From owner-proteins@net.bio.net Sat Mar 01 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!newsfeeds.sol.net!feed1.news.erols.com!cpk-news-hub1.bbnplanet.com!cam-news-hub1.bbnplanet.com!news.bbnplanet.com!howland.erols.net!ix.netcom.com!news From: Larry W Eck Newsgroups: bionet.molbio.proteins Subject: XRCC1 protein Date: Mon, 03 Mar 1997 02:31:45 GMT Organization: Netcom Lines: 5 Message-ID: <5fdcvk$4jl@dfw-ixnews3.ix.netcom.com> NNTP-Posting-Host: ind-in13-20.ix.netcom.com X-NETCOM-Date: Sun Mar 02 8:28:04 PM CST 1997 X-Newsreader: NETCOMplete/3.2 Hello, I need info on this protein (XRCC1) and its interaction with DNA ligase III in the rejoining step of base excision repair. If you have any knowledge in this field please post a reply or e-mail me at eklu@ix.netcom.com hopefully, eklu From owner-proteins@net.bio.net Sat Mar 01 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!newsfeeds.sol.net!newspump.sol.net!mindspring!news3.agis.net!agis!newsgod1.agis.net!agis!newspeer1.agis.net!agis!news.mwci.net!not-for-mail From: lazyhog@mwci.net (Chris R. Hoffman) Newsgroups: bionet.cellbiol,bionet.biology.cardiovascular,bionet.microbiology,sci.bio.misc,bionet.molbio.proteins,bionet.molbio.proteins.7tms_r,bionet.molbio.proteins.fluorescent Subject: TUBULIN USERS PLEASE READ Date: 3 Mar 1997 02:43:51 GMT Organization: Lost Creek Swine Lines: 10 Message-ID: <5fddt7$b71$1@hihat.mwci.net> NNTP-Posting-Host: dbq-dial-3.dbq.mwci.net Mime-Version: 1.0 X-Newsreader: WinVN 0.99.2 Xref: biosci bionet.cellbiol:6815 bionet.biology.cardiovascular:1540 bionet.microbiology:9159 sci.bio.misc:7566 bionet.molbio.proteins:10141 bionet.molbio.proteins.7tms_r:1060 bionet.molbio.proteins.fluorescent:980 Attention all Tubulin buyers Check out microSuppliers new web page on the internet Special pricing for NEW CUSTOMERS!!!! Go to http://users.mwci.net/~microsup CHECK US OUT TODAY From owner-proteins@net.bio.net Sat Mar 01 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!cs.utexas.edu!howland.erols.net!usenet.kornet.nm.kr!newsfeed.dacom.co.kr!korpc!hitel!hbkg From: hbkg@hitel.kol.co.kr () Newsgroups: bionet.molbio.proteins Subject: 1 mM Tris? Date: 3 Mar 1997 04:57:38 GMT Organization: Korea PC Telecom Co.,Ltd. Lines: 7 Message-ID: <5fdlo2$v3@news2.kol.co.kr> NNTP-Posting-Host: hitel.kol.co.kr X-Newsreader: TIN [version 1.2 PL2] We tried to dissolve DTNB, CAT substrate, in Tris buffer, and found that DTNB was not dissolved in Tris buffer at all, which was very strange. We changed water in vain. We found at last the mistake of using 1mM Tris instead of appropriate concentration. I am curious HOW TRIS AFFECTS DTNB SOLUBILITY. From owner-proteins@net.bio.net Sun Mar 02 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!news.sgi.com!news.maxwell.syr.edu!usenet.kornet.nm.kr!ames!purdue!haven.umd.edu!news.umbc.edu!kcarter From: kcarter@umbc.edu (Mr. Ken Carter) Newsgroups: bionet.molbio.proteins Subject: rDNA Techniques & Applications Workshop Date: 3 Mar 1997 15:56:39 GMT Organization: University of Maryland, Baltimore County Lines: 59 Message-ID: <5fesbn$ld4@news.umbc.edu> NNTP-Posting-Host: f-umbc7.umbc.edu NNTP-Posting-User: kcarter X-Newsreader: TIN [version 1.2 PL2] >>>>>>>RECOMBINANT DNA: TECHNIQUES AND APPLICATIONS<<<<<<< American Type Culture Collection (ATCC) April 14-18, 1997, Rockville, MD This five-day, laboratory-intensive, course covers up-to-date recombinant DNA procedures and methodologies both in theory and in practice. This workshop provides introductory and intermediate level instruction in recombinant DNA laboratory techniques. A basic knowledge of nucleic acids is helpful but not necessary. Lecture topics will include Molecular Cloning, Enzymes, Vectors, E. coli Host Genotypes, Library Construction, Nucleic Acid Sequencing, Restriction Mapping, Probes and PCR, Synthesis and Cloning of cDNA. Laboratory sessions will include three simultaneous experiments; characterizing and subcloning a DNA fragment and constructing a eukaryotic genomic library in lambda phage. 1.DNA Characterization: restriction digest/mapping of a selected plasmid, fragment size and number determined by horizontal agarose gel-electrophoresis, DNA transfer to a nylon transfer matrix, probe selection, nick translation of probe with labelled dNTP and characterization of DNA fragments by Southern blot analysis. 2.Subcloning of a DNA Fragment: vector digestion to completion, isolation of DNA fragment by preparative restriction digest, fragment size determination by horizontal gel-electrophoresis, DNA extraction from agarose gel, vector insert ligation, screening for transformants by colony hybridization and analysis of plasmid DNA (miniscreens). 3.Construction of a Genomic Library; isolation of eukaryotic genomic DNA, digestion, ligation, in vitro packaging, plating bacteria, assays of packaged phage and screening by plaque hybridization. Approximately 75% of the course is devoted to laboratory instruction while 25% is lecture-oriented. An intensive manual with protocols and procedures is provided for each participant. Faculty: William Nierman, Ph.D. (Workshop Director), Director, ATCC Program in Molecular Biology and Virology; Donna Maglott, Ph.D., Assoc. Staff Scientist; Tamara Feldblyum, M.S., Collection Scientist. Also, Ramon Jordan, Ph.D., Research Plant Pathologist/Virologist, United States Department of Agriculture. Limited to 25 participants FEE: $1,395.00 **For a full schedule and on-line registration, please visit our Web site: http://www.atcc.org/workshops/workshop.html **Or request a brochure, which includes a full schedule and registration form from: kcarter@atcc.org -- ****************************************************************************** Ken Carter ^ American Type Culture Collection / l \ (301) 231-5525 12301 Parklawn Drive /___l___\__ fax:(301) 816-4364 Rockville, Maryland 20852 \______/ kcarter@atcc.org ~~~~~~~~~~~~~~ From owner-proteins@net.bio.net Sun Mar 02 22:00:00 1997 Newsgroups: bionet.molbio.proteins Path: biosci!ihnp4.ucsd.edu!swrinde!rill.news.pipex.net!pipex!warwick!bris.ac.uk!not-for-mail From: bijgh@zeus.bris.ac.uk (Jared Head) Subject: Re: Protein-protein interaction of His-tag of pET14b? X-Nntp-Posting-Host: zeus.bris.ac.uk Message-ID: Sender: usenet@fsa.bris.ac.uk (Usenet) Organization: University of Bristol, England X-Newsreader: TIN [UNIX 1.3 950824BETA PL0] References: <5f2n2o$cpq2@hkusud.hku.hk> Date: Mon, 3 Mar 1997 14:18:23 GMT Lines: 24 Jimmy S.H. Tsang (jshtsang@hkucc.hku.hk) wrote: : : Dear All, : I have recently fused a bacterial gene downstream of the His-tag : sequence of the pET14b from AMS Biotech. The in vitro transcribed and : translated protein forms a complex which migrates much slower than the wild : type protein in a native PAGE. Did anyone experience a similar : 'multimerisation' problem? The same gene cloned into pET19b produced a : protein similar to that of native protein, i.e. no 'multimerisation'. Any : suggestion and comment will be appreciated. We're just finding the same thing. It seems possible that more than one protein are chelating around one nickel atom. When we cleaved the His.Tag off we made our protein monomeric, and we also found dtt to have the same effect, presumably by reducing the nickel ions. Jared -- Jared Head at the Department of Biochemistry, University of Bristol "A computer lets you make more mistakes faster than any invention in human history - with the possible exceptions of handguns and tequila." Mitch Ratliffe From owner-proteins@net.bio.net Sun Mar 02 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!news.sgi.com!su-news-hub1.bbnplanet.com!cam-news-hub1.bbnplanet.com!news.bbnplanet.com!howland.erols.net!ais.net!ameritech.net!uunet!in1.uu.net!204.96.36.2!wizard.pn.com!news.xensei.com!news From: chi@healthtech.com (Cambridge Healthtech Institute) Newsgroups: bionet.molbio.proteins Subject: INFECTION GENOMICS Date: Mon, 03 Mar 1997 14:48:39 GMT Organization: Cambridge Healthtech Institute Lines: 87 Message-ID: <5feo73$g3i@xensei3.xensei.com> Reply-To: chi@healthtech.com NNTP-Posting-Host: chi.xensei.com X-Newsreader: Forte Free Agent 1.0.82 INFECTION GENOMICS APRIL 1-2, 1997 LOEWS CORONADO BAY RESORT, CORONADO, CALIFORNIA Session Chairs Dr. Craig S. Hill, Gen-Probe, Inc. Dr. Stephen A. Johnston, University of Texas Southwestern Medical Center Dr. Peter A. Schad, National Center for Genome Resources Dr. Richard S. Stephens, University of California, Berkeley Additional Speakers Dr. David S. Bailey, Pfizer Central Research Dr. David T. Beattie, Virus Research Institute, Inc. Dr. Patrick Dillon, Human Genome Sciences, Inc. Dr. Ronald W. Ellis, Astra Research Center Boston Dr. Lance Fors, Third Wave Technologies, Inc. Dr. Richard Goold, Incyte Pharmaceuticals, Inc. Dr. Philippe Gros, McGill University Dr. Stephen L. Hoffman, Naval Medical Research Institute Dr. Jörgen Lönngren, ProGene Lab AB Dr. George Natsoulis, Microcide Pharmaceuticals, Inc. Dr. Steven H. Nye, Pharmacia Biotech, Inc. Dr. C. Kendall Stover, PathoGenesis Corporation Dr. William E. Timberlake, ChemGenics Pharmaceuticals Inc. Dr. Jean-François Tomb, The Institute for Genomic Research Dr. Joaquim Trias, Versicor Inc. Dr. Gerald F. Vovis, Genome Therapeutics Corporation Dr. Terry Walker, Becton Dickinson Research Center Dr. C. Richard Wobbe, SCRIPTGEN Pharmaceuticals, Inc. SEQUENCING Comparative Microbial Genomics Using GSDB at NCGR Genomic Expression Analysis in Pathogenic Bacteria PathSeq: Microbial Genome Database The Malaria Genome Project Role of Nramp1 Gene in Resistance to Infectious Diseases Sequencing and Comparative Genomics of Helicobacter Pylori Genome DIAGNOSTICS Infectious Disease Diagnosis Using Strand Displacement Amplification Transcription-Mediated Amplification for Detection of Microorganisms Fingerprinting Viral and Bacterial Genotypes Using CFLP® HIV-1 Drug Resistance Genotyping by DNA-Sequencing APEX as an Analytic Chip-Based Tool for DNA Sequence Variations VACCINES Genetic Vaccines and Genomics Applying Genomic Sequencing and Analysis to H. Pylori Vaccine Microbial Genomics Approach for the Development of Vaccines THERAPEUTICS AND DRUG DEVELOPMENT Treatment and Prevention Approaches Based on Chlamydia Genome Developing Therapeutic Products from Genomic Sequence of Pathogens Drug Discovery Genomics Microbial and Mammalian Genome Sequences to Validate Drug Screening Targets Pathogen Genomic Regions for Infection and Drug Resistance New Antibiotics from Combichem Libraries and Novel Targets Targeted Genomics Approach to Antibacterials and Antifungals Impact of Genomics on the Diagnosis and Treatment of Infectious Disease. For registration and hotel information, please contact: Cambridge Healthtech Institute 1037 Chestnut Street Newton Upper Falls, MA 02164 USA Phone: 617-630-1300 Fax: 617-630-1325 e-mail: chi@healthtech.com http://www.healthtech.com/conferences/ ______________________________ Cambridge Healthtech Institute 1037 Chestnut Street Newton Upper Falls, MA 02164 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ tel: 617.630.1300 fax: 617.630.1325 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ inquiries@healthtech.com World Wide Web http://www.healthtech.com/conferences From owner-proteins@net.bio.net Sun Mar 02 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!newsfeeds.sol.net!news.maxwell.syr.edu!europa.clark.net!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!rill.news.pipex.net!pipex!warwick!leicester!usenet From: "Dr E. Buxbaum" Newsgroups: bionet.molbio.proteins Subject: Re: Protein precipitation from phenol?? Date: 3 Mar 1997 11:17:28 GMT Organization: University of Leicester (PCFS User) Lines: 5 Message-ID: <5fec08$50m@falcon.le.ac.uk> References: <33175B3C.2951@tcd.ie> NNTP-Posting-Host: pc48.msb.le.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) Phenol extraction of proteins for electrophoresis has been described in a paper in Anal. Biochem. some two or three years ago. You may have to dilute the samples somewhat, so that you get good separation between the phenol and the aquous phase. From owner-proteins@net.bio.net Sun Mar 02 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!newsfeeds.sol.net!news.maxwell.syr.edu!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!rill.news.pipex.net!pipex!warwick!leicester!usenet From: "Dr E. Buxbaum" Newsgroups: bionet.molbio.proteins Subject: Re: Purification of a membrane protein Date: 3 Mar 1997 11:14:01 GMT Organization: University of Leicester (PCFS User) Lines: 33 Message-ID: <5febpp$50m@falcon.le.ac.uk> References: <33168A5C.1AFA@botinst.unizh.ch> NNTP-Posting-Host: pc48.msb.le.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) Karin Wuethrich wrote: >Hi there, >I am trying to purify a plant membrane protein. I can solubilize my >protein with Triton X-100 and I can do an anion-exchange chromatography. >My problem is a great instability of the activity of my protein. I can >elute the activity from the anion-exchanger but the recovery of the >activity is only about 1/10. Also after freezing all my activity has >gone (before the column there is no loss of activity by freezing). I >tried to stabilize my protein by ading 20% ethylene glykol to the buffer >which didn't help. Now I read about ading lipids to the buffer for >stabilization but I couldn't find a protocol on how to do this: What >concentration of lipids, do I have to solubilize the lipids first ect. >Any help (also other suggestions for stabilization) would be >appreciated.Please write to kwue@botinst.unizh.ch >Karin For solubilisation of a membrane bound protein I would start with well defined (and therefore unfortunately expensive) detergents like Octylglucoside, Dodecylmaltoside, Hecameg or similar. You will probably have to add lipids, soy bean asolectin is the cheapest choice that seems to work well with my protein. Keep in mind however, that this is black art, not science. You may have to experiment with different detergents, lipids (and mixtures of them, too). I currently solubilise in 1.4% Octylglucoside, 0.4% asolectin, 1.25 mg/ml protein, just to give you an example. For chromatography I use half the concentration of detergent and lipid. The exact ratios too vary from protein to protein. 10-20% glycerol and/or 250 mM sucrose can protect your protein from denaturation, as may the inclusion of its substrate. Do not freeze solubilised proteins, if you can avoid it. It may be worthwile to reconstitute the protein into proteoliposomes after purification (by dialysis over night against a buffer without detergent and lipid). Good luck (you will need it)! From owner-proteins@net.bio.net Sun Mar 02 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!newsfeeds.sol.net!news.maxwell.syr.edu!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!rill.news.pipex.net!pipex!warwick!leicester!usenet From: "Dr E. Buxbaum" Newsgroups: bionet.molbio.proteins Subject: Re: Antibody purification Date: 3 Mar 1997 11:00:28 GMT Organization: University of Leicester (PCFS User) Lines: 21 Message-ID: <5feb0c$50m@falcon.le.ac.uk> References: NNTP-Posting-Host: pc48.msb.le.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) GMEACHAM@bmg.bhs.uab.edu wrote: >Does anyone have a protocol or reference for precipitating Antisera with >Ammonium Sulfate? Info like optimal concentrations of AmSo4, best buffer >to dialyze in, etc. would be useful. Thanks in advance. > Precipitation is usually carried out with 45% saturation of ammonium sulfate (i.e. a concentrated solution is called 100%). 