From owner-proteins@net.bio.net Tue Jun 01 23:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!dapsun.lif.icnet.uk!macmhc.cryst.bbk.ac.uk!p_travers From: p_travers@icrf.icnet.uk (Paul J Travers) Newsgroups: bionet.molbio.proteins Subject: Refolding proteins in gels/on nitrocellulose membranes Message-ID: <1uhm5cINN1mc@dapsun.lif.icnet.uk> Date: 2 Jun 93 07:51:08 GMT Organization: ICRF Structural Molecular Biology Unit Lines: 14 NNTP-Posting-Host: macmhc.cryst.bbk.ac.uk X-UserAgent: Nuntius v1.1.1d17 X-XXMessage-ID: X-XXDate: Wed, 2 Jun 93 08:52:41 GMT Hello, Does anyone have any information or references about the refolding of proteins either in acrylamide gels or after transfer onto nylon/nitrocellulose membranes. I seem to recall hearing about a protocol for this at one time. My problem is that I have an antibody to a conformational epitope that requires tertiary interactions in the folded protein, yet I can western blot from reducing SDS/PAGE. Are there other examples like this? Does anyone have a good idea what elements of secondary/tertiary structure might be stable in Laemmli sample buffer? Paul Travers From owner-proteins@net.bio.net Tue Jun 01 23:00:00 1993 Path: biosci!ZEUS.AHABS.WISC.EDU!ming From: ming@ZEUS.AHABS.WISC.EDU ("Ding Ming") Newsgroups: bionet.molbio.proteins Subject: Re: isoelectric focusing Message-ID: Date: 2 Jun 93 17:54:26 GMT Sender: daemon@net.bio.net Reply-To: ming@zeus.ahabs.wisc.edu Distribution: bionet Organization: Animal Health & Biomedical Sciences Lines: 31 > I have been using isoelectric focusing gel to analyze a herpes >immediate-early >protein-ICP4. The major problem I encountered was the poor >solubility of the >protein in urea solubilization buffer. Is there anyway to enhance >the solubility >of proteins in this buffer or are there any alternative buffers for >this >purpose? I think probably the concentration of urea in your buffer is not appropriate for your sample protein and you may precipitate your protein by overdenaturation. Try to decrease the conc.of either protein or urea first. If this still does not work, just use whatever buffer you candesolve your protein and use this buffer to make your gel too as far as you can do it. ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ Ding Ming (608)265-3544 ming@calshp.cals.wisc.edu Sweet Protein Section ming@zeus.ahabs.wisc.edu Taste Research Lab Department of Animal Health & Biomedical Sciences University of Wisconsin-Madison, 1655 Linden Dr., Madison, WI53706 ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ Office Location: A215 Babcock Hall ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ From owner-proteins@net.bio.net Tue Jun 01 23:00:00 1993 Path: biosci!POST.ITS.MCW.EDU!fgarbrec From: fgarbrec@POST.ITS.MCW.EDU (Frederick Garbrecht) Newsgroups: bionet.molbio.proteins Subject: Re: Refolding proteins in gels/on nitrocellulose membranes Message-ID: Date: 2 Jun 93 16:24:36 GMT References: <9306021218.AA02650@net.bio.net> Sender: daemon@net.bio.net Distribution: bionet Lines: 30 There is some interesting stuff on renaturation of protein kinases on nitrocellulose blots in "Methods in Enzymology", vol. 200. Fred Garbrecht fgarbrec@post.its.mcw.edu On 2 Jun 1993, Paul J Travers wrote: > Date: 2 Jun 93 07:51:08 GMT > From: Paul J Travers > To: proteins@net.bio.net > Subject: Refolding proteins in gels/on nitrocellulose membranes > > Hello, > Does anyone have any information or references about the refolding of > proteins either in acrylamide gels or after transfer onto > nylon/nitrocellulose membranes. I seem to recall hearing about a > protocol for this at one time. > > My problem is that I have an antibody to a conformational epitope that > requires tertiary interactions in the folded protein, yet I can western > blot from reducing SDS/PAGE. Are there other examples like this? > > Does anyone have a good idea what elements of secondary/tertiary > structure might be stable in Laemmli sample buffer? > > Paul Travers > From owner-proteins@net.bio.net Wed Jun 02 23:00:00 1993 Path: biosci!agate!usenet.ins.cwru.edu!cleveland.Freenet.Edu!ce046 From: ce046@cleveland.Freenet.Edu (Uday Khosla) Newsgroups: bionet.molbio.proteins Subject: Quantitative Analysis on Western Blots Message-ID: <1ulp0k$dkq@usenet.INS.CWRU.Edu> Date: 3 Jun 93 21:04:20 GMT Organization: Case Western Reserve University, Cleveland, Ohio (USA) Lines: 11 NNTP-Posting-Host: hela.ins.cwru.edu I was wondering if anybody knows of a method in which I could perform quantitative analysis on western blots. I know that a densitometer is one way; however, I would like to know of alternative methods of measuring protein concentration from my western blot. Thank you... Uday M. Khosla lime.wustl.edu Washington University St. Louis -- From owner-proteins@net.bio.net Fri Jun 04 23:00:00 1993 Path: biosci!daresbury!daresbury!news From: WALLACE@IRBM.IT (Andrew Wallace, Phone ext. 434) Newsgroups: bionet.molbio.proteins Subject: PEPTIDE-LIBRARIES discussion list proposal Message-ID: <1993Jun5.193354.14040@gserv1.dl.ac.uk> Date: 1 Jun 93 17:42:33 GMT Sender: WALLACE@IT.IRBM Distribution: bionet Lines: 14 X-Vmsmail-To: SMTP%"bioforum@daresbury.ac.uk",SMTP%"proteins@daresbury.ac.uk" Original-To: bioforum@uk.ac.daresbury, proteins@uk.ac.daresbury Dear colleagues, I would like to propose the setting up of a new discussion group concerning phage-displayed and synthetic combinatorial PEPTIDE LIBRARIES, synthesis and use thereof. Anyone who is interested in participating may contact me at the internet address below. TIA, Andrew Wallace Dept. of Biochemistry Istituto di Ricerche di Biologia Molecolare (IRBM) P. Angeletti Pomezia, Italy Internet: wallace@irbm.it From owner-proteins@net.bio.net Tue Jun 08 23:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!agate!darkstar.UCSC.EDU!cats.ucsc.edu!rafael From: rafael@cats.ucsc.edu (David Konerding) Newsgroups: