From owner-proteins@net.bio.net Sun Oct 01 23:00:00 1995 Path: biosci!daresbury!bioftp.unibas.ch!citi2.fr!jussieu.fr!oleane!tank.news.pipex.net!pipex!in2.uu.net!inet.guthrie.org!nchang!nchang From: nchang@guthrie.inet.org (Nan-Shan Chang) Newsgroups: bionet.molbio.proteins Subject: Research Technician Open Followup-To: bionet.molbio.proteins Date: Sun, 1 Oct 95 23:30:46 GMT Organization: Guthrie HealthCare System Lines: 5 Distribution: world Message-ID: NNTP-Posting-Host: pc67.guthrie.org X-Newsreader: VersaTerm Link v1.1.5 A Research Technician position is available immediately in the Guthrie Research Institute. Experience in molecular biology and/or immunology is preferred. BS or MS degree is required. Please submit your resume and 3 references to: Dr. N. Chang, 1 Guthrie Square, Sayre, PA 18840. Fax: (717) 882-5151. From owner-proteins@net.bio.net Sun Oct 01 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!swrinde!news.uh.edu!news.uth.tmc.edu!news From: you@odin.mda.uth.tmc.edu (you) Newsgroups: bionet.molbio.proteins Subject: Re: immunoprecipitation question Date: 2 Oct 1995 22:30:55 GMT Organization: UTMDACC Lines: 36 Message-ID: <44pp70$71o@oac4.hsc.uth.tmc.edu> References: <44gush$hcs@acmex.gatech.edu> NNTP-Posting-Host: tainsky3.mda.uth.tmc.edu Mime-Version: 1.0 X-Newsreader: WinVN 0.99.2 In article <44gush$hcs@acmex.gatech.edu> gt0332b@prism.gatech.edu >(Robert Orlando Scott) writes: > >> I am currently trying to immunoprecipitate a protein with a >> molecular weight of 23 kDa. I assume the immunoprecipitation is >> complete however when I try to detect the protein of interest via >> western blotting the light chain IgG mask the area of interest. I >> am using chemiluminescent detection procedures. I have tried >> non-reducing conditions in my procedures however there still appears >> to be some light chains present. Are there any tricks to avoid the >> IgG bands The same polyclonal antibody is used in both the IP and >> western blot. >-- Hi Robert, I have been having a similar problem, but with IgG heavy chain having the same molecular weight as my protein of interest. You can try the following approaches. First, you can use peptides that have the epitope to competitively elute your IgG bound protein. A ten fold excess of the peptide is usually recommended. However, the elution may not be complete and you may still have IgG coming down with your protein (leaching effect). The second option is to metabolically label your protein with radioactive methionine and then use autoradiography for detection after immunoprecipitation. This is probably the better approach if your protein can be extracted from cultured cells. Good luck. >Okot Nyormoi Department of Tumor Biology Box 79 MD Anderson Cancer Center Houston TX 77030 email: sa88049@odin.mda.uth.tmc.edu > From owner-proteins@net.bio.net Sun Oct 01 23:00:00 1995 Path: biosci!daresbury!nntp-trd.UNINETT.no!news10.sunet.se!news00.sunet.se!sunic!sunic!sunic.sunet.se!columba.udac.uu.se!news From: Jorge Blackhall Newsgroups: bionet.molbio.proteins Subject: Re: IEF standards? Date: 2 Oct 1995 11:15:40 GMT Organization: Uppsala University Lines: 38 Message-ID: <44ohks$ufa@columba.udac.uu.se> References: <1995Sep30.154731.1@icbr2.ifas.ufl.edu> NNTP-Posting-Host: armtwister.medvir.uu.se Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) To: rep@icbr2.ifas.ufl.edu X-URL: news:1995Sep30.154731.1@icbr2.ifas.ufl.edu ----------------------------------------------------------------------- "Has anyone found a good set of isoelectric focusing standards? Are there any prestained IEF markers? I'm running slab gels, blotting them to PVDF and then immunoblotting. Surface pH monitors have been suggested, but transferring that data to the Western blot may cause problems. I would appreciate any information." Robert Peterson Whitney Lab. St. Augustine, FL REP@icbr.ifas.ufl.edu ------------------------------------------------------------------------ I have used the BioRad 2-D SDS PAGE standards (Cat. # 161-0320) many times and they were pretty nice, stained with silver nitrate. I don't have experience with Western blot, but perhaps is possible to see them stained with Ponceau red. I don't know of any prestained IEF markers. Hope it is some help. Jorge Blackhall rep@icbr2.ifas.ufl.edu wrote: >Hello there, > >Has anyone found a good set of isoelectric focusing standards? Are there any >prestained IEF markers? I'm running slab gels, blotting them to PVDF and then >immunoblotting. Surface pH monitors have been suggested, but transferring that >data to the Western blot may cause problems. I would appreciate any >information. > >Robert Peterson >Whitney Lab. >St. Augustine, FL >REP@icbr.ifas.ufl.edu > From owner-proteins@net.bio.net Sun Oct 01 23:00:00 1995 Path: biosci!daresbury!nntp-trd.UNINETT.no!news10.sunet.se!news00.sunet.se!sunic!sunic!sunic.sunet.se!columba.udac.uu.se!news From: Jorge Blackhall Newsgroups: bionet.molbio.proteins Subject: Re: IEF standards? Date: 2 Oct 1995 11:15:22 GMT Organization: Uppsala University Lines: 38 Message-ID: <44ohka$ufa@columba.udac.uu.se> References: <1995Sep30.154731.1@icbr2.ifas.ufl.edu> NNTP-Posting-Host: armtwister.medvir.uu.se Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) To: rep@icbr2.ifas.ufl.edu X-URL: news:1995Sep30.154731.1@icbr2.ifas.ufl.edu ----------------------------------------------------------------------- "Has anyone found a good set of isoelectric focusing standards? Are there any prestained IEF markers? I'm running slab gels, blotting them to PVDF and then immunoblotting. Surface pH monitors have been suggested, but transferring that data to the Western blot may cause problems. I would appreciate any information." Robert Peterson Whitney Lab. St. Augustine, FL REP@icbr.ifas.ufl.edu ------------------------------------------------------------------------ I have used the BioRad 2-D SDS PAGE standards (Cat. # 161-0320) many times and they were pretty nice, stained with silver nitrate. I don't have experience with Western blot, but perhaps is possible to see them stained with Ponceau red. I don't know of any prestained IEF markers. Hope it is some help. Jorge Blackhall rep@icbr2.ifas.ufl.edu wrote: >Hello there, > >Has anyone found a good set of isoelectric focusing standards? Are there any >prestained IEF markers? I'm running slab gels, blotting them to PVDF and then >immunoblotting. Surface pH monitors have been suggested, but transferring that >data to the Western blot may cause problems. I would appreciate any >information. > >Robert Peterson >Whitney Lab. >St. Augustine, FL >REP@icbr.ifas.ufl.edu > From owner-proteins@net.bio.net Sun Oct 01 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!Germany.EU.net!news.dfn.de!news.rwth-aachen.de!newsserver.rrzn.uni-hannover.de!tubsibr!rzlimes.gbf-braunschweig.de!news From: fho@gbf-braunschweig.de (Frank Hoffmann) Newsgroups: bionet.molbio.proteins Subject: metabolic burden of recombinant protein overexpression Date: 2 Oct 1995 09:37:33 GMT Organization: GBF Braunschweig Lines: 12 Message-ID: <44obst$ej8@rzlimes.gbf-braunschweig.de> NNTP-Posting-Host: bvt484.gbf-braunschweig.de X-Newsreader: WinVN 0.92.6+ When a recombinant protein is overexpressed in bacterial cells, building blocs and energy are consumed for that, so cell grow slower. Okay, but after a while they stop production, and also growth ceased, whereas substrat uptake continous. Is anybody out there who can explain me what's up with them? What are they doing with all the substrat? How does the disturbance of metabolism work? Hope you can help me Thank you in advance Frank From owner-proteins@net.bio.net Sun Oct 01 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!swrinde!tank.news.pipex.net!pipex!news.sprintlink.net!news.inc.net!news From: "Paul D. Boyer" Newsgroups: uchi.bio,uchi.general,bionet.molbio.proteins,bionet.molbio.methds-reagnts Subject: Re: Mol Biologists, do any of you use precast gels? Date: 2 Oct 1995 23:11:42 GMT Organization: University of Wisconsin-Parkside Lines: 16 Message-ID: <44prje$f4a@news.inc.net> References: NNTP-Posting-Host: grnq-102.uwp.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Windows; I; 16bit) To: mjfath@midway.uchicago.edu Xref: biosci bionet.molbio.proteins:5811 bionet.molbio.methds-reagnts:34373 I have a lot of experience with Novex' PAGE gels. I love them, although they're a bit pricey. I've never had a problem with them, and all you do is rip open the bag, pull the comb, and run. I can't rave enough about them. I only wish budgets these days allowed for them on a regular basis! -- ************************************************************************* Paul D. Boyer, Ph.D. 900 Wood Road, Box 2000 Assistant Professor Kenosha, WI 53141-2000 Department of Biological Sciences pboyer@cs.uwp.edu University of Wisconsin-Parkside ************************************************************************* From owner-proteins@net.bio.net Sun Oct 01 23:00:00 1995 Path: biosci!daresbury!bioftp.unibas.ch!citi2.fr!jussieu.fr!oleane!tank.news.pipex.net!pipex!uknet!bhamcs!news.ox.ac.uk!news From: "Simon M. Brocklehurst" Newsgroups: bionet.molbio.proteins Subject: NAOMI Version 2.2 Date: 2 Oct 1995 13:54:49 GMT Organization: University of Oxford Lines: 68 Message-ID: <44oqv9$ju5@news.ox.ac.uk> NNTP-Posting-Host: nmrz.ocms.ox.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (X11; I; IRIX 5.3 IP22) X-URL: news:bionet.molbio.proteins NAOMI - Version Upgrade Announcement (Please note, NAOMI is provided at zero charge for academic use) (e-mail contact smb@bioch.ox.ac.uk) _____________________________________________________________________________ The computer program NAOMI Version 2.2 is available _now_ from the NAOMI Web site at: http://www.ocms.ox.ac.uk/~smb/Software/N_details/naomi.html or via anonymous ftp from: nmrz.ocms.ox.ac.uk in directory pub/smb/naomi/ Users of versions older than 2.10 will need new license keys to allow the upgrade to work (please contact the author in this case). _____________________________________________________________________________ Upgrade features : solvent accessibility, symmetry operations, disulphide bonds, new side-chain modelling commands, new molscript output options e.g. see the illustrations on the Cytokines Web: http://www.ocms.ox.ac.uk/~smb/cyt_web/ Fixes of minor bugs in key residues and molscript output commands. _____________________________________________________________________________ What is NAOMI? NAOMI is an easy-to-use, state-of-the-art computer program which is aimed at both specialist and non-specialist researchers who make use of three-dimensional structures of proteins in their work. It has hundreds of users Worldwide. Some facilities offered by the program for working with structure include: automatic 'key' residue identification automatic hydrophobic core/packing analysis automatic hydrogen bonds main-chain and side-chain identification (including high quality energy calculations) automatic secondary structure (helix, strand and turn) classification using fuzzy logic automatic supersecondary structure classification (beta-hairpin loops) conformational parameters: phi,psi,chi1,chi2,chi3,chi4,chi5 etc solvent accessibility (both absolute and percentage) calculations automatic identification of disulphide bonds, salt bridges, chain-breaks side-chain modelling and manipulation applying symmetry operators automatic structure repair (building in missing atoms) NMR structure refinement module interfaces to graphics programs (MOLSCRIPT (and thus Raster3D), INSIGHT, QUANTA to allow automatic preparation of figures More details are available on the Web site. NB NAOMI currently works only on Silicon Graphics workstations running IRIX 5.* _____________________________________________________________________________ | | ,_ o Simon M. Brocklehurst, | / //\, Oxford Centre for Molecular Sciences, Department of Biochemistry, | \>> | University of Oxford, Oxford, UK. | \\, E-mail: smb@bioch.ox.ac.uk | WWW: http://www.ocms.ox.ac.uk/~smb/ |____________________________________________________________________________ From owner-proteins@net.bio.net Sun Oct 01 23:00:00 1995 Path: biosci!daresbury!nntp-trd.UNINETT.no!news10.sunet.se!news00.sunet.se!sunic!sunic!sunic.sunet.se!news.sprintlink.net!howland.reston.ans.net!agate!sunsite.doc.ic.ac.uk!lyra.csx.cam.ac.uk!news From: Manoj Ramjee Newsgroups: bionet.molbio.proteins Subject: Re: Chemical cleavage of cysteine Date: 2 Oct 1995 07:49:01 GMT Organization: University of Cambridge Lines: 20 Message-ID: <44o5hd$qcq@lyra.csx.cam.ac.uk> References: <44i0t2$3i@sake.wwa.com> NNTP-Posting-Host: molgen3.plantsci.cam.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) X-URL: news:44i0t2$3i@sake.wwa.com If you mean at cysteine residues within a protein, rather than cysteine itself then the ref. I have is ....... Jacobson, et al., (1973), Journal of Biological Chemistry, vol. 248, no. 19, 6583-6591 entitled 'Specific chemical cleavage in high yield at the amino peptide bonds of cysteine and cystine residues'. I hope this helps. bYe. -------------------------------------------------------------- ------- Dr. Manoj Ramjee, Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge, CB2 3EA. U.K. E-mail: mkr@mole.bio.cam.ac.uk -------------------------------------------------------------- ------- From owner-proteins@net.bio.net Sun Oct 01 23:00:00 1995 Path: biosci!daresbury!bioftp.unibas.ch!citi2.fr!jussieu.fr!oleane!tank.news.pipex.net!pipex!in2.uu.net!inet.guthrie.org!nchang!nchang From: nchang@guthrie.inet.org (Nan-Shan Chang) Newsgroups: bionet.molbio.proteins Subject: Postdoctoral Position Open Followup-To: bionet.molbio.proteins Date: Sun, 1 Oct 95 23:28:43 GMT Organization: Guthrie HealthCare System Lines: 8 Distribution: world Message-ID: NNTP-Posting-Host: pc67.guthrie.org X-Newsreader: VersaTerm Link v1.1.5 Postdoctoral / Research Associate Position is available at the Guthrie Research Institute to participate in the molecular characterization of extracellular matrix signal in conferring cancer cell resistance to tumor necrosis factor and apoptosis (JBC 270, 7765, 1995). Experience in molecular cloning, sequencing, expression and/or background in immunology is required. Send curriculum vitae and 3 reference letters to: Dr. N.