45% saturation is equivalent to 277 g/l. Add slowly in small portions to your sample, while constantly stirring on ice. Leave stirring for 30 min, then spin, keeping the sample cold all the time. The buffer used for dissolving and dialysing the pellet obviously depends on what you need the antibody for. Phosphate (PBS) or Tris (TBS) buffered saline are probably most commonly used. If however you want to further purify by ion exchange chromatography, you need a low ionic strength buffer (10 mM Tris-HCl pH 7 or similar). If instead you want to use hydrophobic interaction chromatography, you need a high salt buffer instead (add as much ammonium sulfate as possible without precipitating the antibody). From owner-proteins@net.bio.net Sun Mar 02 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!cs.utexas.edu!howland.erols.net!rill.news.pipex.net!pipex!warwick!lyra.csx.cam.ac.uk!news.ox.ac.uk!news From: "Simon M. Brocklehurst" Newsgroups: bionet.molbio.proteins Subject: IMPORTANT ** Cytokines Web Announcement Date: Mon, 03 Mar 1997 10:20:59 +0000 Organization: University of Oxford Lines: 53 Message-ID: <331AA60B.2781E494@bioch.ox.ac.uk> NNTP-Posting-Host: nmrv.ocms.ox.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (X11; I; SunOS 4.1.3_U1 sun4m) ********IMPORTANT ANNOUNCEMENT************* *********** THE CYTOKINES WEB************** MAJOR UPDATE, and CHANGE OF LOCATION The Cytokines Web has moved from it's location at the University of Oxford OCMS Web server. The new URL is: http://www.psynix.co.uk/cytweb/ Please update your links and bookmarks to reflect the new location. The Cytokines Web has been updated and extended, as described below: The Cytokines Web provides leading-edge scientific information about cytokines and their receptors, including 3-D structural information and topological and evolutionary relationships (site info available). It also contains information about potential theraputic uses for some Human cytokines. In addition to updates of previously available sections to include up-to-date structural information, new CytWeb areas for 1997 are. o Journal Watch - Up-to-date surveys of the literature at the click of a mouse button (see the icons in the "known structures" areas) o Hot Targets - recently discovered human cytokines that are targets for structure determination by using X-ray/NMR techniques, and/or prediction by computational techniques. o Clinical Significance - potential uses of Human cytokines in diagnosis and treatment of disease Conference Announcements o Check out upcoming events and conferences in a Calendar format. o Commercial Links - products and services of interest to Cytokines Web users. Thanks for reading this message! _____________________________________________________________ | | Simon M. Brocklehurst, | Oxford Centre for Molecular Sciences, | Department of Biochemistry, | University of Oxford, Oxford, UK. | E-mail: smb@bioch.ox.ac.uk |____________________________________________________________ From owner-proteins@net.bio.net Sun Mar 02 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!news-peer.gsl.net!news.gsl.net!howland.erols.net!newsxfer.itd.umich.edu!uunet!in2.uu.net!198.168.54.138!rcogate.rco.qc.ca!altitude!usenet From: "Achim Recktenwald, PhD" Newsgroups: bionet.molbio.proteins Subject: Re: Purifying a simple protein Date: Mon, 03 Mar 1997 08:10:26 -0500 Organization: IBEX Technologies, Inc., Biochemistry, 5485 Pare, Montreal, PQ, H4P 1P7, Canada Lines: 50 Message-ID: <331ACDC0.7AB1@ibex.ca> References: <33163cb8.90814393@news.concentric.net> NNTP-Posting-Host: ibex.hip.cam.org Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Macintosh; I; PPC) To: Quasi26@concentric.net Quasi26@concentric.net wrote: > > I am a student at WPI in Mass. and was wondering if you could help me > purify this protein. > > The protein has a MW of 6,000, pI @ 4.9 and is intracellular > The contaminants have MW of 113,000; 13,000; and 39,000 > Acetate is an inhibitor and the protein is being used for the food > industry > It is good at temp. from 22 - 62 C, with the highest activity between > 40-50 C > It is good at ph from 4 - 10, with high activity between 6 and 9 > > I am just looking for a simple scheme, using tangental flow, ion > exchanges, HIC, Gel filtration, ultra, PPT etc... > > This is a scheme that I have chosen but I was wondering if you could > help me get more in depth with it or show something that would work > better. I use simple flow sheets, but I will write this out because I > can not think of a way to make a flow sheet on this. > > From the cells, I choose to centrifuge and then break them (French > Pressure cell), Succinate buffer (pKa of 5.6). From the homogenate, I > choose ultrafiltration, using 100K at cutoff (getting rid of 113,000). > then discard pellet and take your low level supernatant and use cation > exchanger with succinate again. pool the active fractions and maybe > use Sephadex G 25 or HIC. > > This is just a simple scheme, but I was hoping that you could critique > it and help me on it. I am taking a purification lab next term, and > this was given to us to show us what we will be seeing in the future. > I also have another question, What ph range should I operate from and > how do you determine that? > > Hope to hear from you soon. My email is davido@wpi.edu Thank > you. I would say, try it, looks good. I don't know how stable your protein is, but if it is not easily deactivated by the proteases in your homogenate, try using a lower cut-off ultrafiltration, e.g., 10000 or 20000. Another possiblility: skip the ultrafiltration step and go directly to the cation exchanger. If the homogenate is too turbid because of colloidal particles, use a 'Big Beads-material' with a larger pore-size; we do this routinely, I am not quite sure, but I think we buy the gel from Pharmacia. Achim From owner-proteins@net.bio.net Sun Mar 02 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!newsfeeds.sol.net!news.maxwell.syr.edu!cpk-news-hub1.bbnplanet.com!cam-news-hub1.bbnplanet.com!news.bbnplanet.com!howland.erols.net!newsxfer.itd.umich.edu!uunet!in2.uu.net!198.168.54.138!rcogate.rco.qc.ca!altitude!usenet From: "Achim Recktenwald, PhD" Newsgroups: bionet.molbio.proteins Subject: Re: Artifact by coomassie blue in amino acid analysis? Date: Mon, 03 Mar 1997 08:01:01 -0500 Organization: IBEX Technologies, Inc., Biochemistry, 5485 Pare, Montreal, PQ, H4P 1P7, Canada Lines: 52 Message-ID: <331ACB8C.1BCB@ibex.ca> References: <5ekd2i$i8u@snunews.snu.ac.kr> <5f4s21$vse@cronkite.ocis.temple.edu> NNTP-Posting-Host: ibex.hip.cam.org Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Macintosh; I; PPC) Stephen P. Driska PhD wrote: > > newera@plaza.snu.ac.kr wrote: > : Can coomassie blue cause an artifact of proline content in > amino acid analysis > : of a protein? > > : I have amino-acid-anayzed my protein 3 times, which is transfered on > : PVDF membrane and stained with coomassie blue. Each time, I have got > : a different result in terms of proline content but the contents of > : the other amino acids have been measured almost the same. > > : What is the possible causes of this problem? > > : -- > : email : newera@plaza.snu.ac.kr > : address : > : Lee, Ji Hyun > : Laboratory of Physical Pharmacy(Prof. Lee, Bong Jin) > > : Seoul National University > : College of Pharmacy > : Shinlim-Dong, Kwanak-Gu > : Seoul 151-742, Korea. > > It's an obvious thing, but could you try removing the coomassie blue > from the PVDF? Also, it might help people suggest an answer if you said what > type of machine or column was being used for the analysis. A few years ago > we were having a problem with coomassie blue and the Waters PicoTag system, > but we had enough protein that we could remove the blue dye with ethanol, I > think 90%. But the protein was freeze dried on the walls of a sample tube, > not stuck to a PVDF membrane. > > Good luck with your project > - Steve > > -- > Steve Driska, Physiology Department, Temple University Medical School > Philadelphia, PA 19140 USA (215) 707-3283 > driska@astro.ocis.temple.edu Check the quality and age of your chemicals. When I did my PhD-thesis, too many years ago, I had similar problems with our protein sequencer. After changing to a new Coomassie and SDS batch of higher quality, the problems disappeared. If you do not want to buy better Coomassie, check Anal. Biochem. during the last two, three years, or so; I have seen at least two papers about prpearing higher quality Coomassie from usual commercial products. Achim From owner-proteins@net.bio.net Sun Mar 02 22:00:00 1997 Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!news.ecn.uoknor.edu!feed1.news.erols.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news.ums.edu!haven.umd.edu!news.umbc.edu!kcarter From: kcarter@umbc.edu (Mr. Ken Carter) Newsgroups: bionet.molbio.proteins Subject: PCR Techniques & Applications Workshop Date: 3 Mar 1997 16:08:59 GMT Organization: University of Maryland, Baltimore County Lines: 62 Message-ID: <5fet2r$ld4@news.umbc.edu> NNTP-Posting-Host: f-umbc7.umbc.edu NNTP-Posting-User: kcarter X-Newsreader: TIN [version 1.2 PL2] >>>>>>>POLYMERSE CHAIN REACTION (PCR) APPLICATIONS/ CYCLE DNA SEQUENCING<<<<<<< American Type Culture Collection (ATCC) April 21-24, 1997, Rockville, MD A four-day, laboratory-intensive, course covering a broad range of applications of the polymerase chain reaction (PCR) for addressing biological problems. Included will be a DNA sequencing experiment using a thermostable DNA polymerase and a temperature cycling protocol (cycle sequencing). The workshop will provide introductory and intermediate level instruction in the application, procedures and trouble-shooting for this extremely useful and versatile technology. A basic knowledge of nucleic acid laboratory procedures is helpful, but not necessary. Approximately 75% of the course is devoted to laboratory instruction while 25% is lecture-oriented. An extensive manual with protocols and procedures is provided for each participant. Lecture topics will include PCR Reaction Basics, Optimizing and Troubleshooting the PCR Reaction, DNA Sequencing, PCR Analysis of Ribosomal DNA to Determine Taxonomic Relatedness, PCR Fingerprinting, PCR in the Diagnosis of Infectious and Genetic Disease, and PCR and Recombinant DNA Methodology. Time will be structured for presentation and discussion of workshop participants' PCR applications/problems. The workshop is intended to be a laboratory-intensive course. Laboratory exercises will include: 1) PCR Diagnosis of Sickle Cell Disease; 2) Fingerprint Analysis of the DNA of Human Individuals; 3) Relatedness of Organisms by Analysis of Amplified Ribosomal DNA; 4) Amplification from mRNA; 5) Optimization of PCR Reactions; 6) Labeling of Hybridization Probes by PCR; 7) Cycle DNA Sequencing; and 8) Fingerprinting of Microorganisms by RAPD and Inter-repeat PCR. Results will be analyzed and discussed. Faculty: William Nierman, Ph.D. (Workshop Director), Director, ATCC Program in Molecular Biology and Virology; Donna Maglott, Ph.D., Research Scientist; Yvonne Reid, Ph.D., Collection Scientist; Francis Molina, Ph.D., Research Scientist; Tamara Feldblyum, M.S., Collection Scientist. * The polymerase chain reaction (PCR) process is covered by patents owned by Hoffmann-La Roche. Use of the PCR process requires a license. Limited to 25 participants FEE: $1,195.00 2.8 CEUs **For a full schedule and on-line registration, please visit our Web site: http://www.atcc.org/workshops/workshop.html **Or request a brochure, which includes a full schedule and registration form from: kcarter@atcc.org -- ****************************************************************************** Ken Carter ^ American Type Culture Collection / l \ (301) 231-5525 12301 Parklawn Drive /___l___\__ fax:(301) 816-4364 Rockville, Maryland 20852 \______/ kcarter@atcc.org ~~~~~~~~~~~~~~ From owner-proteins@net.bio.net Sun Mar 02 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!news.sgi.com!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!dispatch.news.demon.net!demon!netcom.net.uk!nntpfeed.doc.ic.ac.uk!sunsite.doc.ic.ac.uk!charlie.lif.icnet.uk!mac052034.lif.icnet.uk!user From: I.McFarlane@icrf.icnet.uk (Ian McFarlane) Newsgroups: bionet.molbio.proteins Subject: Re: Artifact by coomassie blue in amino acid analysis? Date: Mon, 03 Mar 1997 18:02:54 +0000 Organization: Imperial Cancer Research Fund Message-ID: References: <5ekd2i$i8u@snunews.snu.ac.kr> <5f4s21$vse@cronkite.ocis.temple.edu> NNTP-Posting-Host: mac052034.lif.icnet.uk X-Newsreader: Value-Added NewsWatcher 2.0b27+ Lines: 47 How about staining with amido black instead? Ian mc In article <5f4s21$vse@cronkite.ocis.temple.edu>, driska@astro.ocis.temple.edu (Stephen P. Driska PhD) wrote: > newera@plaza.snu.ac.kr wrote: > : Can coomassie blue cause an artifact of proline content in > amino acid analysis > : of a protein? > > : I have amino-acid-anayzed my protein 3 times, which is transfered on > : PVDF membrane and stained with coomassie blue. Each time, I have got > : a different result in terms of proline content but the contents of > : the other amino acids have been measured almost the same. > > : What is the possible causes of this problem? > > > : -- > : email : newera@plaza.snu.ac.kr > : address : > : Lee, Ji Hyun > : Laboratory of Physical Pharmacy(Prof. Lee, Bong Jin) > > : Seoul National University > : College of Pharmacy > : Shinlim-Dong, Kwanak-Gu > : Seoul 151-742, Korea. > > It's an obvious thing, but could you try removing the coomassie blue > from the PVDF? Also, it might help people suggest an answer if you said what > type of machine or column was being used for the analysis. A few years ago > we were having a problem with coomassie blue and the Waters PicoTag system, > but we had enough protein that we could remove the blue dye with ethanol, I > think 90%. But the protein was freeze dried on the walls of a sample tube, > not stuck to a PVDF membrane. > > Good luck with your project > - Steve > > > -- > Steve Driska, Physiology Department, Temple University Medical School > Philadelphia, PA 19140 USA (215) 707-3283 > driska@astro.ocis.temple.edu From owner-proteins@net.bio.net Sun Mar 02 22:00:00 1997 Newsgroups: bionet.molbio.proteins,bionet.molbio.methds-reagnts,bionet.biophysics Path: biosci!daresbury!yama.mcc.ac.uk!liv!news From: antonio@liverpool.ac.uk Subject: Re: Dissociating agents to prevent protein aggregation Content-Type: text/plain; charset=us-ascii To: newera@plaza.snu.ac.kr Message-ID: <331B1122.28C7@liverpool.ac.uk> Sender: news@liverpool.ac.uk (News System) Nntp-Posting-Host: pc028010.med.liv.ac.uk Content-Transfer-Encoding: 7bit Organization: The University of Liverpool References: <5f1co1$vbu$1@snunews.snu.ac.kr> Mime-Version: 1.0 Date: Mon, 3 Mar 1997 17:57:54 GMT X-Mailer: Mozilla 2.02 (Win16; I) Lines: 49 Xref: biosci bionet.molbio.proteins:10156 bionet.molbio.methds-reagnts:55404 bionet.biophysics:2678 newera@plaza.snu.ac.kr wrote: > > I am purifying a small protein(mw 6100), which tends to aggregate well during its purification process although the completely purified product does not; so I > In choosing dissociating agents, some points should be considered : > > 1. My protein seems to be quite easily renatured, because the last step of its purification is