bionet.molbio.proteins,bionet.molbio.gene-org Subject: a program to list all degenerate codings Message-ID: <1v4129INN20e@darkstar.UCSC.EDU> Date: 9 Jun 93 06:47:37 GMT Organization: University of California; Santa Cruz Lines: 43 Xref: biosci bionet.molbio.proteins:682 NNTP-Posting-Host: am.ucsc.edu Hello. I am an undergraduate major in biochemistry and molecular biology at UC Santa Cruz. As a student and lover of the genetic code, I realized that any possible sequence of amino acids in a protein could have more than one possible codon sequence due to the degeneracy of the code. For example, the amino acid serine has 6 codons, AGA, AGG, AGT, AGC, TCA, and TCG. Therefore, if one were to sequence a polypeptide, there could be many possible sets of codons which would code for that polypeptide. To teach myself how to use recursion, learn more about the genetic code, and solve a homework problem the hard way, I wrote a program which converts a polypeptide sequence into a list of all the possible codon sequences which code for the polypeptide. The program is in C and compiles under Turbo C 1.0. Would this be in any way useful to anybody out there? I can think of one application (bear in mind I'm a sophomore in college and have little understanding of the "reality" of molecular biology): if one sequenced a polypeptide and wanted to find if a sequence coding for that polypeptide (or superset of that sequence) existed somewhere in a large sequence of mRNA (not DNA since there would be introns). So, if this program would be of use to you, please mail me. At current, since it's on my PC, polypeptide sequences of more than about 6 or 7 amino acids crash the program, since the necessary recursion stack and text storage takes up more than the 64k code barrier. However, if anybody expresses any interest in this program, I will either learn how to expand past the 64k data barrier, or recode it so that it compiles under Unix C. If it seems obvious to any of the molecular biologists out here that I am misunderstanding some basic principle of degeneracy, please point it out to me!! I am studying right now so that I may some day research molecular biology, and any pertinent information I don't know yet will be greatly appreciated! -- --- David Konerding rafael@cats.ucsc.edu University of California, Santa Cruz From owner-proteins@net.bio.net Wed Jun 09 23:00:00 1993 Path: biosci!s.u-tokyo!news.u-tokyo.ac.jp!wnoc-tyo-news!sh.wide!sun-barr!cs.utexas.edu!not-for-mail From: arkerlav@tigr.org Newsgroups: bionet.jobs,bionet.molbio.proteins Subject: POSTDOCTORAL POSITION IN PROTEIN STRUCTURE Message-ID: <9306101256.AA20582@bengal.tigr.org> Date: 10 Jun 93 12:52:11 GMT Sender: daemon@cs.utexas.edu Organization: UTexas Mail-to-News Gateway Lines: 51 Xref: biosci bionet.jobs:1933 bionet.molbio.proteins:683 NNTP-Posting-Host: cs.utexas.edu ********************************************* Postdoctoral Research Opportunities in the Study of the Molecular Structure of Proteins at The Institute for Genomic Research (TIGR) ********************************************* TIGR is accepting applications for postdoctoral research associates to assist in structural analysis of gene and protein families. This research will be an integral part of predicting the structure/function of newly discovered genes in a large-scale sequencing effort. Responsibilities include: homology modeling of gene families from a variety of species; structure prediction of newly discovered, uncharacterized gene products; creating and maintaining multiple sequence alignments; gathering information about gene families from relevant scientific literature; and working with other scientists and a staff of programmers to develop sequence analysis tools for manipulating and analyzing nucleotide and amino acid sequence data. The ideal candidate will be a computer literate biochemist with experience in protein structure/function relationships, comparative sequence analysis and secondary and tertiary protein structure prediction methods. TIGR is a recently established, not-for-profit research center devoted to accelerating the sequencing, mapping and functional characterization of human, animal, and plant genomes. The Institute is located in the Washington DC/Baltimore metropolitan area and offers a competitive compensation and benefits package. Qualified applicants are encouraged to send a curriculum vitae and letter of interest to: The Institute for Genomic Research 932 Clopper Road Gaithersburg, MD 20878 Attn. : Human Resources/ark Electronic CV submissions may be sent to: Dr. Anthony Kerlavage, Director Computational Genomics arkerlav@tigr.org From owner-proteins@net.bio.net Thu Jun 10 23:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!agate!spool.mu.edu!torn!thunder!flash!mligr From: mligr@flash.LakeheadU.Ca (Martin Ligr) Newsgroups: bionet.molbio.proteins Subject: Acetone extraction of proteins Message-ID: Date: 11 Jun 93 03:46:05 GMT Sender: news@thunder.LakeheadU.Ca Distribution: bionet Lines: 20 Hello! I have just started working on my thesis and was stopped at the very begining by supposedly ridiculous problem: I'm extracting total proteins of Chlamydomonas using acetone extraction method. The purpose is to quantitate then particular protein by immunoassay. However, I'm not able to redisolve pellet of proteins after extraction completely. I was trying SDS up to 3% conc., Na2CO3 up to 2 M, and NaOH up to 2 M. Only 2M NaOH redisolved the pellet completely, but proteins were partially hydrolized- as was seen on SDS-PAGE. I would appreciate if somebody could post here on send me a protocol describing this method or to give me some hint. N.B.: I prefere acetone extraction to mechanical destruction methods, because it removes chlorophyll which interferes with my fluorescence immunoassay. Thanks. Martin Ligr < mligr@flash.lakeheadu.ca > Dept. of Biology Lakehead University Thunder Bay, Ontario From owner-proteins@net.bio.net Thu Jun 10 23:00:00 1993 Path: biosci!agate!toxic!tyersome From: tyersome@toxic (Randall Tyers) Newsgroups: bionet.molbio.proteins Subject: Re: Acetone extraction of proteins Message-ID: <1vaqg7$bap@agate.berkeley.edu> Date: 11 Jun 93 20:38:31 GMT References: Reply-To: tyersome@toxic.Berkeley.EDU Distribution: bionet Organization: Plant Biology Lines: 15 NNTP-Posting-Host: toxic.berkeley.edu In article mligr@flash.LakeheadU.Ca (Martin Ligr) writes: >[...] However, I'm not able to redisolve pellet of proteins >after extraction completely. I was trying SDS up to 3% conc., Na2CO3 up to >2 M, and NaOH up to 2 M. Only 2M NaOH redisolved the pellet completely, >but proteins were partially hydrolized- as was seen on SDS-PAGE. >I would appreciate if somebody could post here on send me a protocol describing >this method or to give me some hint.