-S. Chang, Guthrie Research Institute, 1 Guthrie Square, Sayre, PA 18840. Fax: (717)882-5151. Email: nchang@inet.guthrie.org An Equal Opportunity Employer. From owner-proteins@net.bio.net Sun Oct 01 23:00:00 1995 Path: biosci!biosci!not-for-mail From: jones@bsm.bioc.ucl.ac.uk (David Jones) Newsgroups: bionet.molbio.proteins,bionet.biology.computational Subject: Re: How to predict transmembrane protein topology? Followup-To: bionet.molbio.proteins,bionet.biology.computational Date: 2 Oct 1995 15:29:23 -0700 Organization: University College London Lines: 20 Sender: biohelp@net.bio.net Approved: comp-bio-moderator@net.bio.net Distribution: world Message-ID: <1995Sep30.143512.85857@ucl.ac.uk> References: <4486ld$dpu@news.ust.hk> NNTP-Posting-Host: net.bio.net Xref: biosci bionet.molbio.proteins:5808 bionet.biology.computational:764 Terence (bclong@uxmail.ust.hk) wrote: > I often read in journals that a certain transmembrane topology is > determined/predicted for a certain transmemnrane protein. How is this done? > Is this done thru some commercial/public domain software? Or is it just by > eyeballing how many hydrophobic segment there? Any hints will be welcomed. Have a look at the following WWW page for one method you can use: http://www.biochem.ucl.ac.uk/~jones/memsat.html alternatively anonymous ftp to ftp.biochem.ucl.ac.uk and look in pub/MEMSAT >---------------------------------------------------------------------------< This message was written, produced and executively directed by Dr David Jones Address: Department of Biochemistry and | Email: jones@bsm.bioc.ucl.ac.uk Molecular Biology, University College, | Tel: +44 171 387 7050 x3879 Gower Street, London WC1E 6BT, U.K. | Fax: +44 171 380 7193 Disclaimer: STANDARD > KEYWORDS : OPINIONS MY OWN NOBODY ELSE'S WHATSOEVER From owner-proteins@net.bio.net Sun Oct 01 23:00:00 1995 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!elroy.jpl.nasa.gov!usc!howland.reston.ans.net!swrinde!tank.news.pipex.net!pipex!news.sprintlink.net!news.inc.net!cs!boyer From: boyer@cs.uwp.edu (Paul Boyer) Newsgroups: bionet.molbio.proteins Subject: Protein manual Date: 2 Oct 1995 17:47:56 GMT Organization: University of Wisconsin - Parkside Lines: 14 Message-ID: <44p8kc$3jl@news.inc.net> NNTP-Posting-Host: cs.uwp.edu X-Newsreader: TIN [version 1.2 PL2] I'm looking for a manual/handbook to use as a text in a lab course called "Working with Proteins". I'd like to find something like the Maniatis bible for molecular biologists, but I can't find anything on the protein level. Can anyone recommend anything? Thanks, Paul Boyer =========================================================================== Paul D. Boyer, Ph.D. University of Wisconsin--Parkside Assistant Professor 900 Wood Road, Box 2000 Department of Biological Sciences Kenosha, WI 53141-2000 ============================================================================ From owner-proteins@net.bio.net Sun Oct 01 23:00:00 1995 Path: biosci!daresbury!nntp-trd.UNINETT.no!news10.sunet.se!news00.sunet.se!sunic!sunic!sunic.sunet.se!columba.udac.uu.se!news From: Jorge Blackhall Newsgroups: bionet.molbio.proteins Subject: Re: IEF standards? Date: 2 Oct 1995 11:15:04 GMT Organization: Uppsala University Lines: 24 Message-ID: <44ohjo$ufa@columba.udac.uu.se> References: <1995Sep30.154731.1@icbr2.ifas.ufl.edu> NNTP-Posting-Host: armtwister.medvir.uu.se Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) To: rep@icbr2.ifas.ufl.edu X-URL: news:1995Sep30.154731.1@icbr2.ifas.ufl.edu ----------------------------------------------------------------------- "Has anyone found a good set of isoelectric focusing standards? Are there any prestained IEF markers? I'm running slab gels, blotting them to PVDF and then immunoblotting. Surface pH monitors have been suggested, but transferring that data to the Western blot may cause problems. I would appreciate any information." Robert Peterson Whitney Lab. St. Augustine, FL REP@icbr.ifas.ufl.edu ------------------------------------------------------------------------ I have used the BioRad 2-D SDS PAGE standards (Cat. # 161-0320) many times and they were pretty nice, stained with silver nitrate. I don't have experience with Western blot, but perhaps is possible to see them stained with Ponceau red. I don't know of any prestained IEF markers. Hope it is some help. Jorge Blackhall From owner-proteins@net.bio.net Sun Oct 01 23:00:00 1995 Path: biosci!daresbury!nntp-trd.UNINETT.no!news10.sunet.se!news00.sunet.se!sunic!sunic!sunic.sunet.se!columba.udac.uu.se!news From: Jorge Blackhall Newsgroups: bionet.molbio.proteins Subject: Re: IEF standards? Date: 2 Oct 1995 11:14:32 GMT Organization: Uppsala University Lines: 24 Message-ID: <44ohio$ufa@columba.udac.uu.se> References: <1995Sep30.154731.1@icbr2.ifas.ufl.edu> NNTP-Posting-Host: armtwister.medvir.uu.se Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) To: rep@icbr2.ifas.ufl.edu X-URL: news:1995Sep30.154731.1@icbr2.ifas.ufl.edu ----------------------------------------------------------------------- "Has anyone found a good set of isoelectric focusing standards? Are there any prestained IEF markers? I'm running slab gels, blotting them to PVDF and then immunoblotting. Surface pH monitors have been suggested, but transferring that data to the Western blot may cause problems. I would appreciate any information." Robert Peterson Whitney Lab. St. Augustine, FL REP@icbr.ifas.ufl.edu ------------------------------------------------------------------------ I have used the BioRad 2-D SDS PAGE standards (Cat. # 161-0320) many times and they were pretty nice, stained with silver nitrate. I don't have experience with Western blot, but perhaps is possible to see them stained with Ponceau red. I don't know of any prestained IEF markers. Hope it is some help. Jorge Blackhall From owner-proteins@net.bio.net Sun Oct 01 23:00:00 1995 Path: biosci!CSHL.ORG!boyce From: boyce@CSHL.ORG (Joan Boyce) Newsgroups: bionet.molbio.proteins Subject: Re: Protein Manual Date: 2 Oct 1995 16:00:31 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 30 Sender: daemon@net.bio.net Distribution: world Message-ID: <9510022256.AA09389@phage.cshl.org> NNTP-Posting-Host: net.bio.net >I'm looking for a manual/handbook to use as a text in a lab course called >"Working with Proteins". >I'd like to find something like the Maniatis bible for molecular biologists, >But I can't find >anything on the protein level. Can anyone recommend anything? > >Thanks, >Paul Boyer The publishers that brought you the "bible" have announced new laboratory course manual entitled "Strategies for Protein Purification and Characterization, a Laboratory Course Manual" It's due anytime now, and the Cold Spring Harbor Laboratory Press catalog states a price of $75.00. Call 1-800-843-4388 for further information Hope this helps in your quest! Joan Boyce ------------------------ Hot Pick of the week: http://www.biosupplynet.com/bsn/ an on-line buyer's guide for reagents, supplies and equipment used in everyday (and not so everyday) research From owner-proteins@net.bio.net Sun Oct 01 23:00:00 1995 Path: biosci!daresbury!bioftp.unibas.ch!citi2.fr!jussieu.fr!centre.univ-orleans.fr!univ-lyon1.fr!in2p3.fr!swidir.switch.ch!swsbe6.switch.ch!scsing.switch.ch!news.rediris.es!acebo.sdi.uam.es!newsmasters From: jayala@mvax.cbm.uam.es (Juan A. Ayala) Newsgroups: bionet.molbio.proteins Subject: Re: IEF standards? Date: 2 Oct 1995 14:20:44 GMT Organization: Centro de Biología Molecular Lines: 23 Message-ID: <44osfs$ils@acebo.sdi.uam.es> References: <1995Sep30.154731.1@icbr2.ifas.ufl.edu> NNTP-Posting-Host: b1011.cbm.uam.es Mime-Version: 1.0 Content-Type: Text/Plain; charset=US-ASCII X-Newsreader: WinVN 0.99.6 Dear nets: We have found new proteins in E.coli with unknown functions. One of them have been shown to be a putative methyltransferase by homology sequence analysis. Do you (some of you) know a defined assay in vitro for this enzymatic activity ? Thank a lot for your help ________________________________________________________________ : Juan A. Ayala : FAX-net: (+34-1)-397.83.44 : PSI-net: (2145)-2120423216 : INTERNET: JAYALA@mvax.cbm.uam.es : TELEX-net: 27810 educi e : PHONE-net: (+34-1)-397.80.83 : Snail-net: : : : ICBM-net: 40 deg 30 min North, 3 deg 42 min West :________________________________________________________________ :The Best Man for the Job is a Wo-man! :________________________________________________________________ From owner-proteins@net.bio.net Sun Oct 01 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!news.cac.psu.edu!psuvm!jps144 Organization: Penn State University Date: Mon, 2 Oct 1995 10:43:18 EDT From: Joe Stains Message-ID: <95275.104318JPS144@psuvm.psu.edu> Newsgroups: bionet.molbio.proteins Subject: Fisher semi dry blotter Lines: 11 Does anyone out there own or use a Fisher semi dry blotter? I am interested in purchasing one, but would first like to know what people's general opinion of it is. In the past I have used the BioRad blotter, and like it very much, but the Fisher model is a better deal for us at the moment, but I know of no one first hand who has one. Any info or opinions on it would be appreciated. Please e-mail me directly at jps144@psuvm.psu.edu. Thanks Joe stains Penn State University From owner-proteins@net.bio.net Mon Oct 02 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!uunet!in1.uu.net!news.sprintlink.net!simtel!swidir.switch.ch!scsing.switch.ch!news.rediris.es!acebo.sdi.uam.es!newsmasters From: jayala@mvax.cbm.uam.es (Juan A. Ayala) Newsgroups: bionet.molbio.proteins Subject: Methyltransferase activity Date: 3 Oct 1995 14:49:23 GMT Organization: Centro de Biología Molecular Lines: 24 Message-ID: <44rihj$5hf@acebo.sdi.uam.es> NNTP-Posting-Host: b1011.cbm.uam.es Mime-Version: 1.0 Content-Type: Text/Plain; charset=US-ASCII X-Newsreader: WinVN 0.99.6 Dear nets: We have found new proteins in E.coli with unknown functions. One of them have been shown to be a putative methyltransferase by homology sequence analysis. Do you (some of you) know a defined assay in vitro for this enzymatic activity ? Thank a lot for your help -- ________________________________________________________________ : Juan A. Ayala : FAX-net: (+34-1)-397.83.44 : PSI-net: (2145)-2120423216 : INTERNET: JAYALA@mvax.cbm.uam.es : TELEX-net: 27810 educi e : PHONE-net: (+34-1)-397.80.83 : Snail-net: : : : ICBM-net: 40 deg 30 min North, 3 deg 42 min West :________________________________________________________________ :The Best Man for the Job is a Wo-man! :________________________________________________________________ From owner-proteins@net.bio.net Mon Oct 02 23:00:00 1995 Path: biosci!daresbury!not-for-mail From: "Andrew, Tel. +396-91093434" Newsgroups: bionet.molbio.proteins Subject: Phosphorylated derivatives of serine, threonine and tyrosine Date: 2 Oct 1995 19:07:40 +0100 Lines: 43 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <44p9pc$7a8@mserv1.dl.ac.uk> Original-To: proteins@dl.ac.uk In bionet.molbio.proteins Msg # 5216 amikhail@btk.utu.fi (Andrey Mikhailow) wrote: >Hi, > >Does anyone know if the tBOC-derivates of phospho-serine, >phospho-threonine and phosphotyrosin are available commercialy? >At what supplier? > >Thanx for attention, >Andrey. As I said in a previous message I don't know if the Boc derivatives are available (or even if they were if they could be protected in a form stable/soluble enough for Boc-based synthesis chemistry) but I am not long back from a conference in Scotland where some people from Novabiochem presented a poster describing the use of monobenzylated Fmoc-phosphoserine, phosphothreonine and phosphotyrosine derivatives in solid-phase peptide synthesis. They said that the peptides are easy to assemble and deprotect, (normal TFA cleavage conditions) even with multiple phospho-residues. You may be able to find a supplier for Fmoc-Ser(PO(OBzl)OH)-OH, etc., as the synthesis was described in 1992 (Lacombe, J.M. et al, Int. J. Peptide Protein Res., 1992, 36, 275) or contact Novabiochem who probably sell/plan to sell these derivatives. Andrew |========================================================================| | Andrew Wallace | Discussion on phage display, | | | combinatorial libraries, etc. - | | IRBM P. Angeletti, | bionet.molecules.repertoires | | Via Pontina KM 30.600 | molreps@daresbury.ac.uk | | 00040 Pomezia, Italy. |-------------------------------------------| | | "It has not escaped our notice that | | Voice: +39-6-91093434 | the specific pairing we have postulated | | Fax: +39-6-91093225 | immediately suggests a possible copying | | Email: wallace@irbm.it | mechanism for the genetic material." | | | | | DISCLAIMER: I do not speak | J.D. Watson and F.H.C. Crick | | on behalf of anyone. | in Nature 171:737-737 (1953). | |========================================================================| From owner-proteins@net.bio.net Mon Oct 02 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!news.sprintlink.net!simtel!swidir.switch.ch!scsing.switch.ch!news.rediris.es!acebo.sdi.uam.es!newsmasters From: jayala@mvax.cbm.uam.es (Juan A. Ayala) Newsgroups: bionet.molbio.proteins Subject: Methyltransferase activity Date: 3 Oct 1995 09:33:19 GMT Organization: Centro de Biología Molecular Lines: 25 Message-ID: <44r00v$huc@acebo.sdi.uam.es> References: <1995Sep30.154731.1@icbr2.ifas.ufl.edu> <44osfs$ils@acebo.sdi.uam.es> NNTP-Posting-Host: b1011.cbm.uam.es Mime-Version: 1.0 Content-Type: Text/Plain; charset=US-ASCII X-Newsreader: WinVN 0.99.6 Dear nets: We have found new proteins in E.coli with unknown functions. One of them have been shown to be a putative methyltransferase by homology sequence analysis. Do you (some of you) know a defined assay in vitro for this enzymatic activity ? Thank a lot for your help ________________________________________________________________ : Juan A. Ayala : FAX-net: (+34-1)-397.83.44 : PSI-net: (2145)-2120423216 : INTERNET: JAYALA@mvax.cbm.uam.es : TELEX-net: 27810 educi e : PHONE-net: (+34-1)-397.80.83 : Snail-net: : : : ICBM-net: 40 deg 30 min North, 3 deg 42 min West :________________________________________________________________ :The Best Man for the Job is a Wo-man! :________________________________________________________________ From owner-proteins@net.bio.net Mon Oct 02 