reverse phase HPLC(water:acetonitrile) and freeze-drying. In add > > 2. I am using a cation exchange column(BioRex 70) and an affinity chromatography(Blue Sepharose); the less the agents affect the chromatographies, the better. > > 3. The buffer throughtout the process is 20mM Na phosphate, pH 6.8, no salt. > > 5% ethylene glycol is considered now. > Is EDTA, DTT or 2-mercaptoethanol helpful? > > If you give me any advice on this matter, I will much appreciate it. > > Thanks. > > Lee, Ji Hyun > > -- > email : newera@plaza.snu.ac.kr > address : > Lee, Ji Hyun > Laboratory of Physical Pharmacy(Prof. Lee, Bong Jin) > > Seoul National University > College of Pharmacy > Shinlim-Dong, Kwanak-Gu > Seoul 151-742, Korea. I am aware of some peptides (MW 4330) which readily aggregate at around mid-range pH but can become very much more soluble and stable at others. So you could try changing the pH to 3 or 8, though this would require some test runs to sort out changes to your cation exhange purification system. Perhaps something as simple say addition of non-ionic detergent, such as triton-X to your buffer systems would overcome your problems. DTT and mercaptoethanol are only likely to be very useful if your target protein has cysteines or methionines in it. I hope this helps, Len Bell. Liverpool University. UK. lgbell@liv.ac.uk From owner-proteins@net.bio.net Sun Mar 02 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!news.sgi.com!su-news-hub1.bbnplanet.com!cam-news-hub1.bbnplanet.com!news.bbnplanet.com!howland.erols.net!news-peer.gsl.net!news.gsl.net!news-paris.gsl.net!news.gsl.net!news.kolumbus.fi!news.funet.fi!mordred.cc.jyu.fi!news From: Olli Heikki Laitinen Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: A BiaCore-like system Date: Mon, 03 Mar 1997 16:24:08 +0200 Organization: Department of Bio and Enviromental science, Jyväskylä, Finland Lines: 24 Message-ID: <331ADF08.7391@dodo.jyu.fi> References: <3315D605.41C6@gandalf.psf.sickkids.on.ca> NNTP-Posting-Host: vap218a.bio.jyu.fi Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (Win95; I) Xref: biosci bionet.molbio.methds-reagnts:55398 bionet.molbio.proteins:10152 Randy Willis wrote: > > Hey everybody (sorry for the X-posting), > > There's a rumour flying around here about a relatively new system for > looking at macromolecular interactions based on a BiaCore like set up of > hooking one molecule to an electronic chip, passing a solution of the > second molecule along the surface and looking for changes in the > potential as the two molecules interact. > > Any ideas as to what this might be and who would manufacture such a > beastie? We've already got access to a BiaCore and just want to make a > comparison. The equipment you possible mean is IAsys that is manufactured by Affinity sensors. The system comes from UK. It's not that new, I think that their biosensor has lifehistory of at least three or four years. The main difference between these two device is that when Biacore has flowthrough system the Iasys is based on cuvette construction. You can find a short "review" about the subject from newspaper Genetic engineering news: november 15, 1996. Cheers, Olli From owner-proteins@net.bio.net Sun Mar 02 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!news.sgi.com!news.maxwell.syr.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: 1 mM Tris? Date: Mon, 03 Mar 1997 17:00:00 -0500 Organization: University of Wisconsin-Madison Lines: 17 Message-ID: References: <5fdlo2$v3@news2.kol.co.kr> NNTP-Posting-Host: alpha.oncology.wisc.edu X-Newsreader: Yet Another NewsWatcher 2.1.8 In article <5fdlo2$v3@news2.kol.co.kr>, hbkg@hitel.kol.co.kr () wrote: :We tried to dissolve DTNB, CAT substrate, in Tris buffer, and :found that DTNB was not dissolved in Tris buffer at all, which :was very strange. We changed water in vain. :We found at last the mistake of using 1mM Tris instead of appropriate :concentration. :I am curious HOW TRIS AFFECTS DTNB SOLUBILITY. My guess is that the low level of Tris was not able to buffer the addition of the DTNB. The benzoate groups lowered the pH so that only some of the compound was in the ionic form and insoluable material was the protonated, non-ionic form. -- Scott McMahan mcmahan@oncology.wisc.edu From owner-proteins@net.bio.net Mon Mar 03 22:00:00 1997 Newsgroups: bionet.molbio.proteins Path: biosci!daresbury!hgmp.mrc.ac.uk!ebi.ac.uk!higgins From: higgins@ebi.ac.uk Subject: Re: (none) Sender: news@ebi.ac.uk (usenet news) Message-ID: <1997Mar4.141815@ebi.ac.uk> Date: Tue, 4 Mar 1997 14:18:15 GMT Lines: 27 References: Organization: European BioInformatics Institute In article , sra@NCBS.TIFRBNG.RES.IN ("S. Ramachandran") writes: > Dear nettters, > I am on the lookout for a fortran or c-program, which on inputting > phi,psi and chi's for an amino acid sequence, would calculate co-ordinates. > I would be thankful if anyone can give me info on a public domain > softwware for doing this. > Thanx a lot. > > ****************************** > S.RAMACHANDRAN > NATIONAL CENTER FOR BIOLOGICAL SCIENCES > TIFR CENTRE, BANGALORE-560012 > FAX:90-80-3343851 > TEL:90-80-3344062 > E-MAIL: sra@ncbs.tifrbng.res.in > If you do get any positive replies, can you please post summary details here (bionet.molbio.proteins) of any software that you find that can do this. Your surname is highly appropriate for this topic :-). Thanks in advance, Des Higgins Biochemistry, UCC, Ireland. > From owner-proteins@net.bio.net Mon Mar 03 22:00:00 1997 Path: biosci!NCBS.TIFRBNG.RES.IN!sra From: sra@NCBS.TIFRBNG.RES.IN ("S. Ramachandran") Newsgroups: bionet.molbio.proteins Subject: (none) Date: 4 Mar 1997 04:44:52 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 16 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Dear nettters, I am on the lookout for a fortran or c-program, which on inputting phi,psi and chi's for an amino acid sequence, would calculate co-ordinates. I would be thankful if anyone can give me info on a public domain softwware for doing this. Thanx a lot. ****************************** S.RAMACHANDRAN NATIONAL CENTER FOR BIOLOGICAL SCIENCES TIFR CENTRE, BANGALORE-560012 FAX:90-80-3343851 TEL:90-80-3344062 E-MAIL: sra@ncbs.tifrbng.res.in From owner-proteins@net.bio.net Mon Mar 03 22:00:00 1997 Path: biosci!eastman.ucl.ac.uk!akirby From: akirby@eastman.ucl.ac.uk (Alun Kirby) Newsgroups: bionet.molbio.proteins Subject: GPI-specific Phospholipase Inhibitors Date: 4 Mar 1997 01:59:42 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 7 Sender: daemon@net.bio.net Distribution: world Message-ID: <331BE735.61B4@eastman.ucl.ac.uk> NNTP-Posting-Host: net.bio.net I am looking for inhibitors of GPI-specific phospholipases C and D. I understand that 2-nitro-4-carboxyphenyl-N,N-diphenylcarbmate (NCDC) may work for GPI-PLC. Can anyone confirm this and suggest an appropriate working concentration? Thanks. Alun Kirby. From owner-proteins@net.bio.net Mon Mar 03 22:00:00 1997 Path: biosci!daresbury!not-for-mail From: yrgan@tju.edu.cn Newsgroups: bionet.molbio.proteins Subject: Stru. information for Interleukin-6(human) Date: 4 Mar 1997 12:59:25 -0000 Lines: 13 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <5fh6bd$h5l@mserv1.dl.ac.uk> Apparently-To: proteins@dl.ac.uk Hi, Dear Netters: Can anyone provide me the structural information for the human Interleukin-6? For example, the PDB code(if any) for it. Thanks in advance! E-mail: yrgan@tju.edu.cn Y. R. Gan From owner-proteins@net.bio.net Mon Mar 03 22:00:00 1997 Path: biosci!biosci!not-for-mail From: biodigm@dial.pipex.com (M J Geisow) Newsgroups: bionet.molbio.proteins Subject: ANNOUNCE: Reduction in registration - Perspectives Conference Date: 4 Mar 1997 21:41:34 -0800 Organization: UUNet PIPEX server (post doesn't reflect views of UUNet PIPEX) Lines: 35 Sender: daemon@net.bio.net Distribution: world Message-ID: <331b829b.57386312@news.dial.pipex.com> NNTP-Posting-Host: net.bio.net The Programme for the Perspectives on Protein Engineering Conference (Norwich Norfolk, UK 28 June - 1 July 1997) is now published, with speaker abstracts received to date at the Web site: http://www.biodigm.com/pope/pope6.htm A new conference rate for non-profit organisations and further reductions for pre-doctoral students is posted at the site Day 1 Genome - Proteome Opening speakers: J Craig Venter (TIGR) Amos Bairoch (Geneva) Mike Bevan (Norwich) Iain Campbell (Oxford) Minisymposia Membrane protein expression & structure Collaboration in bioinformatics via WWW Protein characterisation Metal Centres in Proteins Day 2 Protein Design & Expression Please See site for speakers Day 3 Structure-function: biocatalysis & plant protein engineering Please See site for speakers POPE 6 Secretariat biodigm@dial.pipex.com 64, Langdale Grove Bingham, NG13 8SS, UK Fax +44 1 949 876 156 M Geisow(UK Structural Biology CEC Contact Group representative) From owner-proteins@net.bio.net Mon Mar 03 22:00:00 1997 Path: biosci!biosci!not-for-mail From: Computational Molecular Biology Newsgroups: bionet.software.gcg,bionet.software,bionet.molbio.proteins Subject: US-NC Bioinformatics - Sequence Analysis Position Date: 4 Mar 1997 21:51:00 -0800 Organization: The University of North Carolina at Chapel Hill Lines: 29 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Xref: biosci bionet.software.gcg:2261 bionet.software:18044 bionet.molbio.proteins:10164 The University of North Carolina at Chapel Hill has an immediate opening for a specialist in nucleic acid and protein sequence analysis to provide a leadership role in supporting the community of several hundred users of biocomputational tools. The successful candidate will organize courses for users, prepare documentation, and assist individual users with their sequence analysis problems. Development of publishable teaching materials or articles will be encouraged. Strong knowledge of DNA and protein sequence analysis algorithms and software is essential. Teaching experience and a background in laboratory molecular biology are highly desirable. The position requires excellent oral and written communication skills. Salary is in the range of $33-38K depending on the experience of the applicant. The University of North Carolina is an Equal Opportunity Employer. Applications or additional questions about this position may be sent via regular or email to: Philip Carl, Chair of the Search Committee 101 MBBRL, CB7100 UNC Medical School Chapel Hill, NC 27599-7100 Phone: (919)-966-3544 Fax: 919-966-6821 ====================================================================== Computational Resource for Molecular Sciences and Biotechnology at UNC http://mmlin4.pha.unc.edu/~cmb96/ From owner-proteins@net.bio.net Tue Mar 04 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!cnn.Princeton.EDU!usenet From: Nancy Vogelaar Newsgroups: bionet.molbio.proteins,bionet.xtallography,bionet.structural-nmr Subject: Re: structs vs reality? Date: Thu, 27 Feb 1997 12:14:51 -0500 Organization: Princeton University Lines: 32 Message-ID: <3315C10B.167E@atp.princeton.edu> References: <33134D9A.57D6@spork.niddk.nih.gov> NNTP-Posting-Host: atp.princeton.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (X11; I; IRIX 5.3 IP22) To: John Kuszewski Xref: biosci bionet.molbio.proteins:10168 bionet.xtallography:3256 bionet.structural-nmr:1801 John Kuszewski wrote: > > How well do xray or NMR structures actually model > reality in vivo? > > I've seen some old papers showing that some enzymes > still work in the crystalline state, but there > must be something else about this. > > Can anyone point me toward some references? Hi, John! You may find this paper of interest--especially since it deals with quaternary structure and allostery in crystals vs. solution. D.I. Svergun et al., PROTEINS: Str., Fn., Genet. 27 (1997),pp. 110-117. "Large Differences Are Observed Between the Crystal and Solution Quaternary Structures of Allosteric Aspartate Transcarbamylase in the R State" Nancy --------------------------------------------------------------------- Nancy Vogelaar Department of Chemistry Phone: (609) 258-2927 Princeton University Fax: (609) 258-6746 Princeton, NJ 08544 email: gc@atp.princeton.edu --------------------------------------------------------------------- From owner-proteins@net.bio.net Tue Mar 04 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!gatech!csulb.edu!hammer.uoregon.edu!xfer.kren.nm.kr!www.nntp.primenet.com!nntp.primenet.com!europa.clark.net!cpk-news-hub1.bbnplanet.com!cam-news-hub1.bbnplanet.com!news.bbnplanet.com!howland.erols.net!ais.net!ameritech.net!uunet!in2.uu.net!136.142.185.26!newsfeed.pitt.edu!mv29.pathology.pitt.edu!user From: pxpst2@vms.cis.pitt.edu (THE GREEK) Newsgroups: bionet.molbio.proteins Subject: Protein precipitation methods Date: 5 Mar 1997 13:21:42 GMT Organization: USA Lines: 6 Message-ID: NNTP-Posting-Host: mv29.pathology.pitt.edu I am interested in getting some references to some precipitation methods other than TCA. The method must be suitable for low protein concentrations. Please reply by email and thanks in advance Peter Pediaditakis pxpst2@vms.cis.pitt.edu From owner-proteins@net.bio.net Tue Mar 04 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!cs.utexas.edu!howland.erols.net!rill.news.pipex.net!pipex!warwick!leicester!usenet From: "Dr E. Buxbaum" Newsgroups: bionet.molbio.proteins Subject: Re: questions about protein purification equipment Date: 5 Mar 1997 12:47:30 GMT Organization: University of Leicester (PCFS User) Lines: 5 Message-ID: <5fjq12$77r@falcon.le.ac.uk> References: <19970301200500.PAA15920@ladder02.news.aol.com> NNTP-Posting-Host: pc5.msb.le.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) I have not worked with the ATKA system, but with FPLC and GradiFrac. My general impresion is that Pharmacia columns are the best around, but their pumps are somewhat problematic, particularily with crude feeds. Fraction collectors and monitors appear to be of standard quality. From owner-proteins@net.bio.net Tue Mar 04 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!cs.utexas.edu!geraldo.cc.utexas.edu!usenet From: Brett Phinney Newsgroups: bionet.molbio.proteins Subject: Radius of Gyration Date: Wed, 05 Mar 1997 09:17:42 -0800 Organization: University of Texas at Austin Lines: 11 Message-ID: <331DAAB6.11BF@mail.utexas.edu> Reply-To: brettsp@mail.utexas.edu NNTP-Posting-Host: photo.botany.utexas.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01Gold (Win16; I) Does anyone have a good reference on determining a proteins radius of gyration. I do not know much about this and I do not know what has to be known about the protein before this can be done, ie do you have to know the crystal structure of the protein first? Thanks a lot Brett Phinney Cell Research Institute University of Texas, Austin From owner-proteins@net.bio.net Tue Mar 04 22:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!europa.clark.net!cpk-news-hub1.bbnplanet.com!cam-news-hub1.bbnplanet.com!news.bbnplanet.com!howland.erols.net!torn!newsflash.concordia.ca!sunqbc.risq.net!athena.ulaval.ca!gbp374.ulaval.ca!user From: elauzier@fse.ulaval.ca (Edouard Lauzier) Newsgroups: bionet.metabolic-reg,bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Glycogen Synthase info needed... Date: 5 Mar 1997 20:23:55 GMT Organization: Graduate Student ( LABSAP U. Laval ) Lines: 30 Message-ID: NNTP-Posting-Host: gbp374.ulaval.ca Xref: biosci bionet.metabolic-reg:1078 bionet.molbio.methds-reagnts:55482 bionet.molbio.proteins:10170 First of all sorry if I'm not in the correct newsgroup... I was looking at how to measure glycogen synthase in a muscle extract activity for the first time and a few things are not so clear... They say that to eliminate the influence of endogenous competitive effectors and activators, the enzyme extract is diluted 100-fold. To be sure, you can check the activity with no exogenous G-6-P ( should be real low ). Then you can measure with 0.1mM G-6-P and 10mM G-6-P ( total GS activity ) and use the data as you wish afterward... 