************** From owner-proteins@net.bio.net Wed Sep 29 06:37:00 1999 Path: biosci!newsfeed.stanford.edu!arclight.uoregon.edu!newsfeed.worldnet.att.net.MISMATCH!wn4feed!worldnet.att.net!204.71.34.3!newsfeed.cwix.com!newspeer.monmouth.com!nntp2.deja.com!nnrp1.deja.com!not-for-mail From: Kresten Newsgroups: bionet.molbio.proteins Subject: Re: Non-specific disulphides! Date: Wed, 29 Sep 1999 14:22:31 GMT Organization: Deja.com - Before you buy. Lines: 73 Message-ID: <7st7al$43t$1@nnrp1.deja.com> References: <37F1C043.41C3@lion.imtech.ernet.in> NNTP-Posting-Host: 130.226.183.6 X-Article-Creation-Date: Wed Sep 29 14:22:31 1999 GMT X-Http-User-Agent: Mozilla/4.5 [en] (Win95; I) X-Http-Proxy: 1.0 www2.crc.dk:3128 (Squid/2.1.RELEASE), 1.0 x36.deja.com:80 (Squid/1.1.22) for client 130.226.182.217, 130.226.183.6 X-MyDeja-Info: XMYDJUIDkresten In article <37F1C043.41C3@lion.imtech.ernet.in>, bhupesh@imtech.ernet.in wrote: > Hi all! > This is a pretty naive question and must have been posted on the > newsgroup many times in some way or the other! I am sorry to be > posting > it again but I couldn't find a satisfactory answer in the archives. > My protein has three cysteines. I use 10mM bME during early > stages of > purification. During the last step,I see a very intersting (and > intriguing) observation during purification over a Superdex 75 column. > The protein separates cleanly as a dimer (column buffer containing 3mM > bME as more would affect column stability). However, on storage of > less > than 24 hrs (in buffer+3mM bME), or on concentration, the protein > shows > multimeric forms: 2-mer (native), 4-mer, 6-mer, 8-mer, 10-mer etc on > the > Sup-75 column. Also, some or more of the protein precipates while > using > centricon concentrators with the "purified" dimer. > Can anybody guess what's wrong? Is 3mM bME not enough as a > reducing agent? Not claiming that this is the explanation,- what *could* happen is that your betaME gets oxidised (by air). Not too long ago I incubated some DTT either at pH 7.0 or at pH 8.5 at 37 degC, took samples out at different timepoints and analysed for number of -SH's as a function of time. At pH 7.0 I measured 1.8 -SH's pr. DTT after 66 hours At pH 8.5 0.8 6 hours So if pH is high in your buffer you might not have any reductant left. - and I had purged the solutions with argon. HTH Kresten > Can I resolubilize the precipitate again(unsuccessful with > upto > 50 mM bME in buffer!)? Also, would 10mM bME affect the Superdex-75 > Column (altho' I should probably be posting this question to > Pharmacia)? > Our lab has little experience with proteins with disulhides, > hence the > long mail. For the same reason, all suggestions would be exteremely > helpful. If I inadvertently left out some details that might be > required, I shall be happy to post another mail. > Hope to get help soon! > Radha. 29-9-99. > > **************************** > Radha Chauhan, > Graduate student, > Institute of Microbial Technology, > Sector 39-A, > Chandigarh - 160 036. INDIA. > > e-mail : radha@lion.imtech.ernet.in > radha75@mailcity.com > ***************************** > -- The address kresten@my-dejanews.com is for spambots only. Please mail me at LysLeuLeu@crc.dk transforming the pre@-part into my initials. Kresten Lindorff Larsen, Dept. Yeast Genetics Sent via Deja.com http://www.deja.com/ Before you buy. From owner-proteins@net.bio.net Wed Sep 29 07:29:00 1999 Path: biosci!newsfeed.stanford.edu!arclight.uoregon.edu!logbridge.uoregon.edu!newsfeed2.news.nl.uu.net!sun4nl!newspeer.ebone.net!fu-berlin.de!pc207-60.biochem.uni-potsdam.DE!not-for-mail From: Frank =?iso-8859-1?Q?F=FCrst?