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!news.cac.psu.edu!news.math.psu.edu!psuvax1!news.eecs.nwu.edu!newsfeed.acns.nwu.edu!news.acns.nwu.edu!news From: alanhowe@merle.acns.nwu.edu (Alan K. Howe) Newsgroups: bionet.molbio.proteins Subject: Re: immunoprecipitation question Date: Mon, 02 Oct 95 17:19:02 EST Organization: Northwestern University, Chicago, IL. USA Lines: 50 Message-ID: <44po6r$en9@news.acns.nwu.edu> References: <44gush$hcs@acmex.gatech.edu> Reply-To: alanhowe@merle.acns.nwu.edu NNTP-Posting-Host: pc22.microbio.nwu.edu Mime-Version: 1.0 X-Newsreader: WinVN 0.93.9 In article <44gush$hcs@acmex.gatech.edu>, gt0332b@prism.gatech.edu says... > >I am currently trying to immunoprecipitate a protein with a molecular >weight >of 23 kDa. I assume the immunoprecipitation is complete however when >I try to >detect the protein of interest via western blotting the light chain Ig >G mask >the area of interest. I am using chemiluminescent detection procedure >s. I >have tried non-reducing conditions in my procedures however there stil >l appears >to be some light chains present. Are there any tricks to avoid the Ig >G bands >The same polyclonal antibody is used in both the IP and western blot. > >Any help or advice will be appreciated. > > >Robert I don't think the problem is light chain - most secondary antibodies that I know of are anti-Fc (the constant region), found only on the heavy chain, which should run between 45-60 kDa. However, if the 2' antibody you have is reacting with light chain, you'll have to cross-link your antibody to whatever collection resin (i.e. staph cells or A/G-agarose) you're using. You may also (as a quicker, but probably less effective method) try resuspending your washed immune complex in sample buffer that has plenty of SDS or urea, but no BME or DTT in it. Light chain/heavy chain interactions are through disulfides, mainly, so keeping them oxidized might help the IgG complex stay formed, but let your protein come off. Heck, maybe you should just use 35-S methionine... Good luck. -- Alan K. Howe Northwestern University, Chicago, IL. USA alanhowe@merle.acns.nwu.edu From owner-proteins@net.bio.net Mon Oct 02 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!news.sprintlink.net!news.dgsys.com!kpl From: Mblank@kpl.com (Martin Blankfard) Newsgroups: bionet.molbio.proteins Subject: Re: IEF standards? Date: 3 Oct 1995 20:52:20 GMT Organization: KPL Lines: 30 Message-ID: <44s7is$uto_002@dgs.dgsys.com> References: <1995Sep30.154731.1@icbr2.ifas.ufl.edu> NNTP-Posting-Host: kpl.com X-Newsreader: News Xpress Version 1.0 Beta #4 In article <1995Sep30.154731.1@icbr2.ifas.ufl.edu>, rep@icbr2.ifas.ufl.edu wrote: >Hello there, > >Has anyone found a good set of isoelectric focusing standards? Are there any >prestained IEF markers? I'm running slab gels, blotting them to PVDF and then >immunoblotting. Surface pH monitors have been suggested, but transferring that >data to the Western blot may cause problems. I would appreciate any >information. > >Robert Peterson >Whitney Lab. >St. Augustine, FL >REP@icbr.ifas.ufl.edu > I think NOVEX sells the best IEF standards as this point. I usually treat my IEF gel with SDS running buffer after running it ( bout 30 min.) then transfer my proteins under normal Western Blot conditions. This way all of the proteins transfer evenly. Once transferred to the PVDF or Nitrocellulose the proteins are easily visualized with Pancue stain. The standards will transfer to the membrane as well! Martin Blankfard mblank@kpl.com Director Diagonstics KPL 2 Cessna Court Gaithersburg MD. 20879 From owner-proteins@net.bio.net Mon Oct 02 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!news.sprintlink.net!news.dgsys.com!kpl From: Mblank@kpl.com (Martin Blankfard) Newsgroups: bionet.molbio.proteins Subject: Re: which good anti-mouse IgG light and heavy chain antibody? Date: 3 Oct 1995 21:00:09 GMT Organization: KPL Lines: 32 Message-ID: <44s818$uto_003@dgs.dgsys.com> References: NNTP-Posting-Host: kpl.com X-Newsreader: News Xpress Version 1.0 Beta #4 In article , regina@biotech.ufl.edu (Regina Shaw) wrote: >Dear all, >I have cloned some mouse light and heavy chain fragments into an >expression vector. We have verified that the gene is in-frame, but the >level of expression may be too low to see on a Coomassie-stained gel, so I >would like to do Western blotting and stain with a alkaline-phosphatase >anti-mouse IgG antibody that recognizes both light and heavy chain. I do >have a Sigma goat anti-mouse IgG Fab (AP) secondary antibody which stains >only the light chain fragment though. Does anybody know of a good >secondary antibody that stains both LC and HC? I would welcome all >suggestions. Thank you! Regina Regina... We have great anti mouse conjugates you could try. I would suggest our HRP conjugates with the membrane TMB for the most sensitive assay. However, if you really need AP conjugates we have them too. Our BCIP/NBT substrate for membranes is a great system as well. ------------------------------------- Martin Blankfard Senior Scientist Kirkegaard and Perry Laboratories 2 Cessna Ct. Gaithersburg Md , 20879 Phone: (US) 1-800-638-3167 x141 FAX : 301 948 9442 E-mail: Mblank@kpl.com 10/03/95 16:54:32 This message was sent by Chameleon ------------------------------------- From owner-proteins@net.bio.net Mon Oct 02 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!swrinde!howland.reston.ans.net!xlink.net!rz.uni-karlsruhe.de!news.uni-stuttgart.de!uni-regensburg.de!lrz-muenchen.de!fauern!winx03!wpxx02.toxi.uni-wuerzburg.de!krasel From: krasel@wpxx02.toxi.uni-wuerzburg.de (Cornelius Krasel) Newsgroups: bionet.molbio.proteins Subject: Re: Protein manual Date: 3 Oct 1995 14:14:28 GMT Organization: Dept. of Pharmacology, U Wuerzburg Lines: 23 Message-ID: <44rgg4$jdd@winx03.informatik.uni-wuerzburg.de> References: <44p8kc$3jl@news.inc.net> NNTP-Posting-Host: wpxx02.toxi.uni-wuerzburg.de X-Newsreader: TIN [version 1.2 PL2] Paul Boyer (boyer@cs.uwp.edu) wrote: > I'm looking for a manual/handbook to use as a text in a lab course called > "Working with Proteins". I'd like to find something like the Maniatis > bible for molecular biologists, but I can't find anything on the protein > level. Can anyone recommend anything? Wiley, who publish the "Red Book" (Current Protocols in Mol Bio) have a similar thing about proteins. I have not been able to look at it yet. Other fine books about protein purification (if this is what you are searching for) include a certain Meth. Enz. volume which is called "Guide to protein purification" and edited by Murray Deutscher (unfortunately I forgot the volume number, it *might* be 183) and "Protein Purification: A Practical Approach" from IRL press. Usual disclaimers apply. --Cornelius. -- /* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */ /* D-97078 Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de */ /* "Science is the game we play with God to find out what His rules are." */ From owner-proteins@net.bio.net Mon Oct 02 23:00:00 1995 Path: biosci!agate!lhom From: lhom@OCF.Berkeley.EDU (Louis Hom) Newsgroups: bionet.molbio.proteins Subject: Protein Dot Blots Date: 4 Oct 1995 00:03:20 GMT Organization: U. C. Berkeley Open Computing Facility Lines: 9 Message-ID: <44sj08$26o@agate.berkeley.edu> NNTP-Posting-Host: drought.berkeley.edu I need to isolate a viral clone, and it seems the best approach is to do an endpoint dilution followed by a dot blot to detect viral proteins. Does anyone know of a good protocol for protein dot blots? I will probably want to solubilize the viruses with SDS or something first. -- ______________________________________________________________________________ Lou Hom >K'93 lhom@ocf.berkeley.edu http://www.ocf.berkeley.edu/~lhom/ From owner-proteins@net.bio.net Tue Oct 03 23:00:00 1995 Path: biosci!UTSPH.SPH.UTH.TMC.EDU!gsbs1032 From: gsbs1032@UTSPH.SPH.UTH.TMC.EDU Newsgroups: bionet.molbio.proteins Subject: Seeking postdoctoral position in sequence analysis Date: 4 Oct 1995 13:10:58 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 12 Sender: daemon@net.bio.net Distribution: world Message-ID: <009975FC.BFE5A7B5.2848@utsph.sph.uth.tmc.edu> NNTP-Posting-Host: net.bio.net I am looking for a postdoctoral position in the US in sequence analysis, computational biology, phylogenetic analysis, and other areas of mathematical biology. The position is sought starting in the summer of 1996. I am a foreign student at the University of Texas at Houston with a strong background in sequence analysis, genetics and mathematical biology expecting to obtain a Ph.D. degree in May, 1996. Curriculum vitae, letters of reference, paper reprints and academic transcipts are available upon request. Please respond to gsbs1032@utsph.sph.uth.tmc.edu and sa95081@odin.mda.uth.tmc.edu From owner-proteins@net.bio.net Tue Oct 03 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!news.sprintlink.net!usenet.kornet.nm.kr!agate!spool.mu.edu!howland.reston.ans.net!swrinde!hookup!nstn.ns.ca!ac.dal.ca!ac.dal.ca!nntp Newsgroups: bionet.molbio.proteins Subject: Nuclear Protein Localization Message-ID: <1995Oct5.003028.41924@ac.dal.ca> From: Michael Nichols Date: 5 Oct 95 00:30:27 -0300 Nntp-Posting-Host: slip3.dal.ca X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) MIME-Version: 1.0 X-URL: news:bionet.molbio.proteins Content-Transfer-Encoding: 7bit Content-Type: text/plain; charset=us-ascii Lines: 10 Could someone please steer me toward recent work in nuclear localization of proteins. I am specifically interested in the cytosolic factors involved, and the nature of NLS and associated nucleosolic associated elements. Cheers, Michael Nichols. From owner-proteins@net.bio.net Tue Oct 03 23:00:00 1995 Path: biosci!rutgers!oitnews.harvard.edu!das-news2.harvard.edu!fas-news.harvard.edu!fas.harvard.edu!berriz From: Gabriel Berriz Newsgroups: bionet.molbio.proteins Subject: Re: Phage Display Date: 5 Oct 1995 00:31:26 GMT Organization: Harvard University, Cambridge, Massachusetts Lines: 8 Message-ID: <44v90u$i4b@decaxp.harvard.edu> References: <44ssnp$1sn@sundog.tiac.net> Reply-To: berriz@husc.harvard.edu NNTP-Posting-Host: fas.harvard.edu X-Newsreader: NN version 6.5.0 #3 (NOV) Originator: berriz@fas.harvard.edu dmccoy writes: >Does anyone know anything about phage display? The latest issue of Current Opinion in Structural Biology has a nice review of this technique. Gabriel From owner-proteins@net.bio.net Tue Oct 03 23:00:00 1995 Path: biosci!agate!usenet.kornet.nm.kr!news.kreonet.re.kr!sorak.kaist.ac.kr!kwkang From: kwkang@sorak.kaist.ac.kr (Kang Ke-Won) Newsgroups: bionet.molbio.proteins Subject: Purification Problem in HPLC HELP! Date: 5 Oct 1995 06:59:56 GMT Organization: Korea Research Environment Open Network (KREONet) Lines: 18 Message-ID: <44vvpc$dtb@news.kreonet.re.kr> NNTP-Posting-Host: sorak.kaist.ac.kr X-Newsreader: TIN [version 1.2 PL2] I am purifying a unidentified protein by HPLC. When this protein was eluted through reversed phase HPLC( vydac C18 ), the activity of this protein is detected on wide range of fractions. The buffers were 0.1% TFA in water, and 0.1% TFA in acetonitrile. I tried to elute this protein in neutral condition (pH 7.0), but the resolution was not good. What should I do? The molecular weight of this protein is about 7000Da, and it's pI value is about 4.0. Any comment and any suggestion will be a good help to me. =============================================================================== KAIST : Dept. of Biotech.: ph.D. course : Working on Protein in Leech. Name : Hong Seok Jin : (TEL) 82-42-869-5612, (FAX) 82-42-869-2610 E-Mail address = sjhong@bioneer.kaist.ac.kr wwwadm@bioneer.kaist.ac.kr kwkang@sorak.kaist.ac.kr =============================================================================== From owner-proteins@net.bio.net Tue Oct 03 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!usc!howland.reston.ans.net!swrinde!sdd.hp.com!hamblin.math.byu.edu!news.Arizona.EDU!usenet From: Soaring Bear Newsgroups: bionet.molbio.proteins Subject: Biosym's Apex QSAR program Date: 4 Oct 1995 23:22:08 GMT Organization: The University of Arizona Lines: 27 Message-ID: <44v4v0$pda@news.ccit.arizona.edu> NNTP-Posting-Host: basie.pharm.arizona.edu Mime-Version: 1.0 X-Mailer: Mozilla 1.1N (X11; I; IRIX 5.2 IP22) References: <009975FC.BFE5A7B5.2848@utsph.sph.uth.tmc.edu> X-URL: news:009975FC.BFE5A7B5.2848@utsph.sph.uth.tmc.edu Content-Transfer-Encoding: 7bit Content-Type: text/plain; charset=us-ascii Hello: Despite my posts to CCL & Dibug I have been unable to find a single satisfied user of Biosym's Apex QSAR software. I have received several emails from folks who tried and gave up. Is there anyone out there who is successfully using it? who has published? Please email me at: bear@ellington.pharm.arizona.edu thankyou -- _____ ____ * ___) * _ \ http://ellington.pharm.arizona.edu/~bear : (___ : |_) : Cyber-Chemist: cancer drug design topo-O O-topo \___ \ | _ < Molecular & Nutritional Biochemist 5'*. : : .***. ___) : | |_) : Herbs, Nutrition, Natural Dentistry | *. .* | | | (_____/oaring |____/ear@ellington.pharm.arizona.edu | | *. * | | | * UA New Pharmacy 404, Tucson 85721 3'.