1) What exactly do represent the GS activity with 0.1mM G-6-P added ? Is it the physiological state the GS was in before the muscle extraction ? If so, what happened during the homogenization that causes the 0mM G-6-P measurement to be different from the 0.1mM one ? Did the I-form change toward nearly only D-form during the homogenization ? What's happening to the phosphatases and the kinases during the homogenization and afterward ?? Thanks a million time for your precious help... ps: please reply to my address so I can see your answer for sure... -- Edouard Lauzier, B.Sc. elauzier@fse.ulaval.ca Physical Activity Science Laboratory (418) 656-2131 #2929 (Laval University) G1K 7P4 CANADA From owner-proteins@net.bio.net Tue Mar 04 22:00:00 1997 Path: biosci!daresbury!is.bbsrc.ac.uk!news From: dunnsm@bbsrc.ac.uk (Steve Dunn) Newsgroups: bionet.molbio.proteins Subject: Help! Triton X114 blues.... Date: 5 Mar 1997 17:36:21 GMT Organization: Institute of Arable Crops Research, Rothamsted Lines: 20 Message-ID: <5fkaul$jrd@is.bbsrc.ac.uk> NNTP-Posting-Host: pc773.res.bbsrc.ac.uk X-Newsreader: WinVN 0.92.6+ Dear netters, I've been dabbling with Triton X114 phase-partitioning of my microsomal fraction. After washing the detergent-rich phase a few times with PBS, I (following a procedure in Methods In Enzymology...) precipitate the protein with 10 volumes of ice-cold acetone. This, according to my method, is supposed to get rid of the detergent so that it doesn't interfere with SDS-PAGE gels. After pelleting the protein and removing all the acetone, I resuspend the pellet in 5%SDS and loading buffer for SDS-PAGE. However, after staining the gel with coomassie, there's an artifactual smear obscuring my protein bands which I understand is symptomatic of X114 contamination. Even after processing the acetone pellet through a chloroform/MeOH cleanup (Wessel & Flugge, Anal. Biochem.,1984) the smear persists! If I run just SDS-solubilised microsomes, there's no smear. Any ideas how to successfully combine X114 extraction with SDS-PAGE?? All suggestions gratefully received... Steve Dunn dunnsm@bbsrc.ac.uk From owner-proteins@net.bio.net Tue Mar 04 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!cs.utexas.edu!howland.erols.net!feed1.news.erols.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!cpk-news-feed4.bbnplanet.com!maze.dpo.uab.edu!usenet From: XiaoYin Zhong Newsgroups: bionet.molbio.proteins Subject: how to stop protein degradation? Date: Wed, 05 Mar 1997 18:25:14 -0600 Organization: University of Alabama at Birmingham Lines: 13 Message-ID: <331E0EE9.7141@uabdpo.dpo.uab.edu> Reply-To: medm049@uabdpo.dpo.uab.edu NNTP-Posting-Host: 138.26.20.196 Mime-Version: 1.0 Content-Type: text/plain; charset=gb2312 Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01Gold (Win95; I) Dear Dr. protein: I cloned a protein with His-tag at C-end. After I purified this perotein by Ni-column, SDS-PAGE gel show that the protein was degraded.What is the reason? Is there any other methods which can solve this problem except using proteinase inhibiter coke-tail or EDTA in the buffer? Thank you for your help! qiu@orion.cmc.uab.edu From owner-proteins@net.bio.net Tue Mar 04 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!cs.utexas.edu!howland.erols.net!feed1.news.erols.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!cpk-news-feed4.bbnplanet.com!maze.dpo.uab.edu!usenet From: XiaoYin Zhong Newsgroups: bionet.molbio.proteins Subject: test Date: Wed, 05 Mar 1997 18:27:07 -0600 Organization: University of Alabama at Birmingham Lines: 1 Message-ID: <331E0F5B.6BE5@uabdpo.dpo.uab.edu> Reply-To: medm049@uabdpo.dpo.uab.edu NNTP-Posting-Host: 138.26.20.196 Mime-Version: 1.0 Content-Type: text/plain; charset=gb2312 Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01Gold (Win95; I) test From owner-proteins@net.bio.net Wed Mar 05 22:00:00 1997 Path: biosci!agate!spool.mu.edu!uwm.edu!cs.utexas.edu!howland.erols.net!torn!resunix.sickkids.on.ca!news From: Randy Willis Newsgroups: bionet.molbio.proteins Subject: Re: how to stop protein degradation? Date: Thu, 06 Mar 1997 09:21:32 -0500 Organization: The Hospital For Sick Children Lines: 35 Message-ID: <331ED2EC.167E@gandalf.psf.sickkids.on.ca> References: <331E0EE9.7141@uabdpo.dpo.uab.edu> NNTP-Posting-Host: gollum.psf.sickkids.on.ca Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0 (X11; I; IRIX 5.3 IP12) To: medm049@uabdpo.dpo.uab.edu XiaoYin Zhong wrote: > Is there any other methods which can solve this problem except using proteinase inhibiter coke-tail or EDTA in the buffer? XiaoYin, You may want to be careful as to how much EDTA you add to your system as it may interfere with the chelation of the Ni++ on your column. Otherwise, it is common practice to use a melange of protease inhibitors, at least for the initial steps of purification (ie lysis and column #1). If you are concerned about costs and don't mind a little leg work, borrow some inhibitors from neighbours and try to determine from small lyses and columns (or batch) which specific inhibitor stops the degradation, thus minimizing what you need in the buffer. Boehringer Mannheim biochemicals sells an inhibitor cocktail tablet but they tend to be a little expensive for large scale protein preparations. But as a first step, this may assist you in getting your protein to the point where it has been purified away from the protease. Also, beware of the possibility that your protein is being degraded during expression in the cell. You could look for this by taking whole cell lysates and performing a Western blot with anti-His tag antibodies which are commercially available from a number of companies. Good luck, Randall C Willis, Researcher Biochemistry, Hosp for Sick Children 3522-555 University Ave Toronto, ON M5G 1X8 CANADA 416-813-5933 (ph) -5022 (fax) willis@gandalf.psf.sickkids.on.ca From owner-proteins@net.bio.net Wed Mar 05 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!gatech!csulb.edu!hammer.uoregon.edu!arclight.uoregon.edu!dispatch.news.demon.net!demon!netcom.net.uk!nntpfeed.doc.ic.ac.uk!sunsite.doc.ic.ac.uk!lyra.csx.cam.ac.uk!tnrk1 From: tnrk1@cus.cam.ac.uk (T.N.R. Killick) Newsgroups: bionet.molbio.proteins Subject: Auxotrophic proline strains Date: 6 Mar 1997 18:47:54 GMT Organization: University of Cambridge, England Message-ID: <5fn3gq$ncg@lyra.csx.cam.ac.uk> NNTP-Posting-Host: ursa.cus.cam.ac.uk X-Newsreader: TIN [version 1.2 PL2] Lines: 7 I have been trying for some time now to overexpress my protein in a number of strains that are auxotrophic for proline but without sucess. A number of stains do not transform at all. Other strains express very poorly. DOes anyone out thre have expeience in dealing with these problems. If so please email me at tnrk1@cus.cam.ac.uk cheers Tom From owner-proteins@net.bio.net Wed Mar 05 22:00:00 1997 Path: biosci!aol.com!ANATRAC434 From: ANATRAC434@aol.com Newsgroups: bionet.molbio.proteins Subject: Re: Help! Triton X114 blues.... Date: 6 Mar 1997 07:32:47 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 7 Sender: daemon@net.bio.net Distribution: world Message-ID: <970306103307_1813799831@emout14.mail.aol.com> NNTP-Posting-Host: net.bio.net Anatrace has a purified Triton X114 that is low in peroxides. The catalog number is APX114. Hope that helps. Mel Keyes Anatrace, Inc. From owner-proteins@net.bio.net Wed Mar 05 22:00:00 1997 Path: biosci!daresbury!not-for-mail From: Tim Hubbard Newsgroups: bionet.molbio.proteins Subject: ANNOUNCEMENT: have you a protein to predict? Call for prediction targets Date: 6 Mar 1997 09:23:26 -0000 Lines: 29 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <5fm2ee$edd@mserv1.dl.ac.uk> X-Sender: th@netra.sanger.ac.uk Original-To: pdb-l@pdb.pdb.bnl.gov, bioforum@dl.ac.uk, bionews@dl.ac.uk, biophys@dl.ac.uk, bio-soft@dl.ac.uk, comp-bio@dl.ac.uk, methods@dl.ac.uk, molmodel@dl.ac.uk, proteins@dl.ac.uk, xtal-log@dl.ac.uk, str-nmr@dl.ac.uk Announcement: Call prediction targets ===================================== The previous mail to this list announced the FEBS advanced course: frontiers of protein structure prediction 1997. The aim of the workshop is to predict as much as possible about the structure of a number of proteins of biological interest, taking advantage of the most recent methodologies for fold recognition and ab initio prediction. If you are interested in a structure prediction being made on a protein for which there is no sign of an experimental structure and does not appear to be homologous to any known structure, please consider submitting it as a target for this course. For further information and on-line target submission forms see: http://predict.sanger.cam.ac.uk/irbm-course97/ For the automatic analysis carried out on the 113 targets received for the 1995 course and the predictions made for 17 of them see: http://predict.sanger.cam.ac.uk/irbm-course95/ Tim Hubbard, (Sanger Centre) Anna Tramontano, (IRBM) From owner-proteins@net.bio.net Wed Mar 05 22:00:00 1997 Path: biosci!daresbury!not-for-mail From: Tim Hubbard Newsgroups: bionet.molbio.proteins Subject: ANNOUNCEMENT: FEBS advanced course: frontiers of protein structure prediction 1997 Date: 6 Mar 1997 09:23:17 -0000 Lines: 65 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <5fm2e5$eck@mserv1.dl.ac.uk> X-Sender: th@netra.sanger.ac.uk Original-To: pdb-l@pdb.pdb.bnl.gov, bioforum@dl.ac.uk, bionews@dl.ac.uk, biophys@dl.ac.uk, bio-soft@dl.ac.uk, comp-bio@dl.ac.uk, methods@dl.ac.uk, molmodel@dl.ac.uk, proteins@dl.ac.uk, xtal-log@dl.ac.uk, str-nmr@dl.ac.uk Announcement: Call for applications =================================== FEBS advanced course: frontiers of protein structure prediction 1997 http://predict.sanger.cam.ac.uk/irbm-course97/ The course, which is being run for the second time (see http://predict.sanger.ac.uk/irbm-course95), is directed at young scientists with some experience and a strong interest in protein structure and structure prediction who wish to learn about the latest developments in the field. The aim of the workshop is to predict as much as possible about the structure of a number of proteins of biological interest, taking advantage of the most recent methodologies for fold recognition and ab initio prediction. The participants will be divided into working groups assisted by an instructor. Each group will be equipped with state of the art software and hardware (kindly provided by Silicon Graphics) and assigned the sequences of proteins whose structure has to be predicted. The predictions will be made public as a technical document and also available via World Wide Web. Suggestions for target proteins can also be submitted by non-participants via WWW (see accompanying email) Organizers: Tim Hubbard (Sanger Centre), Anna Tramontano (IRBM) Instructors: G. Barton (Oxford), T. Hubbard (Cambridge), D. Jones (London), M. Sippl (Salzburg), A. Valencia (Madrid) Lecturers: A. Lesk (Cambridge), J. Moult (Rockville), B. Rost (Heidelberg) Dates: 7-20 October 1997 Deadline for applications: 30th June 1997. Registration fee 1200 DM (includes accomodation and meals) Location: IRBM (Istituto di Ricerche di Biologia Molecolare) Pomezia, Rome, Italy Further information and on-line application forms: http://predict.sanger.cam.ac.uk/irbm-course97/ Prof. Anna Tramontano IRBM, Via Pontina Km 30.600 I-00040 Pomezia (Rome) Tel: +39 6 91093207 Fax: +39 6 91093654 email: tramontano@irbm.it Tim Hubbard, Anna Tramontano ------------------------------------------------------------------------- Dr Tim Hubbard email: th@sanger.ac.uk Sanger Centre Tel (direct): +44 1223 494983 Wellcome Trust Genome Campus Tel (switch): +44 1223 834244 Hinxton Fax: +44 1223 494919 Cambridgeshire. CB10 1SA. URL: http://www.sanger.ac.uk/ ------------------------------------------------------------------------- From owner-proteins@net.bio.net Wed Mar 05 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!news-peer.gsl.net!news.gsl.net!EU.net!Austria.EU.net!01-newsfeed.univie.ac.at!03-newsfeed.univie.ac.at!news.univie.ac.at!news-admin@univie.ac.at From: a8803349@unet.univie.ac.at (Martin Offterdinger) Newsgroups: bionet.molbio.proteins Subject: Re: Help! Triton X114 blues.... Date: Thu, 06 Mar 1997 08:09:46 GMT Organization: AKH Lines: 29 Message-ID: <331e7b2b.2169592@news.univie.ac.at> References: <5fkaul$jrd@is.bbsrc.ac.uk> NNTP-Posting-Host: grunt.imed1.akh-wien.ac.at X-Newsreader: Forte Free Agent 1.1/16.230 On 5 Mar 1997 17:36:21 GMT, dunnsm@bbsrc.ac.uk (Steve Dunn) wrote: >Dear netters, > >I've been dabbling with Triton X114 phase-partitioning of my microsomal >fraction. After washing the detergent-rich phase a few times with PBS, >I (following a procedure in Methods In Enzymology...) precipitate the >protein with 10 volumes of ice-cold acetone. This, according to my method, >is supposed to get rid of the detergent so that it doesn't interfere with >SDS-PAGE gels. After pelleting the protein and removing all the >acetone, I resuspend the pellet in 5%SDS and loading buffer for SDS-PAGE. >However, after staining the gel with coomassie, there's an artifactual >smear obscuring my protein bands which I understand is symptomatic of >X114 contamination. Even after processing the acetone pellet through a >chloroform/MeOH cleanup (Wessel & Flugge, Anal. Biochem.,1984) the smear >persists! If I run just SDS-solubilised microsomes, there's no smear. >Any ideas how to successfully combine X114 extraction with SDS-PAGE?? >All suggestions gratefully received... > >Steve Dunn >dunnsm@bbsrc.ac.uk Dear Steve Triton is know to be able to form peroxides in solution , which might partially react with your protein and therefore cause this smear. Check if your Triton is expired, store in the dark(!) , use reducing agents to reduce the peroxides(e.g.:DTT) ... Hope this helps Martin From owner-proteins@net.bio.net Wed Mar 05 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!gatech!csulb.edu!news.sgi.com!esiee.fr!jussieu.fr!univ-lyon1.fr!u-psud.fr!crihan.fr!univ-rennes1.fr!melon.univ-brest.fr!news From: "Fadi.Adas" Newsgroups: bionet.molbio.proteins Subject: DNA/derivatization/TMS/Guanine Date: Thu, 06 Mar 1997 09:05:49 +0100 Organization: faculty of medicine brest france Lines: 12 Message-ID: <331E7ADD.6499@univ-brest.fr> Reply-To: Fadi.Adas@univ-brest.fr NNTP-Posting-Host: rtc7.univ-brest.fr Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (Win95; I) GC analysis DNA bases.TMS derivatization condition? -- Fadi ADAS Equipe d'accueil Alcool-Nutrition-Xenobiotique(EA-948) Faculte de Medecine de BREST Service de Biochimie 22,Avenue Camille Desmoulins 29285-BREST CEDEX FRANCE Tel:(33)02 98 01 64 65 (work) Tel:(33)02 98 42 08 58 (home) Fax:02 98 01 66 03 From owner-proteins@net.bio.net Wed Mar 05 22:00:00 1997 Path: biosci!daresbury!not-for-mail From: Tim Hubbard Newsgroups: bionet.molbio.proteins