[...] You could try gently breaking up the cells and cetrifuging out the organelles and cell wall fragments followed by ammonium sulfate precipitation which is much gentler ... Fraid I don't have a protocol but these are pretty standard techniques. Randall Tyers tyersome@insect.berkeley.edu From owner-proteins@net.bio.net Thu Jun 10 23:00:00 1993 Path: biosci!daresbury!daresbury!not-for-mail From: mbrgw@s-crim1.dl.ac.uk (R.G. Walters) Newsgroups: bionet.molbio.proteins Subject: Re: Acetone extraction of proteins Message-ID: <1v9qr6INN89b@s-crim1.dl.ac.uk> Date: 11 Jun 93 11:38:14 GMT References: Distribution: bionet Organization: Daresbury Lab., Warrington, U.K. Lines: 25 NNTP-Posting-Host: s-crim1.dl.ac.uk In article mligr@flash.LakeheadU.Ca (Martin Ligr) writes: >Hello! >I have just started working on my thesis and was stopped at the very begining >by supposedly ridiculous problem: I'm extracting total proteins of Chlamydomonas >using acetone extraction method. The purpose is to quantitate then particular >protein by immunoassay. However, I'm not able to redisolve pellet of proteins >after extraction completely. I was trying SDS up to 3% conc., Na2CO3 up to >2 M, and NaOH up to 2 M. Only 2M NaOH redisolved the pellet completely, >but proteins were partially hydrolized- as was seen on SDS-PAGE. >I would appreciate if somebody could post here on send me a protocol describing >this method or to give me some hint. I always had this problem with acetone (or TCA) too. The method I use - with no problems - is extraction with methanol/chloroform, as described by Wessel & Fluegge (1984) Anal. Biochem. 138, 141-3 This takes pigment AND lipid out. Just make sure your favourite protein isn't soluble in chloroform. Robin Walters. Robert Hill Institute, Sheffield UK. I got bored with my old .sig So here's a new one From owner-proteins@net.bio.net Sun Jun 13 23:00:00 1993 Path: biosci!agate!howland.reston.ans.net!usc!sdd.hp.com!saimiri.primate.wisc.edu!zazen!news From: nmrdb@vms.macc.wisc.edu (BEVERLY SEAVEY) Newsgroups: bionet.molbio.proteins Subject: Looking for the email address of Michael Nilges at EMBL Message-ID: <1993Jun14.151124.7874@macc.wisc.edu> Date: 14 Jun 93 17:08:58 GMT Sender: news@macc.wisc.edu (USENET News System) Organization: University of Wisconsin Academic Computing Center Lines: 3 Someone in my lab wishes to contact Michael Nilges at EMBL, probably about some protein modeling program. From owner-proteins@net.bio.net Sun Jun 13 23:00:00 1993 Path: biosci!uwm.edu!ux1.cso.uiuc.edu!howland.reston.ans.net!darwin.sura.net!welchgate.welch.jhu.edu!danj From: danj@welchgate.welch.jhu.edu (Dan Jacobson) Newsgroups: bionet.molbio.proteins Subject: Re: Looking for the email address of Michael Nilges at EMBL Message-ID: <1993Jun15.011442.18971@welchgate.welch.jhu.edu> Date: 15 Jun 93 01:14:42 GMT References: <1993Jun14.151124.7874@macc.wisc.edu> Organization: Johns Hopkins Univ. Welch Medical Library Lines: 17 In article <1993Jun14.151124.7874@macc.wisc.edu> nmrdb@vms.macc.wisc.edu (BEVERLY SEAVEY) writes: > Someone in my lab wishes to contact Michael Nilges at EMBL, probably >about some protein modeling program. This should help you get started: MAIL FOR Michael Nilges IS FORWARDED TO nilges@felix.embl-heidelberg.de NOTE: this is a domain mail forwarding arrangement - so mail intended for "nilges" should be addressed to "nilges@embl-heidelberg.de" rather than "nilges@felix.embl-heidelberg.de". Best of luck, Dan Jacobson danj@welchgate.welch.jhu.edu From owner-proteins@net.bio.net Mon Jun 14 23:00:00 1993 Path: biosci!uwm.edu!cs.utexas.edu!uunet!mcsun!news.funet.fi!hydra!klaava!cc.helsinki.fi!louhelainen From: louhelainen@cc.helsinki.fi Newsgroups: bionet.molbio.proteins Subject: Re: Acetone extraction of proteins Message-ID: <1993Jun15.204215.1@cc.helsinki.fi> Date: 15 Jun 93 20:42:15 GMT References: Distribution: bionet Organization: University of Helsinki Lines: 32 NNTP-Posting-Host: hylkb.helsinki.fi In article , mligr@flash.LakeheadU.Ca (Martin Ligr) writes: > Hello! > I have just started working on my thesis and was stopped at the very begining > by supposedly ridiculous problem: I'm extracting total proteins of Chlamydomonas > using acetone extraction method. The purpose is to quantitate then particular > protein by immunoassay. However, I'm not able to redisolve pellet of proteins > after extraction completely. I was trying SDS up to 3% conc., Na2CO3 up to > 2 M, and NaOH up to 2 M. Only 2M NaOH redisolved the pellet completely, > but proteins were partially hydrolized- as was seen on SDS-PAGE. > I would appreciate if somebody could post here on send me a protocol describing > this method or to give me some hint. > > N.B.: I prefere acetone extraction to mechanical destruction methods, because > it removes chlorophyll which interferes with my fluorescence immunoassay. > > Thanks. > > Martin Ligr < mligr@flash.lakeheadu.ca > > Dept. of Biology > Lakehead University > Thunder Bay, Ontario > Maybe methanol/chloroform extraction followed with