| DNA helix| * '***' '***' From owner-proteins@net.bio.net Tue Oct 03 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!lhc.nlm.nih.gov!nih-csl!helix.nih.gov!acmurphy From: "Angela c. Murphy" Subject: Re: peptide recovery in fractions?? In-Reply-To: <44uaom$rod@bubba.ucc.okstate.edu> Content-Type: TEXT/PLAIN; charset=US-ASCII Message-ID: Sender: postman@alw.nih.gov (AMDS Postmaster) Nntp-Posting-Host: helix.nih.gov Organization: National Institutes of Health References: <44uaom$rod@bubba.ucc.okstate.edu> Mime-Version: 1.0 Date: Wed, 4 Oct 1995 21:47:41 GMT Lines: 28 In a study done here at NIH by the late Dr. John Pisano's group, polypropylene tubes were found to be the best "non-stick" tubes. In certain cases, triton X-100 had to be added to each tube to keep the peptide from sticking. Siliconizing glass tubes sometimes works, but in general, I try to stay away from glass, especially when I have something that might react with silanols, or if I want to avoid seeing sodium adducts in the mass spec. analysis. On 4 Oct 1995 price@vms.ocom.okstate.edu wrote: > Are glass 13x100 tubes appropriate for fraction collection generally, or > are there obvious predictable situations (other than post facto 0% yield) > when passivation or plastic tubes would predictably be a better choice? > What are they and what passivation if any do you suggest? > > Do the glass tubes need any other pretreatment before use? > > Thanks; email would be helpful and the most reliable reply mode. > > -- > Joseph A. Price, Ph.D PRICE@VMS.OCOM.OKSTATE.EDU > Ph 918 561-8231 FAX 918 561 8412 > Dept. Biochem. & Micro. > COM-OSU 1111 W.17th. St. > Tulsa OK 74107 > > > From owner-proteins@net.bio.net Tue Oct 03 23:00:00 1995 Path: biosci!bcm.tmc.edu!news.tamu.edu!newshost.comco.com!news.texas.net!news.sprintlink.net!simtel!swidir.switch.ch!scsing.switch.ch!news.belwue.de!fu-berlin.de!zrz.TU-Berlin.DE!cs.tu-berlin.de!fauern!winx03!wpxx02.toxi.uni-wuerzburg.de!krasel From: krasel@wpxx02.toxi.uni-wuerzburg.de (Cornelius Krasel) Newsgroups: bionet.molbio.proteins Subject: Re: Meaning of "B" and "Z" in Swiss-Prot and others Date: 4 Oct 1995 17:02:39 GMT Organization: Dept. of Pharmacology, U Wuerzburg Lines: 14 Message-ID: <44uenf$gaa@winx03.informatik.uni-wuerzburg.de> References: NNTP-Posting-Host: wpxx02.toxi.uni-wuerzburg.de X-Newsreader: TIN [version 1.2 PL2] Matt Parker (at332@FreeNet.Carleton.CA) wrote: > I have been doing protein sequence alignments using the GCG > package. Occasionally, I get a B or a Z listed as a residue. I was > wondering if anybody knows what these are. I suspect that they are asx and > glx. Right. (At least that's what I remember as well :-) --Cornelius. -- /* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */ /* D-97078 Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de */ /* "Science is the game we play with God to find out what His rules are." */ From owner-proteins@net.bio.net Tue Oct 03 23:00:00 1995 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!elroy.jpl.nasa.gov!lll-winken.llnl.gov!enews.sgi.com!decwrl!tribune.usask.ca!duke.usask.ca!prabhuv From: prabhuv@duke.usask.ca (Vikram Prabhu) Newsgroups: bionet.molbio.proteins Subject: Re: Protein manual Date: 4 Oct 1995 19:07:42 GMT Organization: University of Saskatchewan Lines: 17 Message-ID: <44um1u$emp@tribune.usask.ca> References: <44p8kc$3jl@news.inc.net> <44rgg4$jdd@winx03.informatik.uni-wuerzburg.de> NNTP-Posting-Host: duke.usask.ca X-Newsreader: TIN [version 1.2 PL2] : Other fine books about protein purification (if this is what you are : searching for) include a certain Meth. Enz. volume which is called : "Guide to protein purification" and edited by Murray Deutscher : (unfortunately I forgot the volume number, it *might* be 183) and : "Protein Purification: A Practical Approach" from IRL press. The Methods in Enzymology vol referred to above is #182. The classic Robert Scopes 1982 Protein Purification is also invaluable. Another useful book is Bollag and Edelstein's "Protein Methods". -------------------- Vik Prabhu Biology Univ of Saskatchewan Saskatoon. ___________________________________________________________________ From owner-proteins@net.bio.net Tue Oct 03 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!gatech!newsxfer.itd.umich.edu!tank.news.pipex.net!pipex!dispatch.news.demon.net!demon!sunsite.doc.ic.ac.uk!daresbury!not-for-mail From: "Andrew, Tel. +396-91093434" Newsgroups: bionet.molbio.proteins Subject: Repost on how to subscribe to molreps Date: 4 Oct 1995 18:32:12 +0100 Lines: 201 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <44uges$c11@mserv1.dl.ac.uk> Original-To: proteins@dl.ac.uk First off, let me apologise for posting this here. Peter, mail to the address given in your message bounces back to me, so in order to let you see this info I'll have to repost it in the proteins newsgroup. Andrew ------ In Message-Id: <9510041412.AA74160@umabnet.ab.umd.edu> pemanuel@umabnet.ab.edu (Peter) wrote: >My newsgroup does not subscribe to this newsgroup. I do have TELNET and >Netscape capability. Is there another way I can gain access to this >newsgroup? > Peter > >-- >Peter Emanuel Ph.D. >University of Maryland School of Medicine >4-027 Bressler Research Building >655 West Baltimore Street >Baltimore, MD 21201 Here is some information which may help if you are having trouble getting the bionet.molecules.repertoires (molreps@net.bio.net) group. Andrew ---------------------------MOLECULAR-REPERTOIRES------------------------- Subscribing to this group: -------------------------- IF YOU USE USENET NEWS: you need do nothing other than participate in bionet.molecules.repertoires when it appears in your newsreader. Depending upon your news software, this may entail you having to answer a prompt indicating that you want to subscribe. You might also try the command "g bionet.molecules.repertoires" in rn-like newsreaders. IF YOU USE EMAIL AND ARE LOCATED IN EUROPE, AFRICA, OR CENTRAL ASIA: please first be sure that you are sending mail from the address at which you wish to receive news postings, and then send a mail message to the Internet address MXT@dl.ac.uk Leave the Subject: line of the message blank and enter the following line into the body of the mail message: SUB bionet-news.bionet.molecules.repertoires This message will be automatically read by the computer and your e-mail address will be extracted from the mail header and added to the list. IF YOU USE EMAIL AND ARE LOCATED IN THE AMERICAS OR THE PACIFIC RIM: log in to the computer account in which you would like to receive mail (not an account that you use infrequently) and send a mail message to the Internet address biosci-server@net.bio.net Leave the Subject: line of the message blank and enter the following line into the body of the mail message: subscribe molreps This message will be automatically read by our computer and your e-mail address will be extracted from the mail header and added to the list. How to post a message to the group: ----------------------------------- If you use news, simply post a message into bionet.molecules.repertoires. Be sure to set your "distribution" to "world" or else the message might not leave your site!! 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Once again, if you have any administrative questions that require personal assistance, please address them to biosci-help@net.bio.net in the U.S. or biosci@daresbury.ac.uk in the UK. Best wishes for a successful newsgroup! Sincerely, Dave Kristofferson BIOSCI/bionet Manager biosci-help@net.bio.net From owner-proteins@net.bio.net Tue Oct 03 23:00:00 1995 Path: biosci!biosci!not-for-mail From: jmoult@mbisgi.umd.edu (Dr. John Moult) Newsgroups: bionet.molbio.proteins,bionet.jobs.offered Subject: JOB ADVERTISEMENT: PROTEIN STRUCTURE PREDICTION ON THE WEB Date: 4 Oct 1995 17:14:21 -0700 Organization: University of Maryland, College Park Lines: 39 Sender: biohelp@net.bio.net Approved: biojobs-moderator@net.bio.net Distribution: world Message-ID: <44v4bh$cb7@hecate.umd.edu> NNTP-Posting-Host: net.bio.net Xref: biosci bionet.molbio.proteins:5841 bionet.jobs.offered:318 PROTEIN STRUCTURE PREDICTION ON THE WEB Two postdoctoral positions are available immediately to establish a structure prediction database and conduct research in the field of protein sturcture prediction. Following last years's 'Asilomar' experiment in protein structure prediction (Nature, Structural Biology, Vol 2, pages 91-93, 1995), a web based structure prediction facility is being established. The facility will maintain a set of blind prediction targets, collect predictions, establish methods of evaluating predictions and benchmark sets of prediction examples. It will also provide a means of exchange of algorithms, potentials, test cases and other data between researchers in the field. Web and other internet technologies will be exploited to the full, with the goal of finding new ways of interaction and communication to speed development of the prediction field. One position will focus on evaluation of prediction methods and requires a background in protein structure analysis. The second position will focus on the development of tools for structure prediction and analysis as well as web facilities. A strong background in computational and web tool development is required. Work on the facility will be centered at the Lawrence Livermore National Laboratory in Livermore, California. Postdoctoral members will be Research Associates of the Center for Advanced Research in Biotechnology (CARB), University of Maryland Biotechnology Instutute. The University of Maryland is an equal opportunity employer. Send CVs, other material and queries as soon as possible to: jmoult@indigo5.carb.nist.gov (John Moult, CARB) or kaf@sb1.llnl.gov (Krzysztof Fidelis, LLNL) From owner-proteins@net.bio.net Tue Oct 03 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!news.sprintlink.net!qns3.qns.com!news.ecn.uoknor.edu!bubba.ucc.okstate.edu!news From: price@vms.ocom.okstate.edu (Joseph A. Price, Ph.D.) Newsgroups: bionet.molbio.proteins Subject: peptide recovery in fractions?? Date: 4 Oct 1995 15:55:02 GMT Organization: COM-OSU Lines: 16 Message-ID: <44uaom$rod@bubba.ucc.okstate.edu> NNTP-Posting-Host: biochem1.ocom.okstate.edu Mime-Version: 1.0 X-Newsreader: WinVN 0.93.14 Are glass 13x100 tubes appropriate for fraction collection generally, or are there obvious predictable situations (other than post facto 0% yield) when passivation or plastic tubes would predictably be a better choice? What are they and what passivation if any do you suggest? Do the glass tubes need any other pretreatment before use? Thanks; email would be helpful and the most reliable reply mode. -- Joseph A. Price, Ph.D PRICE@VMS.OCOM.OKSTATE.EDU Ph 918 561-8231 FAX 918 561 8412 Dept. Biochem. & Micro. COM-OSU 1111 W.17th. St. Tulsa OK 74107 From owner-proteins@net.bio.net Tue Oct 03 23:00:00 1995 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!cs.utk.edu!gatech!news.sprintlink.net!in2.uu.net!tank.news.pipex.net!pipex!usenet.eel.ufl.edu!news.gmi.edu!zombie.ncsc.mil!cs.umd.edu!haven.umd.edu!umabnet.ab.umd.edu!beaker.ab.umd.edu!user From: pemanuel@umabnet.ab.edu (Peter) Newsgroups: bionet.molbio.proteins Subject: Stability of recom. Fabs? Date: Wed, 04 Oct 1995 10:14:33 -0500 Organization: Bio4 Lines: 13 Message-ID: NNTP-Posting-Host: beaker.ab.umd.edu Has anyone noticed that recombinant Fab antibodies are more or less stable than whole immunoglobulin? I have produced soluble Fabs which are very active during the first use but following a freeze thaw they lose much of their activity. What can be done to avoid this. Peter -- Peter Emanuel Ph.D. University of Maryland School of Medicine 4-027 Bressler Research Building 655 West Baltimore Street Baltimore, MD 21201 From owner-proteins@net.bio.net Tue Oct 03 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!news.sprintlink.net!news00.sunet.se!sunic!mn6.swip.net!plug.news.pipex.net!pipex!tank.news.pipex.net!pipex!dispatch.news.demon.net!demon!sunsite.doc.ic.ac.uk!yama.mcc.ac.uk!news.york.ac.uk!news From: Rana Lee Newsgroups: bionet.molbio.proteins Subject: (no subject) Date: 4 Oct 1995 10:00:32 GMT Organization: University of York, Protein Structure Lines: 5 Message-ID: <44tm00$1c3@netty.york.ac.uk> NNTP-Posting-Host: doc.chem.york.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (X11; I; IRIX 5.2 IP12) X-URL: news:bionet.molbio.proteins does anyone have any information on hydroxyradical footprinting? Any information would be greatfully appreciated. Thanking you. Rana. From owner-proteins@net.bio.net Tue Oct 03 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!swrinde!tank.news.pipex.net!pipex!oleane!jussieu.fr!centre.univ-orleans.fr!univ-lyon1.fr!in2p3.fr!swidir.switch.ch!scsing.switch.ch!news.rediris.es!acebo.sdi.uam.es!newsmasters From: Miguel Angel Andrade Newsgroups: bionet.molbio.proteins Subject: Is there any collection of Protein CD spectra? Date: 4 Oct 1995 12:12:24 GMT Organization: Universidad Autonoma de Madrid, Spain Lines: 8 Message-ID: <44ttn8$28q@acebo.sdi.uam.es> NNTP-Posting-Host: gredos.cnb.uam.es Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (X11; I; IRIX 5.2 IP22) To: k3d@kal-el.ugr.es X-URL: news:bionet.molbio.proteins Does anybody know a recent reference or a database of CD spectra of proteins of known 3D structure? Thanks Miguel A. Andrade CNB, CSIC, Madrid (Spain) From owner-proteins@net.bio.net Tue Oct 03 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!plug.news.pipex.net!pipex!tank.news.pipex.net!pipex!dispatch.news.demon.net!demon!sunsite.doc.ic.ac.uk!daresbury!not-for-mail From: "Andrew, Tel. +396-91093434" Newsgroups: bionet.molbio.proteins Subject: Re: footprinting Date: 4 Oct 1995 13:02:07 +0100 Lines: 41 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <44tt3v$mb1@mserv1.dl.ac.uk> Original-To: proteins@dl.ac.uk In bionet.molbio.proteins Msg # 5382 Rana Lee wrote: >does anyone have any information on hydroxyradical footprinting? >Any information would be greatfully appreciated. >Thanking you. >Rana. You might try looking for papers by the group of Thomas D. Tullius of the Chemistry Department in Johns Hopkins University, Baltimore who was one of the first to describe the use of hydroxyl radicals for studying protein-DNA interactions (in vitro footprinting). One to get you started is: Tullius, T.D. and Dombrowski, B.A. (1986) Proc. Natl. Acad. Sci. USA 83, 5469-5473. He and his coworkers also wrote a chapter in a practical guide to this and other techniques which was published in: "A Laboratory Giude to In Vitro Studies of Protein-DNA Interactions", vol. 5 of the BioMethods series from Birkhaeuser, Basel, eds. J.P. Jost and H.P. Saluz. Hope this helps, Andrew |========================================================================| | Andrew Wallace | Discussion on phage display, | | | combinatorial libraries, etc. - | | IRBM P. Angeletti, | bionet.molecules.repertoires | | Via Pontina KM 30.600 | molreps@daresbury.ac.uk | | 00040 Pomezia, Italy. |-------------------------------------------| | | "It has not escaped our notice that | | Voice: +39-6-91093434 | the specific pairing we have postulated | | Fax: +39-6-91093225 | immediately suggests a possible copying | | Email: wallace@irbm.it | mechanism for the genetic material." | | | | | DISCLAIMER: I do not speak | J.D. Watson and F.H.C. Crick | | on behalf of anyone. | in Nature 171:737-737 (1953). | |========================================================================| From owner-proteins@net.bio.net Tue Oct 03 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!news.sprintlink.net!dispatch.news.demon.net!demon!sunsite.doc.ic.ac.uk!daresbury!not-for-mail From: "Andrew, Tel. +396-91093434" Newsgroups: bionet.molbio.proteins Subject: Re: Meaning of "B" and "Z" in Swiss-Prot and others Date: 4 Oct 1995 12:35:48 +0100 Lines: 139 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <44trik$l12@mserv1.dl.ac.uk> Original-To: proteins@dl.ac.uk In bionet.molbio.proteins Msg # 5376 at332@FreeNet.Carleton.CA (Matt Parker) wrote: >I have been doing protein sequence alignments using the GCG package. >Occasionally, I get a B or a Z listed as a residue. I was wondering if >anybody knows what these are. I suspect that they are asx and glx. > > Thanks, > > Matthew Parker Matthew, The amino acid sequence symbols B and Z used by the GCG package do, as you suspected, mean Asx and Glx, respectively. Here is the complete list of all sequence symbols (nucleotide and amino acid) used by the GCG package as listed in Appendix III of the manual (can also be listed via the online help). Appendix_III SEQUENCE SYMBOLS GCG programs allow all upper- and lowercase letters, periods (.), asterisks (*), pluses (+), ampersands (&), and ats (@) as symbols in biological sequences. Nucleotide symbols, their complements, and the standard one-letter amino acid symbols are shown below in separate lists. The meanings of the symbols +, &, and @ have not been assigned at this writing (March, 1989). GCG uses the letter codes for amino acid codes and nucleotide ambiguity proposed by IUB (Nomenclature Committee, 1985, Eur. J. Biochem. 