Subject: ANNOUNCEMENT2: FEBS advanced course: frontiers of protein structure prediction 1997 Date: 6 Mar 1997 12:34:21 -0000 Lines: 75 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <5fmdkd$1v7@mserv1.dl.ac.uk> X-Sender: th@netra.sanger.ac.uk Original-To: pdb-l@pdb.pdb.bnl.gov, bioforum@dl.ac.uk, bionews@dl.ac.uk, biophys@dl.ac.uk, bio-soft@dl.ac.uk, comp-bio@dl.ac.uk, methods@dl.ac.uk, molmodel@dl.ac.uk, proteins@dl.ac.uk, xtal-log@dl.ac.uk, str-nmr@dl.ac.uk [sorry - with correct web addresses this time] Announcement: Call for applications =================================== FEBS advanced course: frontiers of protein structure prediction 1997 http://predict.sanger.ac.uk/irbm-course97/ The course, which is being run for the second time (see http://predict.sanger.ac.uk/irbm-course95), is directed at young scientists with some experience and a strong interest in protein structure and structure prediction who wish to learn about the latest developments in the field. The aim of the workshop is to predict as much as possible about the structure of a number of proteins of biological interest, taking advantage of the most recent methodologies for fold recognition and ab initio prediction. The participants will be divided into working groups assisted by an instructor. Each group will be equipped with state of the art software and hardware (kindly provided by Silicon Graphics) and assigned the sequences of proteins whose structure has to be predicted. The predictions will be made public as a technical document and also available via World Wide Web. Suggestions for target proteins can also be submitted by non-participants via WWW (see accompanying email) Organizers: Tim Hubbard (Sanger Centre), Anna Tramontano (IRBM) Instructors: G. Barton (Oxford), T. Hubbard (Cambridge), D. Jones (London), M. Sippl (Salzburg), A. Valencia (Madrid) Lecturers: A. Lesk (Cambridge), J. Moult (Rockville), B. Rost (Heidelberg) Dates: 7-20 October 1997 Deadline for applications: 30th June 1997. Registration fee 1200 DM (includes accomodation and meals) Location: IRBM (Istituto di Ricerche di Biologia Molecolare) Pomezia, Rome, Italy Further information and on-line application forms: http://predict.sanger.ac.uk/irbm-course97/ Prof. Anna Tramontano IRBM, Via Pontina Km 30.600 I-00040 Pomezia (Rome) Tel: +39 6 91093207 Fax: +39 6 91093654 email: tramontano@irbm.it Tim Hubbard, Anna Tramontano ------------------------------------------------------------------------- Dr Tim Hubbard email: th@sanger.ac.uk Sanger Centre Tel (direct): +44 1223 494983 Wellcome Trust Genome Campus Tel (switch): +44 1223 834244 Hinxton Fax: +44 1223 494919 Cambridgeshire. CB10 1SA. URL: http://www.sanger.ac.uk/ ------------------------------------------------------------------------- ------------------------------------------------------------------------- Dr Tim Hubbard email: th@sanger.ac.uk Sanger Centre Tel (direct): +44 1223 494983 Wellcome Trust Genome Campus Tel (switch): +44 1223 834244 Hinxton Fax: +44 1223 494919 Cambridgeshire. CB10 1SA. URL: http://www.sanger.ac.uk/ ------------------------------------------------------------------------- From owner-proteins@net.bio.net Wed Mar 05 22:00:00 1997 Path: biosci!daresbury!not-for-mail From: Tim Hubbard Newsgroups: bionet.molbio.proteins Subject: ANNOUNCEMENT2: have you a protein to predict? Call for prediction targets Date: 6 Mar 1997 12:34:17 -0000 Lines: 39 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <5fmdk9$1ul@mserv1.dl.ac.uk> X-Sender: th@netra.sanger.ac.uk Original-To: pdb-l@pdb.pdb.bnl.gov, bioforum@dl.ac.uk, bionews@dl.ac.uk, biophys@dl.ac.uk, bio-soft@dl.ac.uk, comp-bio@dl.ac.uk, methods@dl.ac.uk, molmodel@dl.ac.uk, proteins@dl.ac.uk, xtal-log@dl.ac.uk, str-nmr@dl.ac.uk [sorry - with correct web addresses this time] Announcement: Call prediction targets ===================================== The previous mail to this list announced the FEBS advanced course: frontiers of protein structure prediction 1997. The aim of the workshop is to predict as much as possible about the structure of a number of proteins of biological interest, taking advantage of the most recent methodologies for fold recognition and ab initio prediction. If you are interested in a structure prediction being made on a protein for which there is no sign of an experimental structure and does not appear to be homologous to any known structure, please consider submitting it as a target for this course. For further information and on-line target submission forms see: http://predict.sanger.ac.uk/irbm-course97/ For the automatic analysis carried out on the 113 targets received for the 1995 course and the predictions made for 17 of them see: http://predict.sanger.ac.uk/irbm-course95/ Tim Hubbard, (Sanger Centre) Anna Tramontano, (IRBM) ------------------------------------------------------------------------- Dr Tim Hubbard email: th@sanger.ac.uk Sanger Centre Tel (direct): +44 1223 494983 Wellcome Trust Genome Campus Tel (switch): +44 1223 834244 Hinxton Fax: +44 1223 494919 Cambridgeshire. CB10 1SA. URL: http://www.sanger.ac.uk/ ------------------------------------------------------------------------- From owner-proteins@net.bio.net Wed Mar 05 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!news.sgi.com!csulb.edu!drivel.ics.uci.edu!news.service.uci.edu!rigel.oac.uci.edu!jlhumphr From: Jennifer Humphries Newsgroups: bionet.molbio.proteins Subject: help: enzyme purification Date: Thu, 6 Mar 1997 14:36:34 -0800 Organization: University of California, Irvine Lines: 18 Message-ID: NNTP-Posting-Host: rigel.oac.uci.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII I started with a crude extract from potatoes and have purified the extract through a anion exchange(Q-sepharose) and a hydrophobic interaction(Phenyl-toyopearl) columns. In both cases I eluted the enzyme using either a step wise or linear salt gradient. The activity of the enzyme was monitored after each column and the solution was desalted by ultrafiltration(amicon centriplus conc.). Now I am at the last stage of the purification, a strong anion exchange column(PL-SAX) and again trying to elute the enzyme with a salt gradient. The problem is, when I inject the enzyme onto the column I never see any activity in the eluted fractions. It is only when I don't run a gradient and instead just run the highest salt concentration moblie phase and inject the enzyme that I recover the active enzyme. So I know its not a problem with the injector or the column is mucked up. I don't understand. I appreciate any suggestions. Jennifer From owner-proteins@net.bio.net Wed Mar 05 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!news.sgi.com!su-news-hub1.bbnplanet.com!news.bbnplanet.com!newsout1.alt.net!news1.alt.net!news.u.washington.edu!uw-beaver!entropy1!nwnews.wa.com!brokaw.wa.com!littsj From: littsj@zgi.com (Jim Litts) Newsgroups: bionet.molbio.proteins Subject: Re: Radius of Gyration Date: Thu, 06 Mar 1997 14:05:28 -0800 Organization: Zymogenetics, Inc. Lines: 19 Message-ID: References: <331DAAB6.11BF@mail.utexas.edu> NNTP-Posting-Host: worf.zgi.com Mime-Version: 1.0 Content-Type: text/plain; charset=ISO-8859-1 Content-Transfer-Encoding: 8bit X-Newsreader: Yet Another NewsWatcher 2.3.4 In article <331DAAB6.11BF@mail.utexas.edu>, brettsp@mail.utexas.edu wrote: > Does anyone have a good reference on determining a proteins radius of > gyration. I do not know much about this and I do not know what has to be > known about the protein before this can be done, ie do you have to know > the crystal structure of the protein first? > Try Physical Chemistry of Macromolecules, by Charles Tanford. If I remember correctly, and it's been a while, it's a statistically defined parameter that describes the sphere swept out by the "mass elements" of the peptide chain (or other atoms of whatever molecule). IF I remember correctly it's the radius measured in light scattering experiments, though there are other ways. -- Jim Litts Zymogenetic, Inc. Seattle, WA USA My comments are my own and not my employer's. From owner-proteins@net.bio.net Wed Mar 05 22:00:00 1997 Path: biosci!beri.co.jp!hiroakih From: hiroakih@beri.co.jp ("Hidekazu HIROAKI, PhD") Newsgroups: bionet.molbio.proteins Subject: benzamidine agarose. Date: 6 Mar 1997 18:27:49 -0800 Organization: Biomolecular Engineering Research Institute Lines: 15 Sender: daemon@net.bio.net Distribution: world Message-ID: <331FFE85.5A8D@beri.co.jp> Reply-To: hiroakih@beri.co.jp NNTP-Posting-Host: net.bio.net Dear all, I would be interested whether "benzamidine agarose" is capable to remove proteases (especially trypsin and thrombine) from the reaction mixture of partial proteolytic digestion. In the SIGMA catalor, there're some immobilized benzamidine (A7155, B2768) and seem to be sufficient to trap a couple of mg of trypsin per mL resin. I like to use one of them for separation of recombinant protein from fusion expression system. If anyone can give me some suggestions, I would be very appreciative. Thank you for your time, Hidekazu HIROAKI. From owner-proteins@net.bio.net Wed Mar 05 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!gatech!csulb.edu!hammer.uoregon.edu!arclight.uoregon.edu!su-news-hub1.bbnplanet.com!news.bbnplanet.com!cliffs.rs.itd.umich.edu!bloom-beacon.mit.edu!senator-bedfellow.mit.edu!usenet From: Thomas Schwartz Newsgroups: bionet.molbio.proteins Subject: Re: how to stop protein degradation? Date: Thu, 06 Mar 1997 09:04:21 -0500 Organization: Massachusetts Institute of Technology Lines: 2 Message-ID: <331ECEE5.60A2@MIT.EDU> References: <331E0EE9.7141@uabdpo.dpo.uab.edu> NNTP-Posting-Host: m66-080-8.mit.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (X11; I; SunOS 5.4 sun4m) you could try to express your protein in protease-deficient strains, like BL21 for example. From owner-proteins@net.bio.net Wed Mar 05 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!newsfeeds.sol.net!news.maxwell.syr.edu!usenet.kornet.nm.kr!newsfeed.dacom.co.kr!news.kigam.re.kr!usenet.seri.re.kr!not-for-mail From: "Hong, Minsun" Newsgroups: bionet.molbio.proteins Subject: seek any methods determine and purify mutimeric protein Date: Fri, 07 Mar 1997 16:31:49 +0900 Organization: HIT Lines: 9 Message-ID: <331FC465.7F75@donald.hanho.co.kr> Reply-To: compania@donald.hanhyo.co.kr NNTP-Posting-Host: cobra.hanhyo.co.kr Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01Gold (Win95; I) Hello.. i'm trying to find out that my recombinant protein which is secreted by S. cerevisiae, is multimer or monomer in culture medium. would you show my applicable methods? and if it will be mutimer, is it possible to purify it as it is? i hope your help. From owner-proteins@net.bio.net Wed Mar 05 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!news.sgi.com!news.maxwell.syr.edu!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news-peer.gsl.net!news-hk.gsl.net!news.gsl.net!newsgate.cuhk.edu.hk!hkusud.hku.hk!win95pc From: jshtsang@hkucc.hku.hk (Jimmy S.H. Tsang) Newsgroups: bionet.molbio.proteins Subject: Re: Protein-protein interaction of His-tag of pET14b? Date: Fri, 07 Mar 97 00:20:12 GMT Organization: Botany, University of Hong Kong Lines: 39 Message-ID: <5fnmsi$orv3@hkusud.hku.hk> References: <5f2n2o$cpq2@hkusud.hku.hk> NNTP-Posting-Host: 147.8.185.36 Mime-Version: 1.0 Content-Type: text/plain; charset=US-ASCII X-Newsreader: News Xpress 2.0 Beta #0 In article , bijgh@zeus.bris.ac.uk (Jared Head) wrote: >Jimmy S.H. Tsang (jshtsang@hkucc.hku.hk) wrote: >: >: Dear All, >: I have recently fused a bacterial gene downstream of the His-tag >: sequence of the pET14b from AMS Biotech. The in vitro transcribed and >: translated protein forms a complex which migrates much slower than the wild >: type protein in a native PAGE. Did anyone experience a similar >: 'multimerisation' problem? The same gene cloned into pET19b produced a >: protein similar to that of native protein, i.e. no 'multimerisation'. Any >: suggestion and comment will be appreciated. > >We're just finding the same thing. It seems possible that more than one >protein are chelating around one nickel atom. When we cleaved the His.Tag >off we made our protein monomeric, and we also found dtt to have the same >effect, presumably by reducing the nickel ions. > >Jared > >-- >Jared Head at the Department of Biochemistry, University of Bristol Dear Jared, Thank you for the reply. However in our hands we have already use DTT to stabilise our proteins during the running of the gel. Our native PAGE gel does not contain additional nickel so binding of more than one protein complex around the nickel atom seems unlikely. Our native protein is already a homodimer but expression from pET14b produce more than a dimer and no dimeric protein fraction was detected. We don't think the 'multimerisation' was caused by the His-Tag because expression from pET19b which also contain the His-Tag did not produce such an effect. Thank you ones again for all your comment. J.S.H. Tsang Department of Botany The University of Hong Kong jshtsang@hkucc.hku.hk http://www.hku.hk/botany/staff/tsang.html From owner-proteins@net.bio.net Wed Mar 05 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!news.sgi.com!howland.erols.net!torn!resunix.sickkids.on.ca!news From: Randy Willis Newsgroups: bionet.molbio.proteins Subject: Re: how to stop protein degradation? Date: Thu, 06 Mar 1997 18:52:48 -0500 Organization: The Hospital For Sick Children Lines: 19 Message-ID: <331F58CF.41C6@gandalf.psf.sickkids.on.ca> References: <331E0EE9.7141@uabdpo.dpo.uab.edu> <331ECEE5.60A2@MIT.EDU> NNTP-Posting-Host: fili.sickkids.on.ca Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0 (X11; I; IRIX 5.3 IP20) To: Thomas Schwartz Thomas Schwartz wrote: > you could try to express your protein in protease-deficient strains, > like BL21 for example. This is not always a guarantee against degradation, as we have had experience with proteins which are eaten or nibbled by BL21(DE3). In one case, we found that we could minimize it with lower temperature inductions but still required inhibitors upon cell lysis. Randall C Willis, Researcher Biochemistry, Hosp for Sick Children 3522-555 University Ave Toronto, ON M5G 1X8 CANADA 416-813-5933 (ph) -5022 (fax) willis@gandalf.psf.sickkids.on.ca From owner-proteins@net.bio.net Thu Mar 06 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!gatech!www.nntp.primenet.com!nntp.primenet.com!enews.sgi.com!news.sgi.com!howland.erols.net!surfnet.nl!news-zh.switch.ch!elna.ethz.ch!bclutz2.ethz.ch!user From: hornig@bc.biol.ethz.ch (Ruediger Hornig) Newsgroups: bionet.molbio.proteins Subject: Re: Alkylation of cysteine residues Date: Fri, 07 Mar 1997 16:38:41 +0100 Organization: ETH Zurich Lines: 27 Message-ID: References: <3314A2CC.37DE@charon.dfh.dk> NNTP-Posting-Host: bclutz2.ethz.ch Ulrik Sidenius wrote: > I am looking for a method for reduction and alkylation of proteins > before running SDS-PAGE. Hi Ulrik we do alkylation by adding a final concentration of 25 mM NEM (N-ethyl-maleimide) to the boiled gel samples (reducing agent is 3 mM DTT). This alkylates the DTT and all the cysteins in the protein. As an alternative, you may alkylate the unreduced protein after purification to block free sulfhydryls, dialyze excess of NEM out if needed and boil your gel samples as usual. hope this helps Rudi ---------------------------------------------------------------------- _/_/_/_/_/_/_/_/_/ _/ ETH Zurich _/ _/ _/ _/ Biochemie II _/_/_/ _/ _/_/_/_/ Rüdiger Hornig (hornig@bc.biol.ethz.ch) _/ _/ _/ _/ Universitaetstr. 