ammonium sulphate precipitation would solve your problems ? Jari Louhelainen Department of General Microbiology University of Helsinki FINLAND From owner-proteins@net.bio.net Mon Jun 14 23:00:00 1993 Path: biosci!uwm.edu!ux1.cso.uiuc.edu!howland.reston.ans.net!math.ohio-state.edu!cs.utexas.edu!uunet!news.crd.ge.com!maxwell!ebstokes From: ebstokes@maxwell.crd.ge.com (Ed Stokes) Newsgroups: bionet.molbio.proteins Subject: Looking for Klaus Buff Message-ID: Date: 15 Jun 93 16:57:34 GMT Sender: usenet@crdnns.crd.ge.com (USENET News System) Organization: GE Corp. Research & Development, Schenectady, NY Lines: 9 Nntp-Posting-Host: maxwell.crd.ge.com I am looking for the e-mail address of Dr. Klaus Buff at GSF in Neuherberg, Germany. I wish to discuss a possible application of one of his recent papers. I could (and will, if necessary) write him a letter, but this is so much more fun... Ed Stokes ebstokes@crd.ge.com From owner-proteins@net.bio.net Wed Jun 16 23:00:00 1993 Path: biosci!daresbury!daresbury!news From: sanchez@icgeb.trieste.it (Roberto Sanchez) Newsgroups: bionet.molbio.proteins Subject: loop structure database? Message-ID: <1993Jun17.152119.18865@gserv1.dl.ac.uk> Date: 17 Jun 93 15:03:20 GMT Sender: list-admin@daresbury.ac.uk Distribution: bionet Lines: 18 Original-To: proteins@uk.ac.daresbury X-Mailer: ELM [version 2.3 PL11] Hi all, Does anybody know about a loop structure database? Where, how to get it? Thanks, Roberto -- Roberto J. Sanchez Protein Structure and Function Group UNIDO International Centre for Genetic Engineering and Biotechnology Padriciano 99, 34012 Trieste, Italy Fax +39-40-226555 Internet sanchez@genes.icgeb.trieste.it From owner-proteins@net.bio.net Fri Jun 18 23:00:00 1993 Path: biosci!agate!howland.reston.ans.net!ux1.cso.uiuc.edu!moe.ksu.ksu.edu!matt.ksu.ksu.edu!news From: zhf@matt.ksu.ksu.edu (Feng Zheng) Newsgroups: bionet.molbio.proteins Subject: Purification of recombinant proteins from E.coli Message-ID: <200rciINNl36@matt.ksu.ksu.edu> Date: 20 Jun 93 05:08:34 GMT Organization: Kansas State University Lines: 1 NNTP-Posting-Host: matt.ksu.ksu.edu Hi. Could anybody tell me which is the best method to isolate and purify recombinant proteins expressed in E. coli? Thank you very much. I get a lot of problemswhen I try to purify corn trypsin inhibitor (14 KD). From owner-proteins@net.bio.net Fri Jun 18 23:00:00 1993 Path: biosci!agate!howland.reston.ans.net!darwin.sura.net!haven.umd.edu!uunet!psgrain!library.ucla.edu!ucla-mic!unixg.ubc.ca!cs.ubc.ca!news.UVic.CA!spruce.pfc.forestry.ca!PFC.Forestry.CA!DTAYLOR From: dtaylor@PFC.Forestry.CA Newsgroups: bionet.molbio.proteins Subject: Re: Acetone extraction of proteins Message-ID: <1993Jun18.231343.2651@spruce.pfc.forestry.ca> Date: 18 Jun 93 23:13:43 GMT References: ,<1993Jun15.204215.1@cc.helsinki.fi> Sender: news@spruce.pfc.forestry.ca Reply-To: dtaylor@PFC.Forestry.CA Distribution: bionet Organization: Forestry Canada (Pacific Forestry Centre) Lines: 1 Nntp-Posting-Host: pfc.pfc.forestry.ca In our lab we redissolve in a solution of 4% SDS, 5% Mercaptoethanol, and 5% sucrose. The pellet is resuspended in the solution using a hand-held mixer. This suspension is then vortexed and heated at 100C for 3 minutes. The sequence of vortexing and heating may have to be repeated more than once. The larger the acetone pellet the longer it will take to dissolve. Adding more SDS/MercaptoEtOH/Sucrose to the undissolved pellet might speed up dissolving at the expense of lower protein concentration. From owner-proteins@net.bio.net Sat Jun 19 23:00:00 1993 Path: biosci!agate!howland.reston.ans.net!ux1.cso.uiuc.edu!sdd.hp.com!col.hp.com!csn!hellgate.utah.edu!cc.usu.edu!gr8bn From: gr8bn@cc.usu.edu Newsgroups: bionet.molbio.proteins Subject: insoluble protein--need advise Message-ID: <1993Jun20.124325.69335@cc.usu.edu> Date: 20 Jun 93 18:43:25 GMT Organization: Utah State University Lines: 13 Hi, everyone I am a graduate student working on my thesis. I am trying to purify a protein expressed in Baculovirus system. The protein expressed in SF-9 cells is very insoluble (or aggregate). I have tried many types of nonionic detergents, none of them worked. Anionic detergent made the prtoein lost activity even after dialysis. 8M urea and 6M guanidine-HCl will solubilize the protein, but the same problem that without activity was observed (after dialysis the proteins partially precipitate out). Please give me some suggestions. Thanks ! I-Jen Huang email:gr8bn@cc.usu.edu fax:801-750-1575 From owner-proteins@net.bio.net Mon Jun 21 23:00:00 1993 Path: biosci!daresbury!daresbury!news From: WALLACE@IRBM.IT (Andrew, Phone ext. 434) Newsgroups: bionet.molbio.proteins Subject: Reply to msg # 353 on insoluble recombinant protein Message-ID: <1993Jun22.173056.155@gserv1.dl.ac.uk> Date: 22 Jun 93 17:34:13 GMT Sender: WALLACE@IT.IRBM Distribution: bionet Lines: 28 X-Vmsmail-To: SMTP%"proteins@daresbury.ac.uk" Original-To: proteins@uk.ac.daresbury In bionet.molbio.proteins Msg # 353 I-Jen Huang writes: >Hi, everyone >I am a graduate student working on my thesis. >I am trying to purify a protein expressed in Baculovirus system. The protein >expressed in SF-9 cells is very insoluble (or aggregate). I have tried >many types of nonionic detergents, none of them worked. Anionic detergent >made the prtoein lost activity even after dialysis. 8M urea and 6M guanidine- >HCl will solubilize the protein, but the same problem that without activity was >observed (after dialysis the proteins partially precipitate out). >Please give me some suggestions. Thanks ! Dear I-Jen, I have two suggestions regarding your insolubility problems. 1) Use a reducing agent in your solubilizing buffer, such as 5% beta- mercaptoethanol or 100mM dithiothreitol (DTT). 