150; 1-5). These codes are compatible with the codes used by the EMBL, GenBank, and PIR data libraries. NUCLEOTIDES The meaning of each symbol, its complement, and the Cambridge equivalents are shown below. Cambridge files can be converted into GCG files and vice versa with the programs FromStaden and ToStaden. IUB/GCG Meaning Complement Staden/Sanger A A T A C C G C G G C G T/U T A T M A or C K 5 R A or G Y R W A or T W 7 S C or G S 8 Y C or T R Y K G or T M 6 V A or C or G B not supported H A or C or T D not supported D A or G or T H not supported B C or G or T V not supported X/N G or A or T or C X -/X . not G or A or T or C . not supported The uncertainty and frame ambiguity codes used by Staden are not supported by GCG and are converted by FromStaden as the lowercase single base equivalent. Staden Code Meaning GCG 1 probably C c 2 probably T t 3 probably A a 4 probably G g D C or CC c V T or TT t B A or AA a H G or GG g K C or CX c L T or TX t M A or AX a N G or GX g AMINO ACIDS Here is a list of the standard one-letter amino acid codes and their three-letter equivalents. The synonymous codons and their depiction in the IUB codes are shown. You should recognize that the codons following semicolons (;) are not sufficiently specific to define a single amino acid even though they represent the best possible backtranslation into the IUB codes! All of the relationships in this list can be redefined by you in a local data file, as described below. IUB Symbol 3-letter Meaning Codons Depiction A Ala Alanine GCT,GCC,GCA,GCG !GCX B Asp,Asn Aspartic, Asparagine GAT,GAC,AAT,AAC !RAY C Cys Cysteine TGT,TGC !TGY D Asp Aspartic GAT,GAC !GAY E Glu Glutamic GAA,GAG !GAR F Phe Phenylalanine TTT,TTC !TTY G Gly Glycine GGT,GGC,GGA,GGG !GGX H His Histidine CAT,CAC !CAY I Ile Isoleucine ATT,ATC,ATA !ATH K Lys Lysine AAA,AAG !AAR L Leu Leucine TTG,TTA,CTT,CTC,CTA,CTG !TTR,CTX,YTR;YTX M Met Methionine ATG !ATG N Asn Asparagine AAT,AAC !AAY P Pro Proline CCT,CCC,CCA,CCG !CCX Q Gln Glutamine CAA,CAG !CAR R Arg Arginine CGT,CGC,CGA,CGG,AGA,AGG !CGX,AGR,MGR;MGX S Ser Serine TCT,TCC,TCA,TCG,AGT,AGC !TCX,AGY;WSX T Thr Threonine ACT,ACC,ACA,ACG !ACX V Val Valine GTT,GTC,GTA,GTG !GTX W Trp Tryptophan TGG !TGG X Xxx Unknown !XXX Y Tyr Tyrosine TAT, TAC !TAY Z Glu,Gln Glutamic, Glutamine GAA,GAG,CAA,CAG !SAR * End Terminator TAA, TAG, TGA !TAR,TRA;TRR -------------------------------------------------------------------------- Hope you find this useful. |========================================================================| | Andrew Wallace | Discussion on phage display, | | | combinatorial libraries, etc. - | | IRBM P. Angeletti, | bionet.molecules.repertoires | | Via Pontina KM 30.600 | molreps@daresbury.ac.uk | | 00040 Pomezia, Italy. |-------------------------------------------| | | "It has not escaped our notice that | | Voice: +39-6-91093434 | the specific pairing we have postulated | | Fax: +39-6-91093225 | immediately suggests a possible copying | | Email: wallace@irbm.it | mechanism for the genetic material." | | | | | DISCLAIMER: I do not speak | J.D. Watson and F.H.C. Crick | | on behalf of anyone. | in Nature 171:737-737 (1953). | |========================================================================| From owner-proteins@net.bio.net Tue Oct 03 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!news.sprintlink.net!dispatch.news.demon.net!demon!sunsite.doc.ic.ac.uk!daresbury!not-for-mail From: "Andrew, Tel. +396-91093434" Newsgroups: bionet.molbio.proteins Subject: Re: phage display Date: 4 Oct 1995 12:28:11 +0100 Lines: 31 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <44tr4b$knk@mserv1.dl.ac.uk> Original-To: proteins@dl.ac.uk In bionet.molbio.proteins Msg # 5379 dmccoy@tiac.net wrote: > >Does anyone know anything about phage display? Is there a news group about >phage display? > Yes, there is a newsgroup about phage display, combinatorial libraries and molecular diversity in general which you can find on: bionet.molecules.repertoires Many people who are experts in the field of phage display participate in that newsgroup so if you have any questions you can ask them there. Andrew |========================================================================| | Andrew Wallace | Discussion on phage display, | | | combinatorial libraries, etc. - | | IRBM P. Angeletti, | bionet.molecules.repertoires | | Via Pontina KM 30.600 | molreps@daresbury.ac.uk | | 00040 Pomezia, Italy. |-------------------------------------------| | | "It has not escaped our notice that | | Voice: +39-6-91093434 | the specific pairing we have postulated | | Fax: +39-6-91093225 | immediately suggests a possible copying | | Email: wallace@irbm.it | mechanism for the genetic material." | | | | | DISCLAIMER: I do not speak | J.D. Watson and F.H.C. Crick | | on behalf of anyone. | in Nature 171:737-737 (1953). | |========================================================================| From owner-proteins@net.bio.net Tue Oct 03 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!news.sprintlink.net!news00.sunet.se!sunic!mn6.swip.net!plug.news.pipex.net!pipex!tank.news.pipex.net!pipex!dispatch.news.demon.net!demon!sunsite.doc.ic.ac.uk!yama.mcc.ac.uk!news.york.ac.uk!news From: Rana Lee Newsgroups: bionet.molbio.proteins Subject: footprinting Date: 4 Oct 1995 10:03:50 GMT Organization: University of York, Protein Structure Lines: 5 Message-ID: <44tm66$1c3@netty.york.ac.uk> NNTP-Posting-Host: doc.chem.york.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (X11; I; IRIX 5.2 IP12) X-URL: news:bionet.molbio.proteins#44tm00$1c3@netty.york.ac.uk does anyone have any information on hydroxyradical footprinting? Any information would be greatfully appreciated. Thanking you. Rana. From owner-proteins@net.bio.net Tue Oct 03 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!news5.ner.bbnplanet.net!news3.near.net!yale!usenet From: elena Newsgroups: bionet.molbio.proteins Subject: IgYs and Affi-Gel Hz Date: 4 Oct 1995 00:34:10 GMT Organization: Univ. Connecticut Lines: 16 Message-ID: <44skq2$3nl@babyblue.cs.yale.edu> NNTP-Posting-Host: jpg.mcb.uconn.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) I'm a gradstudent dealing with immunopurification of proteins and I have a question for this newsgroup: Has anyone tried to couple extracted IgYs from eggyolk to Affi-Gel Hz (Biorad)? Any comments, suggestions, pros and cons are highly appreciated. Thanks for your help! Elena Hilario Dept. Mol. Cell Biology Univ. Connecticut Storrs CT 06269-3044 From owner-proteins@net.bio.net Tue Oct 03 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!tank.news.pipex.net!pipex!sunsite.doc.ic.ac.uk!charlie.lif.icnet.uk!usenet From: "Robert B. Russell" Newsgroups: bionet.molbio.proteins,bionet.microbiology,bionet.general Subject: Re: circular permutation of proteins in nature Date: 29 Sep 1995 15:28:27 GMT Organization: Imperial Cancer Research Fund Lines: 60 Message-ID: <44h3ar$43t@charlie.lif.icnet.uk> References: <44gq3e$1j6q@sat.ipp-garching.mpg.de> NNTP-Posting-Host: bonsai.lif.icnet.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (X11; I; IRIX 5.2 IP22) X-URL: news:44gq3e$1j6q@sat.ipp-garching.mpg.de Xref: biosci bionet.molbio.proteins:5825 bionet.microbiology:3475 bionet.general:17546 Dr Lupas, Your posting was very interesting. It would appear that this is another example. I would say that your example was *more* convincing than that of Heinemann and Hahn's, since the sequence motif clearly conserved in the permuted sequence. Are the DNA sequences known? I would assume so, given the location of the papers. We (that's Ponting and Russell) commented that circular permutations within proteins would be aided by the occurence of tandem repeats, since it is easier to extract a single stretch of DNA/protein than to take one piece out and move it to another location. In other words, it requires fewer steps. One could apply this thinking to your example, viz. one SLH repeat is: H1 = helix 1, S= strand, H2 = helix 2 (by your prediction): Consider a hypothetical ancestor of SLH-type domains that contains *four* repeats of helix-strand-helix (labelled a-d): H1a-Sa-H2a--H1b-Sb-H2b--H1c-Sc-H2c--H1d-Sd-H2d (--Omp----) (-----------Slp--------) (----------------XynX-------------) (--------------OlpB-----------------) One could arrive at "homologues" of the four proteins you discussed by merely cutting the ancestral protein as shown above, and obviously adding bits of sequence and mutating as appropriate. For the evolution of OlpB, this would avoid having to, for example, cut off the first helix and stick it on the end: one would only need to take one section out of the ancestral protein as shown. I was particularly intrigued by Heinmann and Hahn's comment that traditional methods of sequence comparison might not be able to detect permutations within the database. Who knows how many of these things are out there and un-discovered? And one thing that strikes me is how permutations can go un-noticed, or at least not spotted as "circular permutations" explictly. I think this applied to the Heinmann and Hahn example, since the original paper showed in a Figure that the sequence was "permuted", but the word "circular permutation" never appeared (to the best of my knowledge) in the manuscript. The description was "only in F. succinogenes the order of these domains is reversed" (Eur. J. Biochem, 204, 3, 13-19, 1992). Again, one wonders how many more examples there are. To all other molbio.proteins readers, I ask for a general discussion of the meaning and significance of these things within nature. I would ask: a) how do they fold? b) what functional significance do they have, and c) does anyone know how many of these things are out there? Yours, etc. Robert B. Russell Biomolecular Modelling, Imperial Cancer Research Fund 44 Lincoln's Inn Fields, P.O. Box 123, London, WC2A 3PX, U.K. Tel: 44 171 269 3583 FAX: 44 171 269 3479 e-mail: russell@icrf.icnet.uk WWW http://bonsai.lif.icnet.uk/people/rob/rob.html From owner-proteins@net.bio.net Tue Oct 03 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!news.sprintlink.net!sundog.tiac.net!usenet From: dmccoy Newsgroups: bionet.molbio.proteins Subject: Phage Display Date: 4 Oct 1995 02:49:29 GMT Organization: The Internet Access Company Lines: 1 Message-ID: <44ssnp$1sn@sundog.tiac.net> NNTP-Posting-Host: dmccoy.tiac.net Does anyone know anything about phage display? Is there a news group about phage display? From owner-proteins@net.bio.net Tue Oct 03 23:00:00 1995 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!sol.ctr.columbia.edu!spool.mu.edu!daily-planet.execpc.com!news.moneng.mei.com!bloom-beacon.mit.edu!newsserver.pixel.kodak.com!news.sprintlink.net!tank.news.pipex.net!pipex!oleane!jussieu.fr!unilim.fr!cict.fr!news From: baudouin@cix.cict.fr (baudouin) Newsgroups: bionet.molbio.proteins Subject: problem with immunoaffinity column Date: 4 Oct 1995 17:04:55 GMT Organization: Univ. P. Sabatier/CNRS URA 1457 Lines: 14 Message-ID: <44uern$qnf@news.cict.fr> NNTP-Posting-Host: 130.120.42.14 X-Posted-From: InterNews 1.0.5@130.120.42.14 X-Authenticated: baudouin on POP host cix.cict.fr Hi netters, I would like to study the composition of a multimeric complex. The strategy I'd plan to use is separating my complex from crude MonoQ fraction using antibodies linked to Affigel Hz -which is supposed to provide the best recognition according to the fact that IgG are orientated-. My problem is that during the elution (pre-elution buffer : Tris 0,01 M pH 7,5 ; elution buffer : glycine 0,1 M pH 2,5) I have a leak of both light and heavy chains of immunoglobulin even after a pre-run to get rid of non covalently linked IgG. So, the question is how to get rid of the leak. I've carried the IgG fixation as told in the instruction manual ; I've also tried mild elution conditions but I wasn't able in this case to get back the protein. Thank you for suggestions Emmanuel From owner-proteins@net.bio.net Wed Oct 04 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!news.sprintlink.net!aimnet.com!news2.aimnet.com!usenet From: xyzzyx@aimnet.com (xyzzyx) Newsgroups: uchi.bio,uchi.general,bionet.molbio.proteins,bionet.molbio.methds-reagnts Subject: Re: Mol Biologists, do any of you use precast gels? Date: 5 Oct 1995 05:55:29 GMT Organization: xyzzyx consultants Lines: 42 Message-ID: <44vs0h$qv2@news2.aimnet.com> References: <44prje$f4a@news.inc.net> NNTP-Posting-Host: dial-bel1-23.iway.aimnet.com Mime-Version: 1.0 Content-Type: Text/Plain; charset=ISO-8859-1 