From owner-proteins@net.bio.net Fri Sep 01 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!usenet.ins.cwru.edu!cleveland.Freenet.Edu!gn686 From: gn686@cleveland.Freenet.Edu (Carmine M. Zingarino) Newsgroups: bionet.molbio.proteins Subject: ONCONASE Enters Phase III Pancreatic Cancer Trial Date: 2 Sep 1995 18:40:32 GMT Organization: Case Western Reserve University, Cleveland, Ohio (USA) Lines: 42 Message-ID: <42a8f0$1nd@usenet.INS.CWRU.Edu> NNTP-Posting-Host: eeyore.ins.cwru.edu Path: hivnet.org!fido.hivnet.org!aegis.hivnet.org!Mary_Elizabeth >Newsgroups: hiv.aids.data Distribution: hivnet Date: Wed, 25 Jan 95 06:38:39 +0100 From: Mary_Elizabeth@aegis.hivnet.org Reply-To: Mary_Elizabeth@aegis.hivnet.org Subject: ONCONASE ENTERS PHASE III FOR PANCREATIC Comment-To: All@f927.n103.z1.fido.hivnet.org Message-ID: X-Fidonet-Comment-To: Mary_Elizabeth@aegis.hivnet.org X-FSC-PATH: 103/927 280/413 Lines: 26 Document 165 DOCN AD950165 TI "ONCONASE Enters Phase III for Pancreatic Cancer" DT 950124 SO Business Wire (01/24/95) AB Alfacell Corp. announced on Tuesday that it will commence Phase III clinical trials for ONCONASE, which is being tested in combination with tamoxifen to treat pancreatic cancer. The FDA approved the company's Phase III protocol design--which calls for a randomized, multi-center trial--on Jan. 23. ONCONASE has been established as a novel enzyme in both structure and function, and is now recognized as the smallest known member of the superfamily of pancreatic ribonucleases. Alfacell is also working with the National Institutes of Health to study promising anti-viral activity exhibited by ONCONASE in in vitro tests against HIV-1. A study recently published in the Proceedings of the National Academy of Sciences found that ONCONASE inhibited HIV in vitro by 99 percent. DISTRIBUTED BY GENA/aegis (714.248.2836 * 8N1/Full Duplex): Copyright (c) 1995 - Information, Inc., Bethesda, MD. This information is provided by the Centers for Disease Control & Prevention (CDC), National AIDS Clearinghouse as a public service. Noncommercial reproduction encouraged. --- FLAME v1.1 * Origin: GENA/aegis-San Juan Capistrano, CA - 714.248.2836 (1:103/927) . -- From owner-proteins@net.bio.net Sat Sep 02 23:00:00 1995 Path: biosci!agate!library.ucla.edu!newsfeed.internetmci.com!usenet.eel.ufl.edu!freenet3.freenet.ufl.edu!afn26055 From: "Edward R. Mason" Newsgroups: bionet.molbio.proteins Subject: Re: removing o-glycosylation Date: Sun, 3 Sep 1995 07:16:53 -0400 Lines: 5 Message-ID: References: <421bvm$8ps@news.uit.no> <4234ph$3jv@ocean.silcom.com> <4241el$e5@news.uit.no> NNTP-Posting-Host: freenet3.afn.org Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII In-Reply-To: <4241el$e5@news.uit.no> Alan, You mentioned a NetOglyc prediction service. Could you follow up with info for accessing this service. Thank you. Ed FERMCO From owner-proteins@net.bio.net Sat Sep 02 23:00:00 1995 Path: biosci!bloom-beacon.mit.edu!newsfeed.internetmci.com!info.ucla.edu!library.ucla.edu!news.bc.net!rover.ucs.ualberta.ca!news.ucalgary.ca!news.ucalgary.ca!not-for-mail From: sun@acs.ucalgary.ca (Yimei Sun) Newsgroups: bionet.molbio.proteins Subject: CFP:Flavins'96 Date: 3 Sep 1995 13:30:46 -0600 Organization: The University of Calgary Lines: 40 Message-ID: <42cvp6$tfm@acs6.acs.ucalgary.ca> NNTP-Posting-Host: sun@acs6.acs.ucalgary.ca Preliminary Announcement -- First Call for Papers 12th International Symposium ON Flavins and Flavoproteins Time: 30th June --- 6th July 1996 Place: University of Calgary Calgary, Alberta, Canada Contact: Dr. Kenneth J. Stevenson Department of Bio. Sci. University of Calgary Calgary, Alberta T2N 1N4 Canada FAX: (403)284-4184 (Conference Office) Ms. Yimei Sun Department of Bio. Sci. University of Calgary Calgary, Alberta T2N 1N4 Canada Email: sun@acs.ucalgary.ca From owner-proteins@net.bio.net Sat Sep 02 23:00:00 1995 Path: biosci!agate!howland.reston.ans.net!vixen.cso.uiuc.edu!uwm.edu!msunews!netnews.upenn.edu!netaxs.com!usenet From: "George A. Heavner" Newsgroups: bionet.molbio.proteins Subject: Re: Peptide Synthesis Date: 3 Sep 1995 22:07:00 GMT Organization: Net Access - Philadelphia's Internet Connection Lines: 5 Message-ID: <42d8u4$o74@netaxs.com> References: <427mk9$1tr@agate.berkeley.edu> NNTP-Posting-Host: 198.69.189.46 The simple answer to your question is Yes, you can synthesize peptide in both directions. From your posting it is obvious that you are totally unfamiliar with peptide synthesis. You might want to learn a little about peptide chemistry and then rephrase your question to get some meaningful information. George A. Heavner Senior Director, Peptide R&D Centocor, Inc. From owner-proteins@net.bio.net Sun Sep 03 23:00:00 1995 Path: biosci!agate!howland.reston.ans.net!usc!news.cerf.net!nntp-server.caltech.edu!sinharoy From: sinharoy@cco.caltech.edu (Ranabir Sinharoy) Newsgroups: bionet.molbio.proteins Subject: Re: Peptide Synthesis Date: 4 Sep 1995 21:42:10 GMT Organization: California Institute of Technology, Pasadena Lines: 17 Message-ID: <42frri$4ch@gap.cco.caltech.edu> References: <427mk9$1tr@agate.berkeley.edu> <42d8u4$o74@netaxs.com> NNTP-Posting-Host: accord.cco.caltech.edu X-Newsreader: NN version 6.5.0 #12 (NOV) "George A. Heavner" writes: >The simple answer to your question is Yes, you can synthesize peptide in both directions. From your posting it is obvious that you are totally unfamiliar with peptide synthesis. You might want to learn a little about peptide chemistry and then rephrase your question to get some meaningful information. >George A. Heavner >Senior Director, Peptide R&D >Centocor, Inc. Could you provide a reference for N to C peptide synthesis? Apart from in vitro translation, I was under the impression that N to C synthesis suffers from extensive racemization due to azlactone formation. Ranabir Sinha Roy Graduate Student From owner-proteins@net.bio.net Sun Sep 03 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!news.sprintlink.net!tank.news.pipex.net!pipex!lyra.csx.cam.ac.uk!news From: jhp20@cus.cam.ac.uk (Jong) Newsgroups: bionet.molbio.proteins Subject: On Telomerase structure Date: 4