2) Change your expression construct to add a "tail" of three lysine or arginine residues at the N- or C-terminus of your protein. Positively charged residues such as these can be very effective in solubilising difficult proteins and preventing aggregation. Some colleagues I am working with are currently preparing a paper on this subject. Hope this helps. Andrew Wallace From owner-proteins@net.bio.net Mon Jun 21 23:00:00 1993 Path: biosci!bcm!cs.utexas.edu!swrinde!emory!nigel.msen.com!math.fu-berlin.de!KR2.CHEMIE.FU-BERLIN.DE!STEFFEN From: steffen@KR2.CHEMIE.FU-BERLIN.DE Newsgroups: bionet.molbio.proteins Subject: Call for Papers - Molecular Bioinformatics Message-ID: <1NOEBDJR@math.fu-berlin.de> Date: 22 Jun 93 16:11:45 GMT Sender: news@math.fu-berlin.de (Math Department) Reply-To: steffen@KR2.CHEMIE.FU-BERLIN.DE Organization: Free University of Berlin (Germany), Institute of Crystallography Lines: 126 CALL FOR PAPERS Colloquium "MOLECULAR BIOINFORMATICS IEE 94" This one day Colloquium is on Applications of Algorithms from Computer Science, including Artificial Intelligence, Machine Learning, Genetic Programming, Evolutionary Algorithms and Neural Nets, to the Analysis, Interpretation and Prediction of Data in Molecular Biology and Protein Engineering. Emphasis should be on why a particular technique seems appropriate for a certain problem. Algorithmic background and biological problem should both be briefly explained for the optimal benefit of an interdisciplinary audience of bioscientists and computer scientists. This announcement is distributed world wide. SCHEDULE Deadline for manuscripts for 30 minute oral presentations (7 - 15 pages, 3 copies, please) ..................... 31. 10. 1993 Notification of acceptance .............................. 31. 12. 1993 Colloquium .............................................. 28. 2. 1994 The Colloquium will be held in association with the ESPRIT III project "Programming Environment for Applications of Parallel Genetic Algorithms" (PAPAGENA). Registration fee will be approximately 60 (BP). Free registration and limited refunding of travel expenses for speakers available. PLACE IEE - Computing and Control Division Professional Group Committe C4 / Artifical Intelligence Savoy Place London WC2R 0BL United Kingdom PLEASE SEND CONTRIBUTIONS AND REGISTRATION TO: Brainware GmbH MBI IEE 1994 Dr. Steffen Schulze-Kremer Gustav-Meyer Allee 25 D-13355 Berlin, Germany email steffen@chemie.fu-berlin.de tel + 49 30 4633040 fax + 49 30 4644097 --- Cut here ------------ For your convenience -------------- Cut here --- Colloquium Registration Form Molecular Bioinformatics IEE 94 * London 28. 2. 94 Name: -------------------------------------------------------------------------- Institution: -------------------------------------------------------------------------- Surface mailing address: -------------------------------------------------------------------------- -------------------------------------------------------------------------- -------------------------------------------------------------------------- -------------------------------------------------------------------------- Country: -------------------------------------------------------------------------- Telephone: -------------------------------------------------------------------------- Fax: -------------------------------------------------------------------------- Email: -------------------------------------------------------------------------- Registration fee will be approximately 60 # (BP). Free for speakers and limited travel funds available. Signature: -------------------------------------------------------------------------- Please indicate your status * I do not want to give a presentation. * I intend to give a 30 minute oral presentation. (Please fill in title of your presentation and send 3 copies of a manuscript of 7 - 15 pages length.) Title: -------------------------------------------------------------------------- -------------------------------------------------------------------------- -------------------------------------------------------------------------- Please return this registration form before 31. 10. 1993 to: Brainware GmbH MBI IEE 1994 Dr. Steffen Schulze-Kremer email steffen@chemie.fu-berlin.de Gustav-Meyer Allee 25 tel + 49 30 4633040 D-13355 Berlin, Germany fax + 49 30 4644097 From owner-proteins@net.bio.net Wed Jun 23 23:00:00 1993 Path: biosci!NET.BIO.NET!kristoff From: kristoff@NET.BIO.NET (David Kristofferson) Newsgroups: bionet.molbio.proteins Subject: IMPORTANT BIOSCI INFORMATION Message-ID: <9306240900.AB09382@net.bio.net> Date: 24 Jun 93 09:00:03 GMT Sender: kristoff@net.bio.net Distribution: bionet Lines: 143 Three important items follow: BIOSCI archive searching by e-mail, the BIOSCI FAQ, and the BIOSCI User Address Directory form. If you have not yet listed yourself in our e-mail address directory, please take a few minutes to complete and return the form below. If your address information has changed since you listed yourself, please send us an updated form. Sincerely, Dave Kristofferson BIOSCI/bionet Manager kristoff@net.bio.net **** SEARCHING BIOSCI ARCHIVES WITH WAISMAIL **** E-mail users can search the BIOSCI archives by using our waismail e-mail server. For instructions send the message help to waismail@net.bio.net. Leave the Subject: line blank. Other methods of searching the archives via WAIS and gopher are described in the BIOSCI FAQ ... **** BIOSCI FREQUENTLY ASKED QUESTIONS (FAQ) SHEET **** New users of BIOSCI/bionet may want to read the "Frequently Asked Questions" or "FAQ" sheet for BIOSCI. The FAQ provides details on how to participate in these forums and is available for anonymous FTP from net.bio.net [134.172.2.69] in pub/BIOSCI/biosci.FAQ. It may also be requested by sending e-mail to biosci@net.bio.net (use plain English for your request). The FAQ is also posted on the first of each month to the newsgroup BIONEWS/bionet.announce immediately following the posting of the BIOSCI information sheet. **** BIOSCI USER