X-Newsreader: WinVN 0.99.5 Xref: biosci bionet.molbio.proteins:5845 bionet.molbio.methds-reagnts:34465 In article <44prje$f4a@news.inc.net>, boyer@cs.uwp.edu says... > >I have a lot of experience with Novex' PAGE gels. I love them, although >they're a bit pricey. I've never had a problem with them, and all you do >is rip open the bag, pull the comb, and run. I can't rave enough about >them. I only wish budgets these days allowed for them on a regular >basis! > >-- > >************************************************************************* >Paul D. Boyer, Ph.D. 900 Wood Road, Box 2000 >Assistant Professor Kenosha, WI 53141-2000 >Department of Biological Sciences pboyer@cs.uwp.edu >University of Wisconsin-Parkside >************************************************************************* > > I too, swear by the Novex gels, but, I also found them expensive. In my last lab, we were running something like 50-100 mini-gels a week. Our solution was a sort of compromise. Novex sells empty cassettes, which you can seal with lab tape. They also sell combs that fit the cassettes (in many different comb numbers) including a "preparative/2nd dimension for IEF" type comb. The latter were great for 2-D electrophoresis and also for semi preparative isolation of single bands for in-situ Edman degradation and protease digestion. We would pre-cast 50-100 gels in a sitting and store them in the refrigerator (with the combs in place) in a ziplock bag with a soaked, wadded up paper towel to keep them moist. They were good for at least two weeks. This gave us a lot of flexibility to concoct new gel formulations to suit our needs. We also ran narrow range IEF in these cassettes by contriving our own ampholyte mixes. (BTW they sell precast IEF gels too). They also sell a Tricine gel and various DNA gels. Once I even ran a thin slab agarose gel just to see if it worked (it did). Finally, They sell a nice semi-dry blot unit that I swear by. I don't work for them, I just like their products. George Trager Matrix Pharmaceuticals, Inc. From owner-proteins@net.bio.net Wed Oct 04 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!swrinde!sdd.hp.com!night.primate.wisc.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!chronos.genetics.wisc.edu!user From: guy@genetics.wisc.edu (Guy Plunkett III) Newsgroups: bionet.molbio.proteins Subject: Re: Meaning of "B" and "Z" in Swiss-Prot and others Date: Thu, 05 Oct 1995 10:24:53 -0600 Organization: University of Wisconsin Lines: 24 Distribution: bionet Message-ID: References: <44trik$l12@mserv1.dl.ac.uk> NNTP-Posting-Host: chronos.genetics.wisc.edu X-Newsreader: Yet Another NewsWatcher 2.0.2 Andrew Wallace posted a summary of the single-letter codes for both bases and amino acids. For those interested in such things, the reference is: Nomenclature Committee of the International Union of Biochemistry (NC-IUB) (1985) Nomenclature for incompletely specified bases in nucleic acid sequences. European Journal of Biochemistry 150, 1-5. My question: I seem to recall that an update was proposed, assigning a letter to represent selenocysteine. Does anyone have the reference for that assignment, or know what the letter assignment was? Thanks, GUY -- Dr.Guy Plunkett III for the E. coli Genome Project Laboratory of Genetics University of Wisconsin Phone: (608) 262-2534 445 Henry Mall Fax: (608) 263-7459 Madison, WI 53706 Internet: guy@genetics.wisc.edu "Life is too short, and DNA too long." -- Michael Crichton, Jurassic Park From owner-proteins@net.bio.net Wed Oct 04 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!plug.news.pipex.net!pipex!dish.news.pipex.net!pipex!tank.news.pipex.net!pipex!oleane!jussieu.fr!univ-lyon1.fr!in2p3.fr!swidir.switch.ch!newsfeed.ACO.net!sbg.ac.at!news From: Anton Hutticher Newsgroups: uchi.bio,uchi.general,bionet.molbio.proteins,bionet.molbio.methds-reagnts Subject: Re: Mol Biologists, do any of you use precast gels? Date: 5 Oct 1995 18:05:32 GMT Organization: Computing Services, University of Salzburg Lines: 27 Message-ID: <4516pc$k6@dwst13.edvz.sbg.ac.at> References: NNTP-Posting-Host: dzoo15.zoo.sbg.ac.at Xref: biosci bionet.molbio.proteins:5848 bionet.molbio.methds-reagnts:34496 mjfath@ellis.uchicago.edu (Michael J. Fath) wrote: > > Hi, > > I was wondering if anyone here uses precast gels for PAGE > or even for agarose gels. (snip) We compared NOVEX precast PAGE gels with our gels. The resolution was quite good but not as good as our own specially crafted gradient gels. However, they were very easy to setup and use. Several people switched to NOVEX, other stayed with their own gels. If you do PAGE only sporadically, precasts are probably a good solution. Take care if you want to do western blots with precasts. Those from NOVEX we used could not stand the blotting conditions we used ( 1 l MeOH, 4,1 g NaHCO3, 1,6 g Na2CO3 ), 40 V, 3 h on Biorad Mini. They fragmented during the run and the fragments were very hard to peel off the nitrocellulose. We have not tested them under any other conditions! Maybe you find blotting conditions which work for them, but test them beforehand! Anton Hutticher (huttich@edvz.sbg.ac.at) (Uni Salzburg / Austria) From owner-proteins@net.bio.net Wed Oct 04 23:00:00 1995 Path: biosci!botany.uq.edu.au!J.Marcus From: J.Marcus@botany.uq.edu.au ("Marcus, Dr J.") Newsgroups: bionet.molbio.proteins Subject: Re: Purification Problem in HPLC HELP! Date: 5 Oct 1995 19:45:34 -0700 Organization: Dept of Botany, Univ of Qld Lines: 54 Sender: daemon@net.bio.net Distribution: world Message-ID: <7E25C090820@botany.uq.edu.au> NNTP-Posting-Host: net.bio.net kwkang@sorak.kaist.ac.kr (Kang Ke-Won) wrote: > I am purifying a unidentified protein by HPLC. When this protein was eluted > through reversed phase HPLC( vydac C18 ), the activity of this protein is > detected on wide range of fractions. The buffers were 0.1% TFA in water, > and 0.1% TFA in acetonitrile. I tried to elute this protein in neutral > condition (pH 7.0), but the resolution was not good. What should I do? > The molecular weight of this protein is about 7000Da, and it's pI value > is about 4.0. Any comment and any suggestion will be a good help to me. Hi Kang Ke-Won, I have also seen very broad peaks eluting off of C-18 HPLC and am not really sure what is causing this phenomena. I do have a few ideas that you might want to check out for yourself: 1) Try a C-8 column. I know that small peptide usually separate better on C-18; but, in your case, it may help to have a matrix that does not bind your protein as strongly. 2) Try reducing your protein with DTT before your separation. Of course, you will probably kill any activity, but you may get a sharp peak which you then can re-oxidize and get your activity back (if your are lucky). In one of our broad-eluting peaks, reduction and alkylation with vinylpyridine dramatically sharpened up the peak, allowing us to purify it. It may be that the protein, in its native form exists in a variety of conformations. 3) Try an anion exchange HPLC separation to purify your protein. Just another approach to think about. Don't do this on a normal HPLC, however, since the stainless steel doesn't get along with ion exchange buffer salts (i.e. chloride ions). Hope that helps. Regards, John _________________________________________________________ John Marcus Marcus@tpp.uq.edu.au (Dr J.Marcus) Cooperative Research Centre for Tropical Plant Pathology 5th Level John Hines Building University of Queensland St. Lucia, QLD 4072 AUSTRALIA Fax: 61-7-365-4771 Phone: 61-7-365-4764 From owner-proteins@net.bio.net Wed Oct 04 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!ix.netcom.com!netnews From: MHAYTHOR@ix.netcom.com (Mark Haythorn) Newsgroups: bionet.molbio.proteins Subject: Polymorphism?? Date: Thu, 05 Oct 1995 23:06:04 GMT Organization: Netcom Lines: 30 Message-ID: <451oe3$608@ixnews5.ix.netcom.com> NNTP-Posting-Host: ix-mor-nj1-11.ix.netcom.com X-NETCOM-Date: Thu Oct 05 4:06:43 PM PDT 1995 X-Newsreader: Forte Free Agent 1.0.82 I have a strange question... True or False A lyophilized protein (just 5-8 amino acids long) does not exhibit polymorphism. Sounds simple, and a few of you might ask "Who cares? When you reconstitute the lyophilized peptide there is no longer an issue of polymorphism". One other point- Is a crystal structure required for polymorphism? If so, does a lyophilized peptide have a crystal structure? Thanks in advance for any thoughts. I've posted here once before and received tremendous help from many people!! Mark Haythorn MHAYTHOR@IX.NETCOM.COM From owner-proteins@net.bio.net Wed Oct 04 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!tank.news.pipex.net!pipex!usenet.eel.ufl.edu!psgrain!iafrica.com!ticsa.com!cstatd.cstat.co.za!ucthpx!wabe.csir.co.za!pukrs7.puk.ac.za!caesar.wits.ac.za!pc40.med.wits.ac.za!075avri From: 075avri@chiron.wits.ac.za (Dr Avri Davidoff) Newsgroups: bionet.molbio.proteins Subject: HIV-1 TAT PROTEIN Date: Tue, 3 Oct 1995 08:26:19 GMT Organization: University of the witwatersrand Lines: 13 Message-ID: <075avri.19@chiron.wits.ac.za> NNTP-Posting-Host: pc40.med.wits.ac.za Dear Colleagues Could anybody advise me as to where purified/recombinant HIV-1 TAT protein can be purchased? The only company that I know of is INTRACEL, and the amount they supply is too large for my needs and thus too expensive for my pocket. Any assistance would be greatly appreciated. Thank you Yours sincerely Avri Davidoff From owner-proteins@net.bio.net Wed Oct 04 23:00:00 1995 Newsgroups: uchi.bio,uchi.general,bionet.molbio.proteins,bionet.molbio.methds-reagnts Path: biosci!bcm.tmc.edu!cs.utexas.edu!news.sprintlink.net!in1.uu.net!nih-csl!fp5-9n.domain!user From: tomasd@bdg10.niddk.nih.gov (Tomas Drgon) Subject: Re: Mol Biologists, do any of you use precast gels? Message-ID: Sender: postman@alw.nih.gov (AMDS Postmaster) Nntp-Posting-Host: fp5-9n.domain Organization: NIH References: <4516pc$k6@dwst13.edvz.sbg.ac.at> Date: Thu, 5 Oct 1995 21:21:18 GMT Lines: 31 Xref: biosci bionet.molbio.proteins:5849 bionet.molbio.methds-reagnts:34506 In article <4516pc$k6@dwst13.edvz.sbg.ac.at>, Anton Hutticher wrote: > > We compared NOVEX precast PAGE gels with our gels. The > resolution was quite good but not as good as our own specially > crafted gradient gels. However, they were very easy to setup > and use. Several people switched to NOVEX, other stayed with > their own gels. If you do PAGE only sporadically, precasts are > probably a good solution. > Take care if you want to do western blots with precasts. Those > from NOVEX we used could not stand the blotting conditions we > used ( 1 l MeOH, 4,1 g NaHCO3, 1,6 g Na2CO3 ), 40 V, 3 h on > Biorad Mini. They fragmented during the run and the fragments > were very hard to peel off the nitrocellulose. We have not tested > them under any other conditions! Maybe you find blotting conditions > which work for them, but test them beforehand! > > Anton Hutticher > (huttich@edvz.sbg.ac.at) > (Uni Salzburg / Austria) I use Novex precast gels for westerns. They run great in Tris/Glycine/SDS and also in CAPS blotting buffers (200 mA for 2 hrs). I also occasionally use precast and pre-EtBr-stained agarose gels from FMC Bioproducts. They are also ok. Tomas Drgon -- Signature under construction... From owner-proteins@net.bio.net Thu Oct 05 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!news.sprintlink.net!in2.uu.net!EU.net!Belgium.EU.net!chaos.kulnet.kuleuven.ac.be!news.sri.ucl.ac.be!usenet From: "B. Knoops" Newsgroups: bionet.molbio.proteins Subject: (no subject) Date: 6 Oct 1995 09:54:00 GMT Organization: Universite Catholique de Louvain, Louvain-la-Neuve, Belgium Lines: 5 Distribution: world Message-ID: <452ubo$gpt@sci3.sri.ucl.ac.be> NNTP-Posting-Host: 130.104.5.80 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Windows; I; 16bit) We are having problems with differential display RT-PCR. We are doing DDRT-PCR and our results are not reproducible. We used the technique described by Liand and Pardee (Science, 257:967-970, 1992). Is there someone somewhere who could give us some advices? From owner-proteins@net.bio.net Thu Oct 05 23:00:00 1995 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!news.sprintlink.net!simtel!swidir.switch.ch!scsing.switch.ch!news.rediris.es!obelix.cica.es!lucano!bc2biced From: "Depto. Biologia Celular" Newsgroups: bionet.molbio.proteins Subject: Malate Dehidrogenase in Native Gel Date: Fri, 6 Oct 1995 16:54:07 +0100 Organization: Centro Informatico Cientifico de Andalucia Lines: 14 Message-ID: NNTP-Posting-Host: lucano.uco.es Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=ISO-8859-1 Content-Transfer-Encoding: QUOTED-PRINTABLE X-Sender: bc2biced@lucano Dear netters, I would like know if exist some assay to detect NADH-Malate= =20 dehydrogenase activity in acrilamide native gels. If you can send me some= =20 reference. =09Thank you=20 =09=09Carlos Santos-Oca=F1a =09=09Dept Biolog=EDa Celular =09=09Facultad de Ciencias =09=09Univ. de C=F3rdoba =09=09SPAIN c - c c From owner-proteins@net.bio.net Thu Oct 05 23:00:00 1995 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!gatech!EU.net!Belgium.EU.net!chaos.kulnet.kuleuven.ac.be!news.sri.ucl.ac.be!usenet From: "B. Knoops" Newsgroups: bionet.molbio.proteins Subject: Problems with DDRT-PCR Date: 6 Oct 1995 17:18:14 GMT Organization: Universite Catholique de Louvain, Louvain-la-Neuve, Belgium Lines: 5 Distribution: world Message-ID: <453ocm$1ee@sci3.sri.ucl.ac.be> NNTP-Posting-Host: 130.104.5.80 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Windows; I; 16bit) To: knoops@nchm.ucl.ac.be We are having problems with differential display RT-PCR. Our results are not reproducible. We use the technique described by Liang and Pardee (Science, 257: 967-971, 1992). Is there someone somewhere who could give us some advices? Thanks a lot. From owner-proteins@net.bio.net Thu Oct 05 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!news.sprintlink.net!simtel!swidir.switch.ch!scsing.switch.ch!news.belwue.de!news.uni-freiburg.de!bio5.chemie.uni-freiburg.de!vonrhein From: vonrhein@bio5.chemie.uni-freiburg.de (Clemens Vonrhein) Newsgroups: bionet.molbio.proteins Subject: [Q] Info about pcWori+/pHSe5 ? Date: 6 Oct 1995 09:42:11 GMT Organization: Rechenzentrum der Universitaet