16, ETH Zentrum _/_/_/_/ _/ _/ _/ CH-8092 Zurich (Switzerland) Phone: +41 1 632 3007 FAX: +41 1 632 12 69 http://www.bc.biol.ethz.ch/BiochemistryII/BioII.html ---------------------------------------------------------------------- From owner-proteins@net.bio.net Thu Mar 06 22:00:00 1997 Path: biosci!daresbury!usenet From: Martyn Winn Newsgroups: bionet.molbio.proteins Subject: Re: Radius of Gyration Date: Fri, 07 Mar 1997 08:37:53 +0000 Organization: Daresbury Laboratory Lines: 24 Distribution: bionet Message-ID: <331FD3E1.155A@dl.ac.uk> References: <331DAAB6.11BF@mail.utexas.edu> NNTP-Posting-Host: ccp4a.dl.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (X11; I; SunOS 5.5 sun4m) Brett Phinney wrote: > > Does anyone have a good reference on determining a proteins radius of > gyration. I do not know much about this and I do not know what has to be > known about the protein before this can be done, ie do you have to know > the crystal structure of the protein first? > > Thanks a lot > > Brett Phinney > Cell Research Institute > University of Texas, Austin Well, not something I knew about until we had a seminar on it yesterday ;-) The radius of gyration can be calculated from the crystal structure or it can be estimated from small angle solution scattering. BUT the two methods disagree by around 10% (number from solution scattering is generally the larger). Also, the solution scattering value varies with pH, and type and concentration of ions in buffer. So yes, you can estimate it without knowing the structure, but don't treat your answer with too much respect..... Martyn Winn From owner-proteins@net.bio.net Thu Mar 06 22:00:00 1997 Path: biosci!daresbury!not-for-mail From: "Alexey M. Eroshkin" Newsgroups: bionet.molbio.proteins Subject: Multiple sequence editor ProMSED2 (Win) available thru IUBio archive Date: 7 Mar 1997 08:01:12 -0000 Organization: State Research Center of Virology and Biotechnology VECTOR Lines: 87 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <5foi08$sq0@mserv1.dl.ac.uk> MIME-Version: 1.0 Original-To: list@gw.itfs.nsk.su Dear all, Multiple sequence editor ProMSED2 (Win 3.x/95) is available now thru IUBio archive as ftp://iubio.bio.indiana.edu/molbio/ibmpc/promsed2.exe and .readme If you have access to e-mail, the program can be obtained via e-mail by sending the following message: To: BITFTP@pucc.Princeton.EDU From: YOUR E-MAIL ADDRESS ftp iubio.bio.indiana.edu uuencode user anonymous cd molbio/ibmpc get promsed2.exe quit Server will return you uuencoded program in several files. Running UUDECODE you'll get the archive with the program. --------------------------------------------------------------- ProMSED2: Protein Multiple Sequence EDitor-2 for Win 3.11/95 State Research Center of Virology an Biotechnology "Vector" Institute of Molecular Biology Koltsovo, Novosibirsk Region, 633159 Russia ProMSED2, Windows application for both automatic and manual DNA and protein sequence alignment, editing, comparison and analysis. The program reads main sequence formats and performs automatic alignments, alignment visualization and editing and it allows sequences to be aligned interactively leaving unchanged previously aligned regions. The program has an user-friendly interface. Manual alignment and sequence analysis are facilitated by coloring schemes reflecting amino acid similarity in mutational, physico-chemical and other properties. Although ProMSED was targeted at protein sequences, it can be used on DNA sequences as well. The program provides flexible tool for sequences alignment, analysis, visualization, edition and presentation preparation. The program does or has (+ - NEW or enhanced features): + inputs DNA and protein sequences in NBRF/PIR, Pearson (Fasta), MSF (GSG), EMBL/SwissProt, Intelligenetics and CLUSTAL formats; o has interface and functions like in others Windows applications (source file view, font changing, marking/unmarking, block and sequence selection, cut and paste, UNDO, etc.); o loads several sequence families in different windows, adds sequences to existing alignment, combines sequences from various files; + outputs the alignment in several popular formats; + makes presentation quality color and black-and-white prints of complete alignment or any selected block; + saves alignment picture as Windows metafile and bitmap; o permits to apply automatic alignment interactively (with options to change the alignment parameters) to any selected part of sequences of marked block; + calculates sequence similarity of complete sequences, of any selected sequence subset or of marked block in % and in PAM250 units (matrix of amino acid similarity); + calculates total (average for %) sequence similarity value - an estimation of alignment quality; + prints sequence similarity matrix; + sorts sequences by similarity of complete sequences or marked block; + displays conserved and semiconserved positions; + has many amino acid coloring schemes aimed to facilitate manual alignment and understanding protein sequence features. Some schemes are: EVOLUTIONARY CONSERVATIVE (reflects amino acid mutational properties), COMPLEX (similarity of amino acids in physico-chemical properties), HYDROPHOBICITY, CHARGE, BIG RESIDUES, ALPHA-HELIX, HELIX-BREAKERS, etc. The options to input user-defined schemes or change the colors of any amino acid groups are available; + searches subsequences and complex sequence patterns; o has complete HELP. Educational version is restricted in number and length of sequences. Comments, bug reports and suggestions for new features are welcome and should be sent by email to eroshkin@vector.nsk.su. We would be happy to get feedback from you. ++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ Anatoly Frolov & Alexey Eroshkin Institute of Molecular Biology E.mail: eroshkin@vector.nsk.su State Research Center of Virology and Tel: +7 (3832) - 647774 Biotechnology "Vector" Fax: +7 (3832) - 328831 Koltsovo, Novosibirsk Region 633159 Russia ++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ From owner-proteins@net.bio.net Thu Mar 06 22:00:00 1997 Path: biosci!imm.ki.se!Andrei.Zhukov From: Andrei.Zhukov@imm.ki.se (Andrei Zhukov) Newsgroups: bionet.molbio.proteins Subject: cycloheximide mechanism Date: 7 Mar 1997 07:03:28 -0800 Organization: Karolinska Institutet Lines: 8 Sender: daemon@net.bio.net Distribution: world Message-ID: <33202E80.D3E@ki.se> NNTP-Posting-Host: net.bio.net Does anybody know how exactly cycloheximide inhibits protein synthesis? Which enzyme or whatever does it act upon? I would very much appreciate an answer by e-mail and, if possible, some reference(s). Sincerely, Andrei Zhukov From owner-proteins@net.bio.net Thu Mar 06 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!gatech!csulb.edu!hammer.uoregon.edu!arclight.uoregon.edu!europa.clark.net!cpk-news-hub1.bbnplanet.com!cam-news-hub1.bbnplanet.com!news.bbnplanet.com!howland.erols.net!torn!resunix.sickkids.on.ca!news From: Randy Willis Newsgroups: bionet.molbio.proteins Subject: Re: benzamidine agarose. Date: Fri, 07 Mar 1997 09:33:57 -0500 Organization: The Hospital For Sick Children Lines: 23 Message-ID: <33202755.41C6@gandalf.psf.sickkids.on.ca> References: <331FFE85.5A8D@beri.co.jp> NNTP-Posting-Host: gollum.psf.sickkids.on.ca Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0 (X11; I; IRIX 5.3 IP12) To: hiroakih@beri.co.jp Hidekazu, We've used the immobilized benzamidine from Sigma off and on for a couple of years now to remove thrombin from our GST-fusion cleavage reactions and it seems to be working well. I couldn't tell you exactly how much we've used per reaction but we have been roughly following the specifics outlined by Sigma and we got the suggestion from another lab in the neighbourhood. Keep in mind that the Sigma resin comes in a slurry with something like 0.5M NaCl, so you may want to pre-rinse the resin with your starting buffer before using it, but that is largely up to what your plans are after removing the thrombin. I would definitely suggest that you give it a try and see what happens. Good luck, Randall C Willis, Researcher Biochemistry, Hosp for Sick Children 3522-555 University Ave Toronto, ON M5G 1X8 CANADA 416-813-5933 (ph) -5022 (fax) willis@gandalf.psf.sickkids.on.ca From owner-proteins@net.bio.net Thu Mar 06 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!newsfeeds.sol.net!feed1.news.erols.com!howland.erols.net!rill.news.pipex.net!pipex!oleane!pasteur.fr!jussieu.fr!univ-lyon1.fr!newsserver.cilea.it!oracle.csi.unimi.it!usenet From: clsmteam@imiucca.csi.unimi.it (Paolo Castano) Newsgroups: bionet.molbio.proteins Subject: IMMUNOFLUORESCENCE COURSE Date: Fri, 07 Mar 1997 13:07:11 GMT Organization: Istitute of Human Anatomy Lines: 26 Message-ID: <332012fb.20206449@news.csi.unimi.it> NNTP-Posting-Host: modem12.csi.unimi.it X-Newsreader: Forte Free Agent 1.1/32.230 Advanced International Immunofluorescence Course Gargnano '97 (Italy) The Advanced International Immunofluorescence Course is a post-doctorate theoretical/practical course, with propedeutical lectures and practical stages on traditional and confocal immunofluorescence microscopy and image and ion analysis. The course will take place in Gargnano (Lake of Garda) from 7 to 10 October 1997. Further information and registration details will be found at the following Web address http://imiucca.csi.unimi.it/endomi/ACIF.html Thank you Paolo Castano _______________________________________________ Prof. Paolo Castano UNIVERSITY of MILAN Institute of Human Anatomy CHAIR OF HUMAN ANATOMY - FACULTY OF PHARMACY Via Mangiagalli 31 - 20133 Milan (Italy) - Tel. 39.2.26.63.683 Fax 39.2.23.64.082 / 39.2.70.63.54.25 e-mail: clsmteam@imiucca.csi.unimi.it http://imiucca.csi.unimi.it/~endomi/confocale.html From owner-proteins@net.bio.net Thu Mar 06 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!newsfeeds.sol.net!newspump.sol.net!howland.erols.net!rill.news.pipex.net!pipex!warwick!leicester!usenet From: "Dr E. Buxbaum" Newsgroups: bionet.molbio.proteins Subject: Re: seek any methods determine and purify mutimeric protein Date: 7 Mar 1997 12:29:39 GMT Organization: University of Leicester (PCFS User) Lines: 16 Message-ID: <5fp1nj$q4q@falcon.le.ac.uk> References: <331FC465.7F75@donald.hanho.co.kr> NNTP-Posting-Host: pc5.msb.le.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) "Hong, Minsun" wrote: >Hello.. > >i'm trying to find out that my recombinant protein which is secreted by >S. cerevisiae, is multimer or monomer in culture medium. > >would you show my applicable methods? >and if it will be mutimer, is it possible to purify it as it is? Get in touch with somebody who has an analytical ultracentrifuge. They can determine the molecular weight in a single run, at least if the sample is monodisperse. Both gel chromatography and rate zonal centrifugation in sucrose or glycerol gradients are getle methods to separate proteins by MW. From owner-proteins@net.bio.net Thu Mar 06 22:00:00 1997 Path: biosci!ihnp4.ucsd.edu!swrinde!howland.erols.net!newsxfer.itd.umich.edu!uunet!in1.uu.net!206.250.118.17!nntp.earthlink.net!usenet From: nestgrp@earthlink.net Newsgroups: bionet.molbio.proteins Subject: Re: help: enzyme purification Date: Fri, 07 Mar 1997 19:33:25 GMT Organization: Earthlink Network, Inc. Lines: 48 Message-ID: <5fpfbq$bj1@bolivia.earthlink.net> References: NNTP-Posting-Host: cust46.max7.washington.dc.ms.uu.net X-Newsreader: Forte Free Agent 1.0.82 Jennifer Humphries wrote: >I started with a crude extract from potatoes and have purified the extract >through a anion exchange(Q-sepharose) and a hydrophobic >interaction(Phenyl-toyopearl) columns. In both cases I eluted the enzyme >using either a step wise or linear salt gradient. The activity of the >enzyme was monitored after each column and the solution was desalted by >ultrafiltration(amicon centriplus conc.). Now I am at the last stage of >the purification, a strong anion exchange column(PL-SAX) and again trying >to elute the enzyme with a salt gradient. The problem is, when I inject >the enzyme onto the column I never see any activity in the eluted >fractions. It is only when I don't run a gradient and instead just run >the highest salt concentration moblie phase and inject the enzyme that I >recover the active enzyme. So I know its not a problem with the injector >or the column is mucked up. I don't understand. >I appreciate any suggestions. Jennifer: Going back to anion exchange chromatography after HIC is rather redundant. For purity, you may be better off with a more orthagonal method since your sequence of steps is anion exchange, HIC, anion exchange. Having said that, it is difficult to answer your question without knowing more about your buffers. What is the pH of the buffers you are using on your strong anion exchange column? If the pH is too low, you definately will need to use greater salt concentrations in order to get elution. For example, if you use the same buffers for both your weak and strong anion exchange columns it will take far greater salt concentrations to elute from the strong column and the problem you are describing would be understandable. Hope this helpds. Let us know your buffer conditions so we can solve this problem. Regards, John ********************************** John K. Troyer, Ph.D. The Nest Group, Inc. nestgrp@earthlink.net http://world.std.com/~nestgrp ********************************** From owner-proteins@net.bio.net Thu Mar 06 22:00:00 1997 Path: biosci!hg.uleth.ca!weselake From: weselake@hg.uleth.ca (Randall Weselake) Newsgroups: bionet.molbio.proteins Subject: [Fwd: post-doctoral position] Date: 7 Mar 1997 21:15:26 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 50 Sender: daemon@net.bio.net Distribution: world Message-ID: <33210092.541D@hg.uleth.ca> NNTP-Posting-Host: net.bio.net Message-ID: Date: Fri, 07 Mar 1997 21:59:13 -0800 From: Randall Weselake X-Mailer: Mozilla 3.01 (Win16; I) MIME-Version: 1.0 To: nobody@net.bio.net CC: Subject: post-doctoral position Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit Post-Doctoral Position, University of Lethbridge Qualifications: Ph.D. in molecular biology. Experience in lipid biochemistry and/or enzymology would be an asset. Project Information: The research will involve molecular genetic and enzymological studies of intramuscular fat deposition in cattle. The project represents a collaboration between the University of Lethbridge and the Agriculture and Agri-Food Canada Research Centre in Lethbridge. The project is supported by the Beef Industry Development Fund of the Alberta Agricultural Research Institute and has the potential for renewal to September, 1999. The annual salary will be in the range of $26,000 to $28,000. Applicants should send a covering letter, curriculum vitae and list of three references by May 30, 1997 to: Dr. Randall J. Weselake] Associate Professor and Chair Department of Chemistry University of Lethbridge 4401 University Drive Lethbridge, Alberta T1K 3M4 For additional information on the position, Dr. Weselake can be contacted through the following channels: Telephone: (403) 329-2303 FAX: (403) 329-2057 E-MAIL: weselake@hg.uleth.ca -------------------------------------------------------------------------------- Return-Path: Received: from tsb08-07.cc.uleth.ca