ADDRESS DIRECTORY **** Please take this opportunity to add you name and address information to the BIOSCI User Address Database if you have not already done so. Below is the address form that we would like each reader of the BIOSCI/bionet newsgroups to complete and return if you would like to be listed in our database. The database will serve as a directory that will enable biologists, who are currently using (or even just reading) the BIOSCI newsgroups, to look up e-mail addresses and other information about our users. The address database will be indexed for WAIS and waismail access (waismail is our WAIS e-mail server, more below) and will also be available for access via other gopher sites if they wish to permit it. The raw unindexed data will be available for FTP from net.bio.net and is atomized sufficiently to allow import into your local RDBMS should you so desire. Please carefully follow the instructions for completing the form below and return it to either of the following two addresses (whichever is more convenient for you). Thanks in advance for taking the time to complete and return the form. Addresses for returning forms Location Network ----------------------------- -------- ------- biovote@net.bio.net U.S.A. Internet/BITNET biovote@daresbury.ac.uk U.K. JANET MAKING SURE THAT YOUR INFORMATION IS CURRENT This notice will be mailed bimonthly to each newsgroup. You should check our WAIS source or waismail e-mail server from time-to-time to see if your address information is still up-to-date. Send the message help to waismail@net.bio.net for instructions on using waismail. Leave the Subject: line in your message blank. (Note as of 5 May 93 - the address database will be updated nightly and will go on-line soon after we have collected and processed the initial rush of data). IMPORTANT INSTRUCTIONS - PLEASE READ CAREFULLY Please enter all responses after the : on each line, leaving one (1) blank space after the : (i.e., before the start of your text). Please do NOT extend your responses past the end of each line (80 characters) or alter any of the field identifiers such as "first name: ". Several lines are provided at the end of the form for comments, but, please adhere to the line length restriction. On the date: line, please enter the date in the DD-MM-YY format, e.g., 05-05-93 for 5 May 1993. This line will tell others when the information was last updated. Please be sure to include the 0's for single digit days or months, e.g., 05-05-93, not 5-5-93. Note that the "e-mail network: " line below is for specifying, e.g., "Internet," "BITNET," "EARN," "JANET," or whatever other network that your computer may be on. If you are uncertain about any field, please feel free to leave it blank, but please DO NOT DELETE the field identifier from the form! In the first field below, "New information or Update ...", please enter "N" if this is the first time that you have registered in the directory or "U" if you are correcting a listing that you sent to us previously. Thanks again for your cooperation! --------------- please cut here and return portion below --------------- New information or Update to old record (enter N or U): date (DD-MM-YY): first name: middle initial: family name: job title: e-mail address: e-mail network: phone number: FAX number: institution: address1: address2: address3: city: state/province: country: postal code: research interest: research interest: comment: comment: comment: comment: comment: From owner-proteins@net.bio.net Wed Jun 23 23:00:00 1993 Path: biosci!daresbury!daresbury!not-for-mail From: mbfw@s-crim1.dl.ac.uk (Frank Wright) Newsgroups: bionet.molbio.proteins Subject: Struct.Predictn software ==> PDB format? Message-ID: <20ccamINNcb8@s-crim1.dl.ac.uk> Date: 24 Jun 93 14:05:10 GMT Organization: Daresbury Lab., Warrington, U.K. Lines: 20 NNTP-Posting-Host: s-crim1.dl.ac.uk I have no access to Molecular Modelling software at the moment. However, I was wondering if there was any PD software that would take in a protein sequence and output a structure prediction (based on various methods) in Brookhaven PDB format. If not software, maybe there is some large number crunching site that can process jobs via a network server? If this was possible then the predicted structure could be viewed using PD software for viewing Brookhaven PDB files (e.g. RasMol). Any ideas? Thanks Frank Wright SASS, University of Edinburgh, J.C.M.B. room 3610, Kings Buildings, Edinburgh EH9 3JZ, Scotland, U.K. frank@sass.sari.ac.uk From owner-proteins@net.bio.net Fri Jun 25 23:00:00 1993 Path: biosci!agate!headwall.Stanford.EDU!rutgers!andromeda.rutgers.edu!hu From: hu@andromeda.rutgers.edu (Zhixiang Hu) Newsgroups: bionet.molbio.proteins Subject: Re: problems renaturing recombinant protein Message-ID: Date: 26 Jun 93 18:46:40 GMT References: Organization: Rutgers Univ., New Brunswick, N.J. Lines: 25 kcrossgr@mail.sas.upenn.edu (Kirsten Crossgrove) writes: >I'm trying to express a zinc-finger containing protein in E. coli. It >expresses well but is very insoluble. I can solubilize it in 6M guanidine So, the protein is expressed as inclussion body. It can only be solublized in denaturant. >HCl and purify it over a nickel-chelate column (it has a 6 histidine >N-terminal fusion). However, when I dialyze to get rid of the GuHCl (or 8M >urea-same result), the protein sometimes precipitates out, and even when it >doesn't, the activity is extremely low to non-existent. The protein is ..... >I get rid of the GuHCl or urea in a stepwise manner (1M, 0.1M, 0M). >I've gotten reasonably active preps before, but it is just not consistent >and lately no matter what I do, I can't get active protein. I think it is a >problem with the renaturation. I'm sure it is. Try to renature the denatured protein (use either acid or GuHCL to denature the active protein) under same conditions. If you would not be able to get any renatured protein, the