Freiburg, Germany Lines: 20 Message-ID: <452tlj$av8@n.ruf.uni-freiburg.de> NNTP-Posting-Host: bio5.chemie.uni-freiburg.de Does anyone out there have a plasmid map or any information about the two plasmids pcWori+ and/or pHSe5 ? Or does anyone know about a reference describing these plasmids ? Thanks for any help Clemens --- *************************************************************************** * Clemens Vonrhein email vonrhein@bio5.chemie.uni-freiburg.de * * WWW http://bio5.chemie.uni-freiburg.de/~vonrhein/ * * * * Institut f. Org. Chemie u. Biochemie * * Albertstr.21 * * D-79104 Freiburg i.Br. (Germany) * * Tel.: 761/203-6061 FAX: 761/203-5987 * *************************************************************************** From owner-proteins@net.bio.net Thu Oct 05 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!news.sprintlink.net!simtel!swidir.switch.ch!scsing.switch.ch!news.belwue.de!news.uni-konstanz.de!news From: Markus Macht Newsgroups: bionet.molbio.proteins Subject: Help on CD-spectra Date: 6 Oct 1995 09:37:53 GMT Organization: I DID NOT CONFIGURE MY ORGANIZATION IN MY NEWSREADER Lines: 13 Distribution: world Message-ID: <452tdh$el6@eurybia.rz.uni-konstanz.de> NNTP-Posting-Host: sg17.chemie.uni-konstanz.de Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.12 (X11; I; IRIX 5.3 IP22) X-URL: news:bionet.molbio.proteins Hello ! Yesterday I recorded a CD-spectrum of an 32 AA peptide. On first glance, the spectrum seems to be a spectrum of an alpha-helix, but the minima at 208 and 222 nm are shifted to 210 and 227 nm. Does anybody of you have experiences with the deviation of the wavelenght of these minima from the classical values ? If this deviation is not within the normal range, what could be it's ex- planation? Any suggestions and/or explanations are welcome ! Yours sincerely Marcus Macht From owner-proteins@net.bio.net Thu Oct 05 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!swrinde!emory!darwin.sura.net!blaze.cs.jhu.edu!RacerX.mse.jhu.edu!news.jhu.edu!news From: Charles Yang Newsgroups: bionet.molbio.proteins Subject: Looking for Cyclin H.... Date: 6 Oct 1995 20:34:57 GMT Organization: Johns Hopkins University Lines: 19 Message-ID: <4543th$fg4@news.jhu.edu> NNTP-Posting-Host: 128.220.54.28 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; PPC) X-URL: news:bionet.molbio.proteins Hello, My name is Charles Yang and I am currently doing some work on yeast RNA pol2 initiation factors in S.Cerevisiae. I am keying up on the TF2H protein specifically the CCL1 gene which codes for a subunit of this protein. My problem: I can't find the nucleotide and amino acid sequences for the Cyclin H gene (human counterpart to CCL1) and its corresponding protein. I've already checked Medline, Swiss-Prot, Genbank, ....etc. to no avail. If someone could e-mail me these sequences or point me to a specific text or article that would have these sequences, I'd very much appreciate it. my address: cyang@jhunix.hcf.jhu.edu Thank You From owner-proteins@net.bio.net Thu Oct 05 23:00:00 1995 Path: biosci!daresbury!sunsite.doc.ic.ac.uk!dispatch.news.demon.net!demon!tank.news.pipex.net!pipex!howland.reston.ans.net!EU.net!Germany.EU.net!nntp.gmd.de!news.ege.edu.tr!news.metu.edu.tr!rorqual.cc.metu.edu.tr!asen From: alaattin sen Newsgroups: bionet.molbio.proteins Subject: Re: Protein manual Date: Fri, 6 Oct 1995 18:02:57 +0400 Organization: Middle East Technical University Lines: 33 Message-ID: References: <44p8kc$3jl@news.inc.net> NNTP-Posting-Host: rorqual.cc.metu.edu.tr Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII In-Reply-To: <44p8kc$3jl@news.inc.net> On 2 Oct 1995, Paul Boyer wrote: > I'm looking for a manual/handbook to use as a text in a lab course called > "Working with Proteins". I'd like to find something like the Maniatis > bible for molecular biologists, but I can't find anything on the protein > level. Can anyone recommend anything? > > Thanks, > Paul Boyer > > > =========================================================================== > Paul D. Boyer, Ph.D. University of Wisconsin--Parkside > Assistant Professor 900 Wood Road, Box 2000 > Department of Biological Sciences Kenosha, WI 53141-2000 > ============================================================================ > >I recommend the book below: "Protein Purification" Methods in Enzymology Vol 182 It is quite good book for handling the proteins Hope this help Alaattin SEN ============================================================================ Department of Biology Tel: +90(312)210-1000/6561 Middle East Technical University /6585 06531 Ankara TURKEY Fax: +90(312)210-1289 E-Mail : alattin-sen@metu.edu.tr ============================================================================ From owner-proteins@net.bio.net Thu Oct 05 23:00:00 1995 Path: biosci!rutgers!gatech!EU.net!Belgium.EU.net!chaos.kulnet.kuleuven.ac.be!news.sri.ucl.ac.be!usenet From: "B. Knoops" Newsgroups: bionet.molbio.proteins Subject: Problems with DDRT-PCR Date: 6 Oct 1995 13:44:29 GMT Organization: Universite Catholique de Louvain, Louvain-la-Neuve, Belgium Lines: 5 Distribution: world Message-ID: <453brt$dt@sci3.sri.ucl.ac.be> NNTP-Posting-Host: 130.104.5.80 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Windows; I; 16bit) To: knoops@nchm.uc.ac.be We are having some problems with differential display RT-PCR. Our results are not reproducible. We use the technique described by Liang and Pardee (Science, 257:967-971, 1992). Is there someone somewhere who could give us some advices? From owner-proteins@net.bio.net Fri Oct 06 23:00:00 1995 Path: biosci!CS.Arizona.EDU!news.Arizona.EDU!hamblin.math.byu.edu!sol.ctr.columbia.edu!spool.mu.edu!usenet.eel.ufl.edu!tank.news.pipex.net!pipex!news.sprintlink.net!in2.uu.net!newsflash.concordia.ca!news.mcgill.ca!news From: hussain Newsgroups: bionet.molbio.proteins Subject: To boil or not to boil? Date: 7 Oct 1995 21:30:10 GMT Organization: McGill University Computing Centre Lines: 5 Message-ID: <456rh2$1c1@sifon.cc.mcgill.ca> NNTP-Posting-Host: g-12.das.mcgill.ca X-Newsreader: AIR News 3.X (SPRY, Inc.) Dear netters.I have been doing western blotting for a year. I have not been boiling my tissue samples before loading. I am not adding b-mercaptoethanol either. I need to know the pros and cons of these two procedures. From owner-proteins@net.bio.net Fri Oct 06 23:00:00 1995 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!info.ucla.edu!news.ucdavis.edu!usenet From: dobates@ucdavis.edu (Dave Bates) Newsgroups: bionet.cellbiol,bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Antibodies to mitotic cells or other cell cycle phases Date: 6 Oct 1995 21:44:17 GMT Organization: Dept of Human Physiology, UC Davis Lines: 23 Sender: -Not-Authenticated-[1543] Message-ID: <4547vh$ct0@mark.ucdavis.edu> References: <44hba5$5bb@ixnews7.ix.netcom.com> NNTP-Posting-Host: norm.hph.ucdavis.edu X-Posted-From: InterNews 1.0.1@norm.hph.ucdavis.edu Xdisclaimer: No attempt was made to authenticate the sender's name. Xref: biosci bionet.cellbiol:3108 bionet.molbio.methds-reagnts:34587 bionet.molbio.proteins:5864 Does anyone know of a good specific antibody to cells in M phase? Preferably one that is known to work in amphibians, but any good antibody would be a start.In addition how about antibodies to G1, G2 or S either specifically to one phase or to all phases. I have PCNA, which picks up S phase transitions (or so I've been told!), and Ki67 which picks up everything. The Ki67 doesn't work too well in my hands in frogs, and I'd like something that worked better. Thanks for your help, Dave Bates ------------------------------------------------------------------------ - Dave Bates PhD Tel: 916 752-7081 Postdoctoral Researcher Fax: 916 758-2554 Dept of Human Physiology email: dobates@ucdavis.edu University of California at Davis drink: anything Davis, California 95616, USA No I don't have a sense of humour now buy me a beer ------------------------------------------------------------------------ ---- From owner-proteins@net.bio.net Sun Oct 08 23:00:00 1995 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!chi-news.cic.net!simtel!harbinger.cc.monash.edu.au!bunyip.cc.uq.oz.au!news From: Nigel McMillan Newsgroups: bionet.molbio.proteins Subject: Re: HIV-1 TAT PROTEIN Date: 9 Oct 1995 22:49:28 GMT Organization: University of Queensland Lines: 24 Message-ID: <45c8to$nls@dingo.cc.uq.oz.au> References: <075avri.19@chiron.wits.ac.za> NNTP-Posting-Host: 130.102.98.21 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) To: 075avri@chiron.wits.ac.za 075avri@chiron.wits.ac.za (Dr Avri Davidoff) wrote: >Dear Colleagues > >Could anybody advise me as to where purified/recombinant HIV-1 TAT protein >can be purchased? The only company that I know of is INTRACEL, and the >amount they supply is too large for my needs and thus too expensive for my >pocket. > >Any assistance would be greatly appreciated. > >Thank you > >Yours sincerely >Avri Davidoff American Biotechnology Inc sells it (+1 617 547 5535 Fax +1 617 4919015) but it is also expensive. You can (or use to be able) get it from the AIDS Resource Program at the NIH fro next to nothing but I dont know anymore and dont have contact number of fax Nigel McMillan PhD University of Queensland. From owner-proteins@net.bio.net Sun Oct 08 23:00:00 1995 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!chi-news.cic.net!simtel!harbinger.cc.monash.edu.au!bunyip.cc.uq.oz.au!news From: Nigel McMillan Newsgroups: bionet.molbio.proteins Subject: Re: HIV-1 TAT PROTEIN Date: 9 Oct 1995 22:48:59 GMT Organization: University of Queensland Lines: 24 Message-ID: <45c8sr$nls@dingo.cc.uq.oz.au> References: <075avri.19@chiron.wits.ac.za> NNTP-Posting-Host: 130.102.98.21 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) To: 075avri@chiron.wits.ac.za 075avri@chiron.wits.ac.za (Dr Avri Davidoff) wrote: >Dear Colleagues > >Could anybody advise me as to where purified/recombinant HIV-1 TAT protein >can be purchased? The only company that I know of is INTRACEL, and the >amount they supply is too large for my needs and thus too expensive for my >pocket. > >Any assistance would be greatly appreciated. > >Thank you > >Yours sincerely >Avri Davidoff American Biotechnology Inc sells it (+1 617 547 5535 Fax +1 617 4919015) but it is also expensive. You can (or use to be able) get it from the AIDS Resource Program at the NIH fro next to nothing but I dont know anymore and dont have contact number of fax Nigel McMillan PhD University of Queensland. From owner-proteins@net.bio.net Sun Oct 08 23:00:00 1995 Path: biosci!rutgers!concert!news-server.ncren.net!decwrl!its.hooked.net!usenet From: Newsgroups: bionet.molbio.proteins Subject: Re: To boil or not to boil? Date: 7 Oct 1995 22:49:12 GMT Organization: Hooked Online Services Lines: 14 Message-ID: <457058$g7d@its.hooked.net> References: <456rh2$1c1@sifon.cc.mcgill.ca> NNTP-Posting-Host: tuna.hooked.net Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) X-URL: news:456rh2$1c1@sifon.cc.mcgill.ca This depends on what information you wish to gain about the particular protein you are identifying from your western blots. If your protein contains disulfide bonds, and you wish to denature it completely, you must add a reducing agent such as BME or DTT(I reccommend DiThioThreitol because it is just as effective and much less offensive than BME). For total denaturation, there must also be SDS in your sample buffer and running buffer. Boiling in the presence of DTT and SDS ensures that the protein is completely denatured. However, if you wish to isolate isoforms of your protein based on differential disulfide bonding, do not include a reducing agent and do not boil, but keep the SDS in your buffers. If you wish to run a "completely" non-denaturing gel, do not boil, use a rducing agent, and do not use SDS. From owner-proteins@net.bio.net Sun Oct 08 23:00:00 1995 Path: biosci!rutgers!concert!news-server.ncren.net!decwrl!its.hooked.net!usenet From: Newsgroups: bionet.molbio.proteins Subject: Re: metabolic burden of recombinant protein overexpression Date: 7 Oct 1995 22:18:47 GMT Organization: Hooked Online Services Lines: 11 Message-ID: <456uc7$g7d@its.hooked.net> References: <44obst$ej8@rzlimes.gbf-braunschweig.de> NNTP-Posting-Host: tuna.hooked.net Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) X-URL: news:44obst$ej8@rzlimes.gbf-braunschweig.de It's called "stationary phase". Cell division stops almost completely however "reagents" from the media are still used for a kind of house-keeping activity to keep the cell alive. This may be a bad analogy since people are quite different from E. coli but I'll put it forth anyway: You have reached adulthood and have completed your growth phase (except for adipose tissue in many cases :-) however you have a continuos "turn-over" of carbon etc. in your body. Some estimates claim that a person completely "turns over" every atom in their body every six months... From owner-proteins@net.bio.net Sun Oct 08 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!newsxfer.itd.umich.edu!chi-news.cic.net!simtel!zombie.ncsc.mil!news.missouri.edu!muccmail.missouri.edu!bctrna From: bctrna@muccmail.missouri.edu (tRNA lab) Newsgroups: bionet.molbio.proteins Subject: His-protein purification from reticulocyte lysate Date: Mon, 9 Oct 1995 15:41:58 GMT Organization: University of Missouri Lines: 5 Message-ID: NNTP-Posting-Host: bchem46.biochem.missouri.edu Summary: copurification of hemoglobin? with his-protein Keywords: his-protein, in vitro translation, reticulocyte lysate X-Newsreader: Trumpet for Windows [Version 1.0 Rev B final beta #4] I am trying to purify his-protein after in vitro translation in reticulocyte lysate and getting copurification of hemoglobin? (resin turns red). As a result the yield and purity of desired protein are not satisfactory. Any suggestions will be more than welcome. Alexander Kenzior From owner-proteins@net.bio.net Sun Oct 08 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!newsjunkie.ans.net!Rezonet.net!titane!comback!nntphost!usenet From: marite@login.net Newsgroups: bionet.molbio.proteins Subject: Sugar and cholesterol level Date: 9 Oct 1995 15:30:56 GMT Organization: Login Communication Lines: 6 Message-ID: <45bf7g$ptl@tonka.login.qc.ca> NNTP-Posting-Host: m1l4.login.net X-Newsreader: AIR News 3.X (SPRY, Inc.) I heard about the report on an experience, the result of wich shows a strong correlation between sugar ingestion and the cholesterol level. If you happen to know something on this subject, I would appreciate you tell me about it. Thanks in advance. (Georges Chauvette) marite@login.net From owner-proteins@net.bio.net Sun Oct 08 23:00:00 1995 Path: biosci!UFCC.UFL.EDU!kewugator From: kewugator@UFCC.UFL.EDU Newsgroups: bionet.molbio.proteins Subject: stripping western blot Date: 9 Oct 1995 08:34:21 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 5 Sender: daemon@net.bio.net Distribution: world Message-ID: <009979C2.F7351D80.4@UFCC.UFL.EDU> NNTP-Posting-Host: net.bio.net Hi, there: I did a western blot using native gel. Dose anyone know a method to strip the western blot so I can reprobe it? Thank you in advance. From owner-proteins@net.bio.net Sun Oct 08 23:00:00 1995 Path: biosci!UFCC.UFL.EDU!kewugator From: kewugator@UFCC.UFL.EDU Newsgroups: bionet.molbio.proteins Subject: Logon Date: 9 Oct 1995 08:30:39 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 3 Sender: daemon@net.bio.net Distribution: world Message-ID: <009979C2.79528D80.4@UFCC.UFL.EDU> NNTP-Posting-Host: net.bio.net Dear sir: Could you please put my name on your mailing list. Thanks. From owner-proteins@net.bio.net Sun Oct 08 23:00:00 1995 Path: biosci!agate!spool.mu.edu!uwm.edu!chi-news.cic.net!newsfeed.internetmci.com!howland.reston.ans.net!newsjunkie.ans.net!Rezonet.net!titane!comback!nntphost!usenet From: marite@login.net Newsgroups: bionet.molbio.proteins Subject: Sugar and cholesterol level Date: 9 Oct 1995 15:06:41 GMT Organization: Login Communication Lines: 6 Message-ID: <45bdq1$oe7@tonka.login.qc.ca> NNTP-Posting-Host: m1l4.login.net X-Newsreader: AIR News 3.X (SPRY, Inc.) I heard about the report on an experience, the result of wich shows a strong correlation between sugar ingestion and the cholesterol level. If you happen to know something on this subject, I would appreciate you tell me about it. Thanks in advance. (Georges Chauvette) marite@login.net From owner-proteins@net.bio.net Sun Oct 08 23:00:00 1995 Path: biosci!agate!howland.reston.ans.net!tank.news.pipex.net!pipex!sunsite.doc.ic.ac.uk!hgmp.mrc.ac.uk!news From: pdezoysa@hgmp.mrc.ac.uk (Dr. P.A. de Zoysa) Newsgroups: bionet.molbio.proteins Subject: Custom peptide/antibody service Date: 9 Oct 1995 12:18:13 GMT Organization: UK HGMP Resource Centre Lines: 13 Distribution: world Message-ID: <45b3u5$b93@mercury.hgmp.mrc.ac.uk> Reply-To: pdezoysa@hgmp.mrc.ac.uk NNTP-Posting-Host: iron.hgmp.mrc.ac.uk Can anyone out there recommend a custom peptide/antibody service that they have successfully tried and tested. Thanks, Priyal. ---------------------------------------------------------------------------------------------- Dr. Priyal A. de Zoysa E:MAIL-pdz@rfhsm.ac.uk Academic Dept. of Medicine, Tel-0171-7940-500x5059 10th Floor, RFHSM, Pond St., Fax-0171-794-3472 Hampstead, London, UK, NW3 2PF. ---------------------------------------------------------------------------------------------- From owner-proteins@net.bio.net Sun Oct 08 23:00:00 1995 Path: biosci!ns1.faseb.org!lamarck.sura.net!newsfeed.internetmci.com!tank.news.pipex.net!pipex!dispatch.news.demon.net!demon!mail2news.demon.co.uk!genesys.demon.co.uk From: Duncan Clark Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Nucleotide triphosphates Date: Mon, 09 Oct 1995 11:42:51 +0100 Organization: GeneSys Ltd. Lines: 22 Message-ID: <137064904wnr@genesys.demon.co.uk> Reply-To: Duncan@genesys.demon.co.uk X-NNTP-Posting-Host: genesys.demon.co.uk X-Newsreader: Newswin Alpha 0.9 Xref: biosci bionet.molbio.methds-reagnts:34603 bionet.molbio.proteins:5865 Hi folks, A couple of questions regarding nucleotide triphosphates. 1. Can they be dialysed? I know v.short oligos can even though by mwt it shouldn't be possible. 2. Anyone know of a reference for their purification or failing that a rough idea as to the binding capacity of DEAE or QAE exchangers for them. It gets expensive to keep loading a 1ml column just to see how much binds. If I have to do it so be it but it would be nice to avoid. Many thanks Duncan -- --------------------------------------------------------------------------- -- My mind's made up. Don't confuse me with the facts! From owner-proteins@net.bio.net Sun Oct 08 23:00:00 1995 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!chi-news.cic.net!simtel!harbinger.cc.monash.edu.au!bunyip.cc.uq.oz.au!news From: Nigel McMillan Newsgroups: bionet.molbio.proteins Subject: Re: HIV-1 TAT PROTEIN Date: 9 Oct 1995 22:52:11 GMT Organization: University of Queensland Lines: 24 Message-ID: <45c92r$nls@dingo.cc.uq.oz.au> References: <075avri.19@chiron.wits.ac.za> NNTP-Posting-Host: 130.102.98.21 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) To: 075avri@chiron.wits.ac.za 075avri@chiron.wits.ac.za (Dr Avri Davidoff) wrote: >Dear Colleagues > >Could anybody advise me as to where purified/recombinant HIV-1 TAT protein >can be purchased? The only company that I know of is INTRACEL, and the >amount they supply is too large for my needs and thus too expensive for my >pocket. > >Any assistance would be greatly appreciated. > >Thank you > >Yours sincerely >Avri Davidoff American Biotechnology Inc sells it (+1 617 547 5535 Fax +1 617 4919015) but it is also expensive. You can (or use to be able) get it from the AIDS Resource Program at the NIH fro next to nothing but I dont know anymore and dont have contact number of fax Nigel McMillan PhD University of Queensland. From owner-proteins@net.bio.net Sun Oct 08 23:00:00 1995 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!tank.news.pipex.net!pipex!swrinde!howland.reston.ans.net!news-e1a.megaweb.com!newstf01.news.aol.com!newsbf02.news.aol.com!not-for-mail From: alussow@aol.com (ALussow) Newsgroups: bionet.cellbiol,bionet.metabolic-reg,bionet.molbio.proteins Subject: Re: HSP - unique? Date: 9 Oct 1995 19:07:20 -0400 Organization: America Online, Inc. (1-800-827-6364) Lines: 6 Sender: root@newsbf02.news.aol.com Message-ID: <45c9v8$o6t@newsbf02.news.aol.com> References: <45c3fa$o2m$1@mhafm.production.compuserve.com> Reply-To: alussow@aol.com (ALussow) NNTP-Posting-Host: newsbf02.mail.aol.com Xref: biosci bionet.cellbiol:3122 bionet.metabolic-reg:553 bionet.molbio.proteins:5879 The answer is no. HSP's are in fact misnamed. They are better known as stress proteins, heat being only one form of stress. The glucocorticoid pathway is another response to stress (including heat) that is upregulated in an organism under "attack." If you don't ask much from life, that's what you'll get. From owner-proteins@net.bio.net Sun Oct 08 23:00:00 1995 Path: biosci!reading.ac.uk!ssu94071 From: ssu94071@reading.ac.uk (Darran Hayes) Newsgroups: bionet.molbio.proteins Subject: Bio-Comp References Date: 9 Oct 1995 17:08:24 -0700 Organization: University of Reading, U.K. Lines: 13 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Has anyone got any information, references, prefereably ftp sites for the use of Bacteriorhodopsin as an optical storage device. I need papers and preferably online. In addition, does anyone know Prof R. R. Birge of the University in Syracuse or how I could get his e-mail address? Thanks Darran Hayes --- Cybernetics and Computer Science Reading Uni UK From owner-proteins@net.bio.net Sun Oct 08 23:00:00 1995 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!chi-news.cic.net!simtel!oleane!jussieu.fr!news.forth.gr!news-ath.forthnet.gr!news.compulink.gr!athena!tzic From: tzic@athena.compulink.gr (Nikos Tzitzikostas) Newsgroups: bionet.molbio.proteins Subject: soy proteins info Date: 10 Oct 1995 02:34:57 GMT Organization: CompuLink Network Lines: 4 Message-ID: <45cm4h$pj1@news.compulink.gr> NNTP-Posting-Host: athena.compulink.gr X-Newsreader: TIN [version 1.2 PL2] I am looking for information about soy proteins so if anyone can suggest to me a www or http or gopher adress he could be very helpfull From owner-proteins@net.bio.net Sun Oct 08 23:00:00 1995 Path: biosci!rutgers!uwm.edu!chi-news.cic.net!simtel!news.sprintlink.net!in2.uu.net!news.compuserve.com!news.production.compuserve.com!news From: Edan Foley <100711.732@CompuServe.COM> Newsgroups: bionet.cellbiol,bionet.metabolic-reg,bionet.molbio.proteins Subject: HSP - unique? Date: 9 Oct 1995 21:16:26 GMT Organization: CompuServe, Inc. (1-800-689-0736) Lines: 11 Message-ID: <45c3fa$o2m$1@mhafm.production.compuserve.com> Xref: biosci bionet.cellbiol:3120 bionet.metabolic-reg:552 bionet.molbio.proteins:5875 Recently as part of my studies I came across heat shock proteins. After a lengthy period of head-scratching and asking around I couldn't get a satisfactory answer to my question. The Question? Oh yeah..the question. Does anyone know if the production of HSPs is the only examples of a biological pathway that is 'kicked' into action as a direct response to an external heat stimulus? From owner-proteins@net.bio.net Sun Oct 08 23:00:00 1995 Path: biosci!agate!howland.reston.ans.net!swrinde!sdd.hp.com!night.primate.wisc.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!alpha.oncology.wisc.edu!user From: mcmahan@oncology.wisc.edu (Scott McMahan) Newsgroups: bionet.molbio.proteins Subject: Re: His-protein purification from reticulocyte lysate Date: Mon, 09 Oct 1995 22:24:14 -0600 Organization: University of Wisconsin-Madison Lines: 24 Message-ID: References: NNTP-Posting-Host: alpha.oncology.wisc.edu In article , bctrna@muccmail.missouri.edu (tRNA lab) wrote: > I am trying to purify his-protein after in vitro translation in reticulocyte > lysate and getting copurification of hemoglobin? (resin turns red). As a > result the yield and purity of desired protein are not satisfactory. Any > suggestions will be more than welcome. > Alexander Kenzior I take it you mean a protein with a histidine tail that you are purifying over a Ni(II) chelate column. I've never worked with the reticulocyte resin, but the red color could be due to oxidized iron, I guess. (There are other metals that could give a red/pink color, too.) If all this is true, my guess would be that the chelator on the column is complexing with the iron (or other metal). To prevent this, try loading more nickel onto the column before you load to make sure all metal binding sites are occupied with nickel. If this doesn't eliminate the problem, add nickel to the load in case leaching of the nickel is a problem. If neither of these work, your problem may be that some protein with a affinity for nickel is binding. Try increasing the level of imidazole in your washes. -- Scott McMahan mcmahan@oncology.wisc.edu From owner-proteins@net.bio.net Mon Oct 09 23:00:00 1995 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!chi-news.cic.net!simtel!swidir.switch.ch!scsing.switch.ch!news.belwue.de!news.uni-ulm.de!rz.uni-karlsruhe.de!news.uni-stuttgart.de!uni-regensburg.de!lrz-muenchen.de!fauern!faui0n.informatik.uni-erlangen.de!uni-erlangen.de!winx03!wpxx02.toxi.uni-wuerzburg.de!krasel From: krasel@wpxx02.toxi.uni-wuerzburg.de (Cornelius Krasel) Newsgroups: bionet.molbio.proteins Subject: Re: Purification Problem in HPLC HELP! Date: 10 Oct 1995 14:54:38 GMT Organization: Dept. of Pharmacology, U Wuerzburg Lines: 22 Message-ID: <45e1fe$dau@winx03.informatik.uni-wuerzburg.de> References: <44vvpc$dtb@news.kreonet.re.kr> NNTP-Posting-Host: wpxx02.toxi.uni-wuerzburg.de X-Newsreader: TIN [version 1.2 PL2] Kang Ke-Won (kwkang@sorak.kaist.ac.kr) wrote: > I am purifying a unidentified protein by HPLC. When this protein was eluted > through reversed phase HPLC( vydac C18 ), the activity of this protein is > detected on wide range of fractions. The buffers were 0.1% TFA in water, > and 0.1% TFA in acetonitrile. I tried to elute this protein in neutral > condition (pH 7.0), but the resolution was not good. What should I do? > The molecular weight of this protein is about 7000Da, and it's pI value > is about 4.0. Any comment and any suggestion will be a good help to me. I have heard that using C8 instead of C18 might give sharper resolution in the case of proteins. There are a lot of other purification methods in addition to reversed phase HPLC. Ion exchange is often a good method to separate proteins and would probably work in your case (assuming that the protein is stable at neutral pH). To get rid of high-MW contaminations you could try gel filtration. --Cornelius. -- /* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */ /* D-97078 Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de */ /* "Science is the game we play with God to find out what His rules are." */ From owner-proteins@net.bio.net Mon Oct 09 23:00:00 1995 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!howland.reston.ans.net!vixen.cso.uiuc.edu!uwm.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!alpha.oncology.wisc.edu!user From: mcmahan@oncology.wisc.edu (Scott McMahan) Newsgroups: bionet.molbio.proteins Subject: Re: His-protein purification from reticulocyte lysate Date: Wed, 11 Oct 1995 00:46:32 -0600 Organization: University of Wisconsin-Madison Lines: 18 Message-ID: References: NNTP-Posting-Host: alpha.oncology.wisc.edu In article <45ff0s$i1p@charm.magnus.acs.ohio-state.edu>, rsaldanh@magnus.acs.ohio-state.edu (Roland J Saldanha) wrote: > While your first suggestion may be true I think adding nickel to the load will > guarantee the tagged protein wont bind since it binds by virtue of its affinity > to the nickel immobilised on the resin. > > > Roland Of course, how foolish of me. -- Scott McMahan mcmahan@oncology.wisc.edu From owner-proteins@net.bio.net Mon Oct 09 23:00:00 1995 Path: biosci!rutgers!uwm.edu!chi-news.cic.net!simtel!zombie.ncsc.mil!cs.umd.edu!haven.umd.edu!purdue!lerc.nasa.gov!magnus.acs.ohio-state.edu!rsaldanh From: rsaldanh@magnus.acs.ohio-state.edu (Roland J Saldanha) Newsgroups: bionet.molbio.proteins Subject: Re: His-protein purification from reticulocyte lysate Date: 11 Oct 1995 03:51:56 GMT Organization: The Ohio State University Lines: 28 Message-ID: <45ff0s$i1p@charm.magnus.acs.ohio-state.edu> References: > lysate and getting copurification of hemoglobin? (resin turns red). As a >> result the yield and purity of desired protein are not satisfactory. Any >> suggestions wi