by acad2.cc.uleth.ca (MX V4.2 AXP) with SMTP; Fri, 07 Mar 1997 22:15:03 MST Message-ID: <33210031.377C@hg.uleth.ca> From owner-proteins@net.bio.net Thu Mar 06 22:00:00 1997 Path: biosci!STARWAVE.COM!registration-l From: registration-l@STARWAVE.COM Newsgroups: bionet.molbio.proteins Subject: Welcome to Sportszone Date: 7 Mar 1997 07:30:13 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 70 Sender: daemon@net.bio.net Distribution: world Message-ID: <199703071534.HAA02552@helios.starwave.com> NNTP-Posting-Host: net.bio.net Congratulations! You have now registered for your Free ESPNET SportsZone account. Keep this confirmation email as a record of your user name and password. You'll need this information to take advantage of these and any future SportsZone registered user activities. Your password is: SharkHeat As a registered user you have instant access to these great SportsZone games and features: - Men's and Women's Tournament Challenge (See below) - ZoneGames - Chat rooms and - ZoneMail Weekly Here's where to go to get the most from your SportsZone registration: ****************** Tournament Challenge Registrants Thanks for registering early! The game begins on March 9th. Start working on your game plan with Rob Neyer's historical analysis of tourney results at: http://espnet.sportszone.com/editors/zoned/statclass/ncaahoops1.html ********************* ZoneGames Registrants You're eligible now to play any of our FREE minigames or join a SportsZone Fantasy League. Just check out the latest offerings at: http://espnet.sportszone.com/zonegames/ ********************** Chat Rooms To start talking sports with some of the Web's most knowledgeable fans, check out: http://ESPNET.SportsZone.com/editors/talk/chatter.html User Names and Passwords To change your password, go to Subscriber Services at: http://espnet.sportszone.com/subscriber/index.html Premium Subscribers Becoming a subscriber will give you access to all premium content areas on the service (denoted by ticket symbol). For more information about becoming an ESPNET SportsZone Subscriber, go to Subscriber Benefits at: http://espnet.sportszone.com/tour/index.info. The Legal Stuff Before you log in to ESPNET SportsZone's registered areas please be sure to read the Terms of Service Agreement at: http://espnet.sportszone.com/terms.html. By using the Service, you agree that you will not post any of the following material in Service Chat rooms, bulletin boards, or other forums ("postings"): * Material which defames, abuses or threatens others. * Statements that are bigoted, hateful or racially offensive. * Material that advocates illegal activity or discusses illegal activities with the intent to commit them. * Unauthorized copyrighted material. * Material that contains vulgar, obscene or indecent language or images. * Advertising or any form of commercial solicitation. Thanks again for registering with SportsZone! From owner-proteins@net.bio.net Thu Mar 06 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!news.sgi.com!enews.sgi.com!arclight.uoregon.edu!newsfeed.uk.ibm.net!ibm.net!newsfeed.de.ibm.net!ibm.net!02-newsfeed.univie.ac.at!paladin.american.edu!news.jhu.edu!welchlink!stebby From: stebby@welchlink.welch.jhu.edu (Stephen C. Dahl) Newsgroups: bionet.molbio.proteins Subject: Re: Help! Triton X114 blues.... Date: 7 Mar 1997 17:46:03 GMT Organization: HCF - Johns Hopkins University, Baltimore, Maryland, USA Lines: 15 Message-ID: <5fpk8r$fn2@news.jhu.edu> References: <5fkaul$jrd@is.bbsrc.ac.uk> NNTP-Posting-Host: 128.220.59.78 X-Newsreader: TIN [version 1.2 PL2] Steve Dunn (dunnsm@bbsrc.ac.uk) wrote: : Any ideas how to successfully combine X114 extraction with SDS-PAGE?? : All suggestions gratefully received... Steve: I have limited experience with TX114, but did not experience the problem you described. I followed Bordier's J. Biol. Chem. vol. 256: 1604--1607 paper including equilibration of the detergent and the TX114/sucrose cushion spins. I didn't see what I wanted to see, but the gel looked ok! Regards, Steve Dahl From owner-proteins@net.bio.net Thu Mar 06 22:00:00 1997 Path: biosci!agate!howland.erols.net!EU.net!enews.sgi.com!news.sgi.com!csulb.edu!drivel.ics.uci.edu!news.service.uci.edu!taurus.oac.uci.edu!jlhumphr From: Jennifer Humphries Newsgroups: bionet.molbio.proteins Subject: Re: help: enzyme purification Date: Fri, 7 Mar 1997 15:09:09 -0800 Organization: University of California, Irvine Lines: 28 Message-ID: References: <5fpfbq$bj1@bolivia.earthlink.net> NNTP-Posting-Host: taurus.oac.uci.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII To: nestgrp@earthlink.net In-Reply-To: <5fpfbq$bj1@bolivia.earthlink.net> > > Going back to anion exchange chromatography after HIC is rather > redundant. For purity, you may be better off with a more orthagonal > method since your sequence of steps is anion exchange, HIC, anion > exchange. Having said that, it is difficult to answer your question > without knowing more about your buffers. What is the pH of the > buffers you are using on your strong anion exchange column? I am using a 20mM Tris-Cl, 5mM 2-mercaptoethanol soln, pH 7.5 and eluting with a linear gradient of 150mM NaCl-400mM NaCl in the same buffer. The enzyme is also in a 20mM Tris-Cl, 5mM mercaptoethanol solution, pH 7.5. I know that the enzyme will elute with a 400mM NaCl soln because I injected the enzyme onto the column with this high salt concentration and was able to recover the enzyme. It is only when I try to inject the enzyme onto the column at the lower salt conc.(150mM) and then increase the concentration to 400mM that I no longer recover the enzyme, which doesn't make sense because it should at least be coming out at the very end of the gradient with the 400mM salt solution. The only thing I've been able to come up with is maybe the enzyme is coming off the column but it is coming off in such a broad band that it is being diluted out of the assay detection range. Thank you for your response. I work in the chem dept and no one can help me here. Jennifer From owner-proteins@net.bio.net Fri Mar 07 22:00:00 1997 Path: biosci!agate!newsgate.cuhk.edu.hk!hammer.uoregon.edu!xfer.kren.nm.kr!snunews.snu.ac.kr!plaza.snu.ac.kr!newera From: newera@plaza.snu.ac.kr () Newsgroups: bionet.molbio.proteins,bionet.molecules.peptides,sci.chem.analytical,bionet.biophysics,bionet.molbio.methds-reagnts Subject: Ion Exchange Chro/HPLC with 6M Guanidine or Some Detergent? Date: 8 Mar 1997 13:59:17 GMT Organization: Seoul National University, Republic of Korea Lines: 26 Message-ID: <5frrbl$e16$1@snunews.snu.ac.kr> NNTP-Posting-Host: plaza.snu.ac.kr X-Newsreader: TIN [version 1.2 PL2] Xref: biosci bionet.molbio.proteins:10204 bionet.molecules.peptides:699 sci.chem.analytical:7841 bionet.biophysics:2705 bionet.molbio.methds-reagnts:55577 Do not 6M ganidine HCl, 8M urea or some zwitter-ionic detergent make any difference to cation exchange chromatography or affinity chromatography? My protein tends to be aggregated during some purification steps such as dialysis or concentration with a Centriprep. In the midst of misfortune, my protein has no cysteines and seems to be easily renatured; so I plan to add 6M guanidine HCl/8M urea and 0.5~2% or more zwitter-ionic detergents such as CHAPS, sarkosyl to my current buffer system(20mM Na Phosphate, pH 6.8). I am deeply concerned that they severely interfere with my protein's binding ability to BioRex70 cation exchange resin, BlueSepharose affinity chromatography resin, to which my protein seems to quite tightly bind since it begins to elute at around 0.5M NaCl, and C18 reverse phase HPLC. Thank you very much. Key word: 6M Guanidine HCl/8M Urea CHAPS/Sarkosyl Cation Exchange Chromatography Affinity Chromatography Reverse Phase HPLC -- email : newera@plaza.snu.ac.kr address : Lee, Ji Hyun Laboratory of Physical Pharmacy(Prof. Lee, Bong Jin) Seoul National University College of Pharmacy Shinlim-Dong, Kwanak-Gu Seoul 151-742, Korea. From owner-proteins@net.bio.net Fri Mar 07 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!news-peer.gsl.net!news.gsl.net!news.sprintlink.net!news-peer.sprintlink.net!newsfeed.internetmci.com!metro.atlanta.com!uunet!in1.uu.net!198.168.54.138!rcogate.rco.qc.ca!altitude!usenet From: "Achim Recktenwald, PhD" Newsgroups: bionet.molbio.proteins Subject: Re: help: enzyme purification Date: Fri, 07 Mar 1997 08:27:50 -0500 Organization: IBEX Technologies, Inc., Biochemistry, 5485 Pare, Montreal, PQ, H4P 1P7, Canada Lines: 25 Message-ID: <332017D5.6852@ibex.ca> References: NNTP-Posting-Host: ibex.hip.cam.org Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Macintosh; I; PPC) To: Jennifer Humphries Jennifer Humphries wrote: > > I started with a crude extract from potatoes and have purified the extract > through a anion exchange(Q-sepharose) and a hydrophobic > interaction(Phenyl-toyopearl) columns. In both cases I eluted the enzyme > using either a step wise or linear salt gradient. The activity of the > enzyme was monitored after each column and the solution was desalted by > ultrafiltration(amicon centriplus conc.). Now I am at the last stage of > the purification, a strong anion exchange column(PL-SAX) and again trying > to elute the enzyme with a salt gradient. The problem is, when I inject > the enzyme onto the column I never see any activity in the eluted > fractions. It is only when I don't run a gradient and instead just run > the highest salt concentration moblie phase and inject the enzyme that I > recover the active enzyme. So I know its not a problem with the injector > or the column is mucked up. I don't understand. > > I appreciate any suggestions. > > Jennifer You enzyme might bind too strongly on the column to be eluted. Try decreasing the pH, to reduce the charge of your protein. Achim From owner-proteins@net.bio.net Fri Mar 07 22:00:00 1997 Path: biosci!daresbury!lyra.csx.cam.ac.uk!uknet!usenet1.news.uk.psi.net!uknet!EU.net!cpk-news-hub1.bbnplanet.com!cam-news-hub1.bbnplanet.com!news.bbnplanet.com!howland.erols.net!newspump.sol.net!newsfeeds.sol.net!hammer.uoregon.edu!news.ironhorse.com!nntp.portal.ca!van-bc!n1van.istar!van.istar!west.istar!news-w.ans.net!newsfeeds.ans.net!lantana.singnet.com.sg!newsfeed.singnet.com.sg!nuscc.nus.sg!scip3090 From: scip3090@leonis.nus.sg (Tan Nguan Soon) Newsgroups: bionet.molbio.proteins Subject: Yeast KEX2 recognition sequences Date: 9 Mar 1997 02:58:18 GMT Organization: National University of Singapore Lines: 4 Message-ID: <5ft90a$qm5@nuscc.nus.sg> NNTP-Posting-Host: scip3090@leonis.nus.sg X-Newsreader: TIN [version 1.2 PL2] Does anyone know the recognition peptide sequences for S. cerevisiae KEX2 endopeptidase? I know that it cleave at Lys-Arg site, but certainly a longer sequence is necessary for recognition. Please help. Any references? From owner-proteins@net.bio.net Sat Mar 08 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!gatech!arclight.uoregon.edu!enews.sgi.com!news.sgi.com!cgl!espinoza From: espinoza@cgl.ucsf.edu (Hernan Espinoza) Newsgroups: bionet.molbio.proteins Subject: Re: Why APS & TEMED? Date: 10 Mar 97 00:25:05 GMT Organization: UCSF Computer Graphics Lab Lines: 8 Message-ID: References: <01bc2ccd$d98dd4c0$33019386@schmidtdw.rz.ruhr-uni-bochum.de> NNTP-Posting-Host: socrates.ucsf.edu "Thorsten Schmidt" writes: >To polymerize acrylamide one uses APS and TEMED to start and continue >the polymerization. >But why? Which are the functions of APS (ammoniumpersulfate) and TEMED? >Okay, APS produces the radicals for the reaction but TEMED? It's a catalyst. -Hernan From owner-proteins@net.bio.net Sat Mar 08 22:00:00 1997 Path: biosci!RUTCHEM.RUTGERS.EDU!hall From: hall@RUTCHEM.RUTGERS.EDU ("G.S. Hall") Newsgroups: bionet.molbio.proteins Subject: Cerebrospinal fluid protein collaboration Date: 9 Mar 1997 06:57:19 -0800 Organization: Chemistry Department, Rutgers University Lines: 14 Sender: daemon@net.bio.net Distribution: world Message-ID: <3322FCDF.696@rutchem.rutgers.edu> Reply-To: hall@rutchem.rutgers.edu NNTP-Posting-Host: net.bio.net I am looking for a source of cerebrospinal fluid (human) for a collaborative research project using HPLC-ICPMS to fractionate proteins and determine the concentrations of Fe, Cu, and Zn bound to specific proteins. I only need about 1-mL of the fluid. Also, samples will be analyzed for 10-15 trace elements. Thanks for your time, Gene Hall -- Gene S. Hall, PhD 908-445-2590 Professor of Analytical Chemistry e-mail hall@rutchem.rutgers.edu Rutgers University, P.O. Box 939 Piscataway, NJ 08855-0939 From owner-proteins@net.bio.net Sat Mar 08 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!newsfeeds.sol.net!feed1.news.erols.com!howland.erols.net!news.nacamar.de!news.apfel.de!news-fra1.dfn.de!news-koe1.dfn.de!news.ruhr-uni-bochum.de!not-for-mail From: "Thorsten Schmidt" Newsgroups: bionet.molbio.proteins Subject: Why APS & TEMED? Date: 9 Mar 1997 21:07:05 GMT Organization: Ruhr-Universitaet Bochum, Rechenzentrum Lines: 10 Message-ID: <01bc2ccd$d98dd4c0$33019386@schmidtdw.rz.ruhr-uni-bochum.de> NNTP-Posting-Host: dialppp-1-51.rz.ruhr-uni-bochum.de X-Newsreader: Microsoft Internet News 4.70.1155 Dear reader! To polymerize acrylamide one uses APS and TEMED to start and continue the polymerization. But why? Which are the functions of APS (ammoniumpersulfate) and TEMED? Okay, APS produces the radicals for the reaction but TEMED? Thank you for your answer Thorsten Schmidt From owner-proteins@net.bio.net Sun Mar 09 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!cs.utexas.edu!news.sprintlink.net!news-peer.sprintlink.net!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!rill.news.pipex.net!pipex!warwick!leicester!usenet From: "Dr E. Buxbaum" Newsgroups: bionet.molbio.proteins,bionet.molecules.peptides,sci.chem.labware Subject: Re: Help me! AIR BUBBLES trapped in HPLC column... Date: 10 Mar 1997 14:19:51 GMT Organization: University of Leicester (PCFS User) Lines: 16 Message-ID: <5g15a7$jbq@falcon.le.ac.uk> References: <5g0tvl$ehu$1@snunews.snu.ac.kr> NNTP-Posting-Host: pc47.msb.le.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: quoted-printable X-Mailer: Mozilla 1.22 (Windows; I; 16bit) Xref: biosci bionet.molbio.proteins:10211 bionet.molecules.peptides:702 sci.chem.labware:3149 newera@plaza.snu.ac.kr () wrote: >*Urgent!* > >Some or probably quite many air bubbles seem to be trapped in my HPLC >column: 18C reverse phase column, pore size 7um. Could you g= ive me any >suggestions to remedy this problem as quickly as possible? >I am much disappointed not to be able to proceed my experiments any >more. After some nasty remarks about prevention being better than cure I would suggest to run the column with degassed solvent at the highest possible flow rate (i. e., just before pressure cut out) for some 30 min or so. The column should be upside down, so that the flow is upward. Alternatively, if column construction allows, you could also try to run it in reverse direction (i.e., feed the buffer into the exit of the column). From owner-proteins@net.bio.net Sun Mar 09 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!newsfeeds.sol.net!europa.clark.net!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!rill.news.pipex.net!pipex!warwick!leicester!usenet From: "Dr E. Buxbaum" Newsgroups: bionet.molbio.proteins Subject: Re: Protein precipitation methods Date: 10 Mar 1997 11:30:31 GMT Organization: University of Leicester (PCFS User) Lines: 11 Message-ID: <5g0rcn$cv0@falcon.le.ac.uk> References: NNTP-Posting-Host: pc5.msb.le.