renaturation becomes another project. You may try to use chaperons like hsp60 though. Zhixiang From owner-proteins@net.bio.net Fri Jun 25 23:00:00 1993 Path: biosci!uwm.edu!msuinfo!netnews.upenn.edu!gradmac.bio.upenn.edu!user From: kcrossgr@mail.sas.upenn.edu (Kirsten Crossgrove) Newsgroups: bionet.molbio.proteins Subject: problems renaturing recombinant protein Message-ID: Date: 26 Jun 93 17:32:18 GMT Sender: news@netnews.upenn.edu Followup-To: bionet.molbio.proteins Organization: University of Pennsylvania Lines: 21 Nntp-Posting-Host: gradmac.bio.upenn.edu Hi everyone, I'm trying to express a zinc-finger containing protein in E. coli. It expresses well but is very insoluble. I can solubilize it in 6M guanidine HCl and purify it over a nickel-chelate column (it has a 6 histidine N-terminal fusion). However, when I dialyze to get rid of the GuHCl (or 8M urea-same result), the protein sometimes precipitates out, and even when it doesn't, the activity is extremely low to non-existent. The protein is eluted off the column at low pH (usu. 5.9-6.3). The dialysis buffer is: 10mM HEPES pH 7.9 80mM KCl 1mM DTT 0.1% Triton X100 5-20% glycerol 10uM ZnCl2 I get rid of the GuHCl or urea in a stepwise manner (1M, 0.1M, 0M). I've gotten reasonably active preps before, but it is just not consistent and lately no matter what I do, I can't get active protein. I think it is a problem with the renaturation. Any suggestions? Thanks. Kirsten Crossgrove From owner-proteins@net.bio.net Sun Jun 27 23:00:00 1993 Path: biosci!daresbury!daresbury!not-for-mail From: mbfw@s-crim1.dl.ac.uk (Frank Wright) Newsgroups: bionet.molbio.proteins Subject: Re: Struct.Predictn software ==> PDB format? Message-ID: <20n4nvINNra3@s-crim1.dl.ac.uk> Date: 28 Jun 93 16:03:11 GMT Organization: Daresbury Lab., Warrington, U.K. Lines: 50 NNTP-Posting-Host: s-crim1.dl.ac.uk In reply to my (rather vague) question asking about Public Domain (PD) software for protein structure prediction... My hope had been to get a list of software that implemented different methods of structure prediction. I was aware that current methods were not very accurate and that they varied in accuracy. My intention was to try out the software on proteins for which both sequence and structure was known so as to get an idea of the accuracy of the available methods, before applying the software to sequences of unknown structure. Summary of replies.... For secondary structure prediction, the neural net PHD method of Rost and Sander (1993; in press) was suggested. This has a reported accuracy of 70% with water-soluble proteins (i.e. accuracy in predicting whether helix, strand, or loop). For information, send a one line e-mail message with the word "help" to the network server at predictprotein@embl-heidelberg.de. Jobs can also be submitted to this address by sending a properly formatted input sequence (details are given in the reply that you will recieve from your "help" message). Several replies suggested even more caution in using tertiary prediction methods. The need for considerable computing speed (e.g. greater than my SPARCstation) was also a likely requirement. Although there wasn't much PD software around, there were several packages available for a modest fee (usually about 100 dollars) to educational groups. One reply suggested I have a look at the COMPOSER program produced by the Birbeck group in London - this program uses a "homology modelling" method (this program may be an option in the SYBIL package too). The PROFILE set of programs (by David Eisenberg's group in LA) were also suggested for checking the "quality" of a model. I've not had time to check out COMPOSER or SYBIL (a commercial package by Tripos Ass., San Diego, CA) or PROFILE software, but I thought I should summarise the replies that I have had so far. I have previously tried the "predictprotein" server - the server is easy to use and B.Rost has kindly sent me a copy of his paper in press describing the PHD method. Frank Wright SASS, University of Edinburgh, J.C.M.B. room 3610, Kings Buildings, Edinburgh EH9 3JZ, Scotland, U.K. frank@sass.sari.ac.uk From owner-proteins@net.bio.net Mon Jun 28 23:00:00 1993 Path: biosci!agate!howland.reston.ans.net!torn!nott!emr1!weathere From: weathere@emr1.emr.ca (Carl Weatherell) Newsgroups: bionet.molbio.proteins Subject: Collagen Preconcentration Message-ID: <1993Jun29.175152.20800@emr1.emr.ca> Date: 29 Jun 93 17:51:52 GMT Organization: Dept. of Energy, Mines, and Resources, Ottawa Lines: 4 X-Newsreader: TIN [version 1.1 PL8] I am trying to pre-concentrate collagen (0.1-10 mg/L) from solutions of 2M H2SO4 an 50g/L Zn prior to SEC. I have tried anionic, cationic and HIC modes of solid phase extraction using BioRad MacroPrep resins. Is there a simple quantitative procedure to pre-concentrate collagens from these solutions? Email weathere.emr1.emr.ca From owner-proteins@net.bio.net Mon Jun 28 23:00:00 1993 Path: biosci!daresbury!daresbury!news From: thiery@orstom.fr (THIERY Jean) Newsgroups: bionet.molbio.proteins Subject: Searching antibodies Message-ID: <1993Jun29.074653.14477@gserv1.dl.ac.uk> Date: 29 Jun 93 07:27:13 GMT Sender: list-admin@daresbury.ac.uk Distribution: bionet Lines: 7 Original-To: proteins@uk.ac.daresbury Searching antibodies raised against plant HSPs (LMW and HSP70). Please contact Christian TRIANTAPHYLIDES, Care of Jean M. THIERY (thiery at orstom.orstom.fr) Best regards, JMT From owner-proteins@net.bio.net Tue Jun 29 23:00:00 1993 Path: biosci!MAYO.EDU!morbeck From: morbeck@MAYO.EDU Newsgroups: bionet.molbio.proteins Subject: PAGE of peptides Message-ID: <9306301922.AA15473@fermat.Mayo.EDU> Date: 30 Jun 93 19:22:53 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 12 We need to be able to determine if our synthetic peptides of 1.5 to 2.5 kD are forming dimers or polymers at physiologic pH. Are there any methods for obtaining decent separation/resolution