= Newsgroups: bionet.molbio.proteins Subject: Re: Disulphide or aggregation? Date: Wed, 29 Sep 1999 17:26:09 +0200 Lines: 58 Message-ID: <7staro$cg2$1@fu-berlin.de> References: <37F2AAFC.3C58@lion.imtech.ernet.in> NNTP-Posting-Host: pc207-60.biochem.uni-potsdam.de (141.89.207.60) Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Access: 16 49 955 X-Trace: fu-berlin.de 938618552 12802 (none) 141.89.207.60 X-Mailer: Mozilla 4.51 [de]C-CCK-MCD QXW03200 (Win95; I) X-Accept-Language: de-DE,en ewald wrote: > > Dear Dalby (Pablo)! > Thank you for your reply to my query posted on the the bionet > newsgroup. Here's a query as a followup to your mail which I hope you > would help me clarify. > During overexpression the protein is perfectly soluble and no inclusion > bodies occur. Although the anomaly occurs only after further > concentrating the protein, I feel it is due to non-specific disulphide > formation as I clearly see 2-mer, 4-mer, 6-mer and so on, whereas I > thought that in non-specific aggregations, the majority population would > be higher aggregates. Aggregation usually is also not a completely non-specific reaction. In refolding experiments, it is often one particular, clearly defined folding intermediate that aggregates. And also then one can observe the formation of every multimer on the aggregation pathway. In your case, of course, the n-mers with odd n are missing, but this might be because a dimeric species which exposes some nonpolar surface might be the one that aggregates. Also the observation that the protein is soluble upon recombinant (I suppose) overexpression does not rule out aggregation. The buffers used can never completely mimic the cytosolic environment, and perhaps it even interacts with bacterial chaperones that keep it soluble. To distinguish between the two possibilies I would - perform SDS-PAGE with the usual heating step before loading the sample on the gel, but without any reducing agent in the sample buffer. Then you should see multimers on the gel if it's really intermolecular disulfide bridges, but simple aggregates usually can be dissolved by SDS in the heat, and if not, only the higher aggregates stay insoluble and that won't migrate at all. - try to dissolve it in 6 or 8 M Guanidinium Chloride. This should work with any aggregates, I think even if crosslinked by SS-bonds. Then again do nonreducing SDS-PAGE. If you then don't find oligomers, there was no disulfide linkage. (But of course if you do find some, they could also have been formed after re-disolving the protein). > to be involved in the active site. Also, we would have liked to use > higher concentrations of the protein to try crystallizations. > Are my arguments good enough? Sometimes the choice of the right buffer is crucial. High salt, low salt, changed buffer ion, additives - it's just alchemy. Good luck. -- Frank Fuerst, Institute of Biochemistry of Potsdam University Im Biotechnologiepark, D-14943 Luckenwalde Tel.: +49-3371-681334; Fax: +49-3371-681339 I'm SignatureVirus 99! Copy me into your signature and join the fun! From owner-proteins@net.bio.net Wed Sep 29 08:46:00 1999 Newsgroups: bionet.molbio.proteins Path: biosci!newshost.lanl.gov!logbridge.uoregon.edu!newsfeed.icl.net!newsfeed.icl.net!nntp.news.xara.net!xara.net!gxn.net!server6.netnews.ja.net!leeds.ac.uk!news From: BMBRNL@leeds.ac.uk (R.N. Leach) Subject: protein binding to immobilised ligand Message-ID: <7stdvb$7ic_001@leeds.ac.uk> NNTP-Posting-Host: bmbpc033.leeds.ac.uk Organization: University of Leeds Date: Wed, 29 Sep 1999 17:17:02 +0100 (BST) X-Newsreader: News Xpress Version 1.0 Beta #4 Lines: 21 I have a transmembrane protein which is hypothesised to bind a ligand which has been shown to bind to other (unrelated) transmembrane proteins. The ligand is commercially available in an immobilised form, covalently attached to agarose @ 7.3umole per ml of packed gel. Now, am I being simplistic in assuming that to compete with immobilised ligand bound to protein binding sites, the free ligand must be applied to the column @ > 7.3mM (i.e 7.3umole/ml gel = 7.3mM) ? My reason for asking is that, when reading through the literature, I came across a paper where the authors used this immobilised ligand, from the same suppliers, in a one-step purification method of an unrelated protein and (assuming they used the same amount of immobilised ligand / ml gel) were able to elute bound protein using 2mM free ligand, whereas I can only elute bound protein using 100mM free ligand. Any suggestions gratefully received, Rob Leach, School of Biochemistry & Molecular Biology, University of Leeds From owner-proteins@net.bio.net Wed Sep 29 11:53:00 1999 Newsgroups: bionet.molbio.proteins Path: biosci!newsfeed.stanford.edu!paloalto-snf1.gtei.net!news.gtei.net!lynxgen.com!news From: "John E. Wiktorowicz" Subject: Re: Non-specific