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) pxpst2@vms.cis.pitt.edu (THE GREEK) wrote: >I am interested in getting some references to some precipitation methods >other than TCA. The method must be suitable for low protein >concentrations. The most common method is probably the one described by Wessel and Fluegge in Anal. Biochem., somewhere in the early 80's, which uses chloroform/methanol. Also in Anal. Biochem a few years ago was a method that uses phenol/ether extraction. The nice thing about this method is that the protein is never actually precipitated, but extracted into the phenolic phase. Therefore, there is no trouble solubilizing the protein afterwards. From owner-proteins@net.bio.net Sun Mar 09 22:00:00 1997 Newsgroups: bionet.molbio.proteins Path: biosci!rutgers.rutgers.edu!uwm.edu!newsfeeds.sol.net!europa.clark.net!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!rill.news.pipex.net!pipex!warwick!bris.ac.uk!ssa.bris.ac.uk!ogsdw From: "S.D. Wainwright" Subject: Storage of alkylation reagents X-Nntp-Posting-Host: ssa.bris.ac.uk Content-Type: TEXT/PLAIN; charset=US-ASCII Message-ID: Sender: usenet@fsa.bris.ac.uk (Usenet) Organization: University of Bristol, England Mime-Version: 1.0 Date: Mon, 10 Mar 1997 14:33:35 GMT Lines: 25 Dear All My antibody only seems to detect its target protein when it is reduced with 2-ME or DTT. I have to perform immunoprecipitations and immunohistochemistry with this antibody and therefore have to alkylate the reduced proteins and reducing agent. I have two questions 1. Has anyone performed immunoprecipitations and immunohistochemitry on reduced and alylated proteins. and 2. How do you store stock solutions of N-ethyl-maleimide, iodoacetamide and iodoacetate. Thanks in advance Shane Wainwright From owner-proteins@net.bio.net Sun Mar 09 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!gatech!www.nntp.primenet.com!nntp.primenet.com!europa.clark.net!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news.sprintlink.net!news-peer.sprintlink.net!EU.net!enews.sgi.com!news.sgi.com!cgl!espinoza From: espinoza@cgl.ucsf.edu (Hernan Espinoza) Newsgroups: bionet.molbio.proteins Subject: Fellow Biochemists : Advice needed Date: 10 Mar 97 18:45:32 GMT Organization: UCSF Computer Graphics Lab Lines: 11 Message-ID: NNTP-Posting-Host: socrates.ucsf.edu Howdy, A friend of mine is trying to separate out very basic proteins on polyacrylamide using urea as a denaturant under non-reducing, acidic conditions. It has worked before, but not consistently. He was hoping to talk to someone who has worked with these kind of gels before so he can get an idea of where to begin troubleshooting and optimization. Any advice, references, and/or pointers to an expert welcome. Thanks in advance. -Hernan From owner-proteins@net.bio.net Sun Mar 09 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!gatech!EU.net!Germany.EU.net!wizard.pn.com!news.gte.com!news-in.tiac.net!posterchild!news@tiac.net From: chi@healthtech.com (Cambridge Healthtech Institute) Newsgroups: bionet.molbio.proteins Subject: From Genes to Proteins June 9-10, 1997 San Francisco, CA Date: Mon, 10 Mar 1997 19:43:22 GMT Organization: Cambridge Healthtech Institute Lines: 43 Message-ID: <5g1o3k$rfd@news-central.tiac.net> Reply-To: chi@healthtech.com NNTP-Posting-Host: 207.60.238.8 X-Newsreader: Forte Free Agent 1.0.82 BEYOND THE HUMAN GENOME PROJECT From Genes to Proteins June 9-10, 1997 San Francisco, CA The goal of the Human Genome Project is to sequence the entire genome, with subsequent determination of the functionality of all genes. There is an analog of this project, which focuses on characterization, gene linkage, and functional understanding of expressed proteins. This effort is critical for recognizing the potential value of the information being generated by the Human Genome Project. Many similar tools that have been developed for use in genomics, including rapid automated screening and powerful bioinformatic capabilities, will need to be applied to this effort. Major differences, such as the inability to amplify proteins as can be done with genes, must also be dealt with. Protein expression patterns, determination of protein-protein interactions, and correlation with phenotype are all steps essential for advancing diagnostics and drug development. If you wish to receive more information, please contact: Cambridge Healthtech Institute 1037 Chestnut Street Newton Upper Falls, MA 02164 USA Phone: 617-630-1300 Fax: 617-630-1325 e-mail: chi@healthtech.com http://www.healthtech.com/conferences/ ______________________________ Cambridge Healthtech Institute 1037 Chestnut Street Newton Upper Falls, MA 02164 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ tel: 617.630.1300 fax: 617.630.1325 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ inquiries@healthtech.com World Wide Web http://www.healthtech.com/conferences From owner-proteins@net.bio.net Sun Mar 09 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!news.sgi.com!howland.erols.net!usenet.kornet.nm.kr!xfer.kren.nm.kr!snunews.snu.ac.kr!plaza.snu.ac.kr!newera From: newera@plaza.snu.ac.kr () Newsgroups: bionet.molbio.proteins,bionet.molecules.peptides,sci.chem.labware Subject: Help me! AIR BUBBLES trapped in HPLC column... Date: 10 Mar 1997 12:14:45 GMT Organization: Seoul National University, Republic of Korea Lines: 22 Message-ID: <5g0tvl$ehu$1@snunews.snu.ac.kr> NNTP-Posting-Host: plaza.snu.ac.kr X-Newsreader: TIN [version 1.2 PL2] Xref: biosci bionet.molbio.proteins:10210 bionet.molecules.peptides:701 sci.chem.labware:3147 *Urgent!* Some or probably quite many air bubbles seem to be trapped in my HPLC column: 18C reverse phase column, pore size 7um. Could you give me any suggestions to remedy this problem as quickly as possible? I am much disappointed not to be able to proceed my experiments any more. :-( Thanks... -- email : newera@plaza.snu.ac.kr address : Lee, Ji Hyun Laboratory of Physical Pharmacy(Prof. Lee, Bong Jin) Seoul National University College of Pharmacy Shinlim-Dong, Kwanak-Gu Seoul 151-742, Korea. From owner-proteins@net.bio.net Sun Mar 09 22:00:00 1997 Path: biosci!agate!spool.mu.edu!uwm.edu!cs.utexas.edu!swrinde!howland.erols.net!cam-news-hub1.bbnplanet.com!news.bbnplanet.com!news.maxwell.syr.edu!news.bc.net!torn!news.unb.ca!usenet From: elf8365@umoncton.ca (Levon Fendekian) Newsgroups: bionet.molbio.proteins Subject: Re: cycloheximide mechanism Date: Tue, 11 Mar 1997 02:37:32 GMT Organization: University of New Brunswick Lines: 15 Message-ID: <3324c524.51963229@news.unb.ca> References: <33202E80.D3E@ki.se> NNTP-Posting-Host: 139.103.29.50 X-Newsreader: Forte Free Agent 1.1/32.230 Andrei.Zhukov@imm.ki.se (Andrei Zhukov) wrote: > Does anybody know how exactly cycloheximide inhibits protein synthesis? >Which enzyme or whatever does it act upon? I would very much appreciate >an answer by e-mail and, if possible, some reference(s). > >Sincerely, > >Andrei Zhukov > Cyclohexamide blocks the eucaryotic protein synthesis, by inhibiting the translocation process (binds to EF2). From owner-proteins@net.bio.net Sun Mar 09 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!cs.utexas.edu!howland.erols.net!psinntp!news.nstn.ca!coranto.ucs.mun.ca!news.unb.ca!usenet From: elf8365@umoncton.ca (Levon Fendekian) Newsgroups: bionet.molbio.proteins Subject: Re: Protein precipitation methods Date: Tue, 11 Mar 1997 02:33:52 GMT Organization: University of New Brunswick Lines: 17 Message-ID: <3324c469.51776674@news.unb.ca> References: NNTP-Posting-Host: 139.103.29.50 X-Newsreader: Forte Free Agent 1.1/32.230 pxpst2@vms.cis.pitt.edu (THE GREEK) wrote: >I am interested in getting some references to some precipitation methods >other than TCA. The method must be suitable for low protein >concentrations. >Please reply by email and thanks in advance > > Peter Pediaditakis pxpst2@vms.cis.pitt.edu try the phenol : ether method ... analytical biochemistry 226;382-383 (1995) hope this helps, From owner-proteins@net.bio.net Sun Mar 09 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!cs.utexas.edu!howland.erols.net!torn!news.unb.ca!usenet From: elf8365@umoncton.ca (Levon Fendekian) Newsgroups: bionet.molbio.proteins Subject: Guanidinium hydrochloride Date: Tue, 11 Mar 1997 02:28:10 GMT Organization: University of New Brunswick Lines: 10 Message-ID: <3324c2cf.51366703@news.unb.ca> NNTP-Posting-Host: 139.103.29.50 X-Newsreader: Forte Free Agent 1.1/32.230 Hi, I would like to know how guanidinium hydrochloride denatures proteins ? thanks for the help, From owner-proteins@net.bio.net Sun Mar 09 22:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!europa.clark.net!news.clark.net!news.clark.net From: Harvard Morehead Newsgroups: bionet.molbio.proteins Subject: RE:Help me! AIR BUBBLES trapped in HPLC column... Date: 10 Mar 1997 22:03:06 GMT Organization: Clark Internet Services, Inc. Lines: 38 Message-ID: <5g20eq$dlo@clarknet.clark.net> References: 1160 <5g0tvl$ehu$1@snunews.snu.ac.kr> NNTP-Posting-Host: harvard.clark.net Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=ISO-8859-1 Content-Transfer-Encoding: 8bit > > > *Urgent!* > > Some or probably quite many air bubbles seem to be trapped in my HPLC column: 18C reverse phase column, pore size 7um. > Could you give me any suggestions to remedy this problem as quickly as possible? > I am much disappointed not to be able to proceed my experiments any more. > :-( > > Thanks... > > > > -- > email : newera@plaza.snu.ac.kr > address : > Lee, Ji Hyun > Laboratory of Physical Pharmacy(Prof. Lee, Bong Jin) > > Seoul National University > College of Pharmacy > Shinlim-Dong, Kwanak-Gu > Seoul 151-742, Korea. > I have had some luck by running overnight at a slow rate (0.1 ml/min for an analytical column), with the column with the outlet up, so that any bubbles will float trhough the column, and using very thouroughly degassed water. The water will dissolve any air, and any bubbles will be pushed up through the column. Good luck! -Harvard Morehead harvardm@aol.com -no cool .sig . From owner-proteins@net.bio.net Sun Mar 09 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!cs.utexas.edu!swrinde!howland.erols.net!torn!news.unb.ca!usenet From: elf8365@umoncton.ca (Levon Fendekian) Newsgroups: bionet.molbio.proteins Subject: Re: Protein precipitation methods Date: Tue, 11 Mar 1997 02:45:45 GMT Organization: University of New Brunswick Lines: 16 Message-ID: <3324c369.51519941@news.unb.ca> References: NNTP-Posting-Host: 139.103.29.50 X-Newsreader: Forte Free Agent 1.1/32.230 pxpst2@vms.cis.pitt.edu (THE GREEK) wrote: >I am interested in getting some references to some precipitation methods >other than TCA. The method must be suitable for low protein >concentrations. >Please reply by email and thanks in advance > > Peter Pediaditakis pxpst2@vms.cis.pitt.edu You can try the phenol : ether method ! Analytical biochemistry 226: 382-383 (1995) hope it is usefull ! From owner-proteins@net.bio.net Sun Mar 09 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!cs.utexas.edu!swrinde!howland.erols.net!feed1.news.erols.com!news.maxwell.syr.edu!news.bc.net!torn!news.unb.ca!usenet From: elf8365@umoncton.ca (Levon Fendekian) Newsgroups: bionet.molbio.proteins Subject: Re: how to stop protein degradation? Date: Tue, 11 Mar 1997 02:40:15 GMT Organization: University of New Brunswick Lines: 18 Message-ID: <3324c5f9.52176193@news.unb.ca> References: <331E0EE9.7141@uabdpo.dpo.uab.edu> NNTP-Posting-Host: 139.103.29.50 X-Newsreader: Forte Free Agent 1.1/32.230 XiaoYin Zhong wrote: >Dear Dr. protein: > >I cloned a protein with His-tag at C-end. After I purified this perotein >by Ni-column, SDS-PAGE gel show that the protein was degraded.What is >the reason? Is there any other methods which can solve this problem >except using proteinase inhibiter coke-tail or EDTA in the buffer? > >Thank you for your help! > > > > >qiu@orion.cmc.uab.edu Are you working at 4 C ? From owner-proteins@net.bio.net Sun Mar 09 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!gatech!csulb.edu!hammer.uoregon.edu!news-xfer.netaxs.com!cronkite.ocis.temple.edu!astro.ocis.temple.edu!driska From: driska@astro.ocis.temple.edu (Stephen P. Driska PhD) Newsgroups: bionet.molbio.proteins Subject: Re: 1 mM Tris? Date: 10 Mar 1997 19:43:56 GMT Organization: Temple University, Academic Computer Services Lines: 20 Message-ID: <5g1o9s$7h9@cronkite.ocis.temple.edu> References: <5fdlo2$v3@news2.kol.co.kr> NNTP-Posting-Host: astro.ocis.temple.edu X-Newsreader: TIN [version 1.2 PL2] hbkg@hitel.kol.co.kr wrote: : We tried to dissolve DTNB, CAT substrate, in Tris buffer, and : found that DTNB was not dissolved in Tris buffer at all, which : was very strange. We changed water in vain. : We found at last the mistake of using 1mM Tris instead of appropriate : concentration. : I am curious HOW TRIS AFFECTS DTNB SOLUBILITY. What is the pH of the solution? 1 mM is not a very high concen- tration and may not hold the pH to a pre-set value if you add a significant amount of a solute that may be acidic or basic. Good luck, someone should be able to help you in this group. Steve -- Steve Driska, Physiology Department, Temple University Medical School Philadelphia, PA 19140 USA (215) 707-3283 driska@astro.ocis.temple.edu From owner-proteins@net.bio.net Mon Mar 10 22:00:00 1997 Path: biosci!agate!howland.erols.net!news.mathworks.com!news.kei.com!news.thenet.net!uunet!in1.uu.net!138.133.17.7!amgen!usenet From: John Philo Newsgroups: bionet.molbio.proteins,bionet.molecules.peptides,sci.chem.analytical,bionet.biophysics,bionet.molbio.methds-reagnts Subject: Re: Ion Exchange Chro/HPLC with 6M Guanidine or Some Detergent? Date: Tue, 11 Mar 1997 08:59:05 -0800 Organization: Amgen Inc. Lines: 37 Message-ID: <33258F59.7A92@amgen.com> References: <5frrbl$e16$1@snunews.snu.ac.kr> Reply-To: jphilo@amgen.com Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01Gold (Win95; I) To: newera@plaza.snu.ac.kr Xref: biosci bionet.molbio.proteins:10225 bionet.molecules.peptides:706 sci.chem.analytical:7871 bionet.biophysics:2723 bionet.molbio.methds-reagnts:55649 newera@plaza.snu.ac.kr wrote: > > Do not 6M ganidine HCl, 8M urea or some zwitter-ionic detergent make any difference to cation exchange chromatography or affinity chromatography? > > My protein tends to be aggregated during some purification steps such as dialysis or concentration with a Centriprep. In the midst of misfortune, my protein has no cysteines and seems to be easily renatured; so I plan to add 6M guanidine HCl/8M urea and 0.5~2% or more zwitter-ionic detergents such as CHAPS, sarkosyl to my current buffer system(20mM Na Phosphate, pH 6.8). > I am deeply concerned that they severely interfere with my protein's binding ability to BioRex70 cation exchange resin, BlueSepharose affinity chromatography resin, to which my protein seems to quite tightly bind since it begins to elute at around 0.5M NaCl, and C18 reverse phase HPLC. You will certainly not be able to use ion exchange chromatography in 6M GdnHCl! The extrememly high ionic strength from that addition would completely shield the electrostatic interactions of proteins with the column and thus prevent binding/separation. It is possible to use ion exchange with (non-ionic) urea, although I don't think I have ever heard of anyone doing it with urea concentrations that hig