using PAGE at that MW? We have tried a number of different things, including analytical HPLC and HPCE, but can't identify the size of the species with these techniques. Thanks, Dean Morbeck Department of Biochemistry Mayo Clinic Morbeck@Mayo.edu From owner-proteins@net.bio.net Tue Jun 29 23:00:00 1993 Path: biosci!agate!doc.ic.ac.uk!warwick!news.dcs.warwick.ac.uk!mrccrc!dmartin From: dmartin@crc.ac.uk (David Martin x3175) Newsgroups: bionet.molbio.proteins Subject: Re: PAGE of peptides Message-ID: <1993Jun30.200707.11608@crc.ac.uk> Date: 30 Jun 93 20:07:07 GMT References: <9306301922.AA15473@fermat.Mayo.EDU> Sender: news@crc.ac.uk Distribution: bionet Organization: MRC Human Genome Resource Centre Lines: 10 Nntp-Posting-Host: tin You could try using gel filtration with superose 12. I am currently having similar problems with a peptide (trying to reform 3 disulphide bridges and most of what I get is HMW polymer) I have been using gel filtration to analyse the refolding results with quite reasonable results. ...d From owner-proteins@net.bio.net Tue Jun 29 23:00:00 1993 Path: biosci!uwm.edu!ux1.cso.uiuc.edu!howland.reston.ans.net!agate!darkstar.UCSC.EDU!orchid.UCSC.EDU!quinones From: quinones@ucsc.edu (Cathy Quinones) Newsgroups: bionet.molbio.proteins Subject: glycoprotein stains Message-ID: <20t7s8INNbj2@darkstar.UCSC.EDU> Date: 30 Jun 93 23:33:28 GMT Distribution: usa Organization: Santa Cruz Lines: 15 NNTP-Posting-Host: orchid.ucsc.edu Originator: quinones@orchid.UCSC.EDU I want to stain glycoproteins and then counterstain them with Coomasie Blue. The glycoprotein stain protocol I have chosen (Thymol-Sulfuric acid stain) mentions that the gel will become dehydrated in the process, and will rehydrate back to the original size; the problem is the stain will fade within several hours (I assume sometime between dehydration and rehydration). My idea was to stain the glycoproteins, photograph the gel, stain with Coomasie Blue, photograph gel again, and see which bands corres- pond to glycoproteins. BUT, if photo#1 depicts a shrunken gel, how can I match up its bands to those in photo#2 ("normal" gel size)??? I chose the thymol-sulfuric acid stain for its increased sensitivity (compared to phosphotunfstic acid/Schiff's reagent stain) and because I want to keep things simple. Hints/suggestions are desperately welcome! From owner-proteins@net.bio.net Wed Jun 30 23:00:00 1993 Path: biosci!agate!howland.reston.ans.net!darwin.sura.net!nih-csl!csax From: csax@helix.nih.gov (Carl Saxinger) Newsgroups: bionet.molbio.proteins Subject: Re: PAGE of peptides Message-ID: <1993Jul1.164509.8948@alw.nih.gov> Date: 1 Jul 93 16:45:09 GMT References: <9306301922.AA15473@fermat.Mayo.EDU> Sender: postman@alw.nih.gov (AMDS Postmaster) Distribution: bionet Organization: (National Institutes of Health, Bethesda, MD) Lines: 6 In article <9306301922.AA15473@fermat.Mayo.EDU> morbeck@MAYO.EDU writes: >We need to be able to determine if our synthetic peptides of 1.5 to 2.5 kD are >forming dimers or polymers at physiologic pH. Are there any methods for We have been able to do this with a 20cm GPC-peptide (50A) column from Synchrom, Inc. From owner-proteins@net.bio.net Wed Jun 30 23:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!agate!overload.lbl.gov!dog.ee.lbl.gov!network.ucsd.edu!dialin1-23-1.extern.ucsd.edu!rhughes From: rhughes@ucsd.edu (Richard J. Hughes) Newsgroups: bionet.molbio.proteins Subject: Trivia question...why do carrots turn green in cakes? Message-ID: Date: 1 Jul 93 04:26:50 GMT Organization: ucsd.edu Lines: 21 NNTP-Posting-Host: dialin1-23-1.extern.ucsd.edu Hi netters! Here is a trivia question. Why do carrot shreds turn green when cooked in carrot cake? Is this due to oxidation of the beta-carotene? Is beta- carotene responsible for the orange colour in the first place? Could the colour change be prevented by baking with ascorbic acid for a healthier, cold-preventing, carrot cake? Many thanks. Richard +------------------------------------------------------------------------+ | Dr. Richard J. Hughes rhughes@ucsd.edu | | Department of Pharmacology <> | | University of California at San Diego Tel (619) 534-2298 | | La Jolla, CA 92093-0636 FAX (619) 534-7461 | +------------------------------------------------------------------------+ From owner-proteins@net.bio.net Wed Jun 30 23:00:00 1993 Path: biosci!agate!howland.reston.ans.net!noc.near.net!saturn.caps.maine.edu!dartvax!grafton.dartmouth.edu!knight From: knight@grafton.dartmouth.edu (John Boswell) Newsgroups: bionet.molbio.proteins Subject: Protein Purification Protocols Message-ID: Date: 1 Jul 93 17:53:09 GMT Sender: news@dartvax.dartmouth.edu (The News Manager) Organization: Dartmouth College, Hanover, NH Lines: 27 Hi, I posted a similar request on another group, then found this one :) Could someone in the field please recommend some good texts/references on protein purification protocols? I have about 15 liters of media from cell culture, and I need to isolate and purify an expressed protein from this. I can detect the protein via Western blot, and I also have an activity assay. What I need are good ways to start to purify the protein, i.e. protocols for salt-pption., gel-filtration, and other chromatography; maybe elution via an ethylene glycol gradient. I don't have much experience with protein purification past a short course I took a bunch of years ago in graduate school. Any pointers in the right direction would be greatly appreciated! I'm off to look in the library now... :):) Thanks, -John Boswell, Ph.D. Dept. of Chemistry, Biochemistry, and Molecular Biology Oregon Graduate Institute Portland, OR 97229 (knight@grafton.dartmouth.edu) -- **************************************************************************** Dr. John Boswell knight@grafton.dartmouth.edu Dept. of Medicine/GI MCN C2104 Vanderbilt University, Nashville, TN 37232 615-322-6367 or 615-343-4747