disulphides! X-Nntp-Posting-Host: johnw Content-Type: text/plain; charset=us-ascii; x-mac-type="54455854"; x-mac-creator="4D4F5353" Message-ID: <37F20AF2.29EF8032@lynxgen.com> Sender: news@lynxcalif.com (The News System) Reply-To: johnw@lynxgen.com Content-Transfer-Encoding: 7bit Organization: Lynx Therapeutics, Inc. X-Accept-Language: en References: <37F1C043.41C3@lion.imtech.ernet.in> Mime-Version: 1.0 Date: Wed, 29 Sep 1999 12:49:56 GMT X-Mailer: Mozilla 4.5 (Macintosh; I; PPC) Lines: 51 My first question is: What makes you think the multimeric forms are due to sulfhydryl oxidation? The concentration-dependent aggregation might just as well be due to hydrophobic interaction (particularly since resolublization with bMe doesn't work). Try increasing denaturant concentrations to resolubilize. It is not unusual to lose 3mM bME overnight, especially in a smoggy (high ozone) environment (I don't know how smoggy your air is). Should have no effect on the column. Try higher concentrations, with degassed buffers, etc. Hydrophobic aggregation might be prevented by any of the many neutral detergents (Triton, Tween, etc). Also try a HIC column such as phenyl sepharose to establish the presence of surface hydrophobic patches; might also serve as an effective purification step. Can you assay for function? Is the dimeric form active? Might shed some light on the nature and specificity of the aggregation. Good luck > > My protein has three cysteines. I use 10mM bME during early stages of > purification. During the last step,I see a very intersting (and > intriguing) observation during purification over a Superdex 75 column. > The protein separates cleanly as a dimer (column buffer containing 3mM > bME as more would affect column stability). However, on storage of less > than 24 hrs (in buffer+3mM bME), or on concentration, the protein shows > multimeric forms: 2-mer (native), 4-mer, 6-mer, 8-mer, 10-mer etc on the > Sup-75 column. Also, some or more of the protein precipates while using > centricon concentrators with the "purified" dimer. > Can anybody guess what's wrong? Is 3mM bME not enough as a reducing > agent? Can I resolubilize the precipitate again(unsuccessful with upto > 50 mM bME in buffer!)? Also, would 10mM bME affect the Superdex-75 > Column (altho' I should probably be posting this question to Pharmacia)? > Our lab has little experience with proteins with disulhides, hence the > long mail. For the same reason, all suggestions would be exteremely > helpful. If I inadvertently left out some details that might be > required, I shall be happy to post another mail. > Hope to get help soon! > Radha. 29-9-99. > > **************************** > Radha Chauhan, > Graduate student, > Institute of Microbial Technology, > Sector 39-A, > Chandigarh - 160 036. INDIA. > > e-mail : radha@lion.imtech.ernet.in > radha75@mailcity.com > ***************************** From owner-proteins@net.bio.net Wed Sep 29 16:47:00 1999 Path: biosci!newshost.lanl.gov!arclight.uoregon.edu!hammer.uoregon.edu!newsfeed.direct.ca!news.maxwell.syr.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!Home From: klenchin@REMOVE_TO_REPLY.facstaff.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins Subject: Re: protein binding to immobilised ligand Date: Thu, 30 Sep 1999 00:33:21 GMT Organization: UW Lines: 29 Message-ID: <7sub4n$mh8$1@news.doit.wisc.edu> References: <7stdvb$7ic_001@leeds.ac.uk> NNTP-Posting-Host: t-14-181-50.dialup.wisc.edu X-Newsreader: News Xpress 2.01 X-No-Archive: yes BMBRNL@leeds.ac.uk (R.N. Leach) wrote: :I have a transmembrane protein which is hypothesised to bind a ligand which :has been shown to bind to other (unrelated) transmembrane proteins. The ligand :is commercially available in an immobilised form, covalently attached to :agarose @ 7.3umole per ml of packed gel. : :Now, am I being simplistic in assuming that to compete with immobilised ligand :bound to protein binding sites, the free ligand must be applied to the column :@ > 7.3mM (i.e 7.3umole/ml gel = 7.3mM) ? You are. Optimal elution conditions will depend mainly not just on concentration of the ligand but on Kd of your protein to that ligand and have to be determined empirically. :My reason for asking is that, when reading through the literature, I came :across a paper where the authors used this immobilised ligand, from the same :suppliers, in a one-step purification method of an unrelated protein and :(assuming they used the same amount of immobilised ligand / ml gel) were able :to elute bound protein using 2mM free ligand, whereas I can only elute bound :protein using 100mM free ligand. Your protein binds much tighter than theirs - disoociation event occurs with lower probability, and thus more soluble competitor is required to move the protein zone along the mobile phase. You may try to fill the column with ~ 15 mM, stop the flow and wait _some_ time, then collect what is eluted. This might help to elute with lower concentrations if koff is limiting. - Dima From owner-proteins@net.bio.net Wed Sep 29 21:33:00 1999 Path: biosci!IMTECH.ERNET.IN!bhupesh From: bhupesh@IMTECH.ERNET.IN (ewald) Newsgroups: bionet.molbio.proteins Subject: disulphide aggregation!! Date: 29 Sep 1999 22:32:59 -0700 Organization: Institute of Microbial Technology, Sector 39-A, Chandigarh-160036 Lines: 8 Sender: daemon@net.bio.net Distribution: world Message-ID: <37F3A2F5.533E@lion.imtech.ernet.in> Reply-To: bhupesh@imtech.ernet.in NNTP-Posting-Host: net.bio.net Dear John E. Wiktorowicz, Multimeric forms are due to the nonspecifis disulphide bond formation, this is checked on non reducing SDS-PAGE in presence of bME,DTT and in absence of both. I found several bands of higher molecular wt. in bME untreated protein samples while single band when protein was treated by bME. I think this will help you to guess what kind of aggregation are formed by protein. hope to get reply soon. From owner-proteins@net.bio.net Thu Sep 30 00:52:00 1999 Path: biosci!newsfeed.stanford.edu!newsfeed.berkeley.edu!news.algonet.se!algonet!f.de.uu.net!news.uni-stuttgart.de!news.belwue.de!news.uni-freiburg.de!not-for-mail From: Jens Lohrmann Newsgroups: bionet.molbio.proteins Subject: ProteinASepharose Purification of Antibodies Date: Thu, 30 Sep 1999 08:43:20 +0100 Organization: Rechenzentrum der Universitaet Freiburg, Germany Lines: 28 Message-ID: <37F31498.86006BEF@sun2.ruf.uni-freiburg.de> NNTP-Posting-Host: tomate.biologie.uni-freiburg.de Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Mailer: Mozilla 4.5 [de] (Win95; I) X-Accept-Language: de Dear Netters! Just a short question: Can you please send me or quote an easy and reliable protocol for proteinAsepharose purification of antibodies? Especially I'm wondering if there are alternatives to the acidic elution or how long you've got to incubate the sepharose/antibody with the acid. Thanks a lot! Sincerely Jens ____________________________________________________________ Jens Lohrmann lohrmann@sun2.ruf.uni-freiburg.de http://www.biologie.uni-freiburg.de/data/schaefer/jl01.html http://www.biologie.uni-freiburg.de/data/schaefer/jl02.html ____________________________________________________________ Schänzlestr. 1 Institut für Biologie II / Botanik 79104 Freiburg Fon: 0761/203-2619 Fax: 0761/203-2612 ____________________________________________________________ From owner-proteins@net.bio.net Thu Sep 30 17:41:00 1999 Path: biosci!newsfeed.stanford.edu!skynet.be!hermes.visi.com!news-out.visi.com!cam-news-hub1.bbnplanet.com!news.gtei.net!newscon02!prodigy.com!not-for-mail From: "Ming-Ching Hsieh" Newsgroups: bionet.molbio.proteins Subject: concentration of human neutrophil elastase Date: Thu, 30 Sep 1999 21:31:02 -0400 Organization: Prodigy Internet http://www.prodigy.com Lines: 11 Message-ID: <7t132o$ehg$1@newssvr04-int.news.prodigy.com> NNTP-Posting-Host: phl2b510-46.splitrock.net X-Trace: newssvr04-int.news.prodigy.com 938741656 3588633 209.254.92.149 (1 Oct 1999 01:34:16 GMT) X-Complaints-To: abuse@prodigy.net NNTP-Posting-Date: 1 Oct 1999 01:34:16 GMT X-Newsreader: Microsoft Outlook Express 4.72.3110.1 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3110.3 Dear Netters: Would someone kindly tell me the concentration of human neutrophil elastase in serum either in normal state or in acute phase? Or refer me to some references? I know that the concentration of HNE is high in the cells (about 1.5 ug/10^6 cells), but not sure that in serum or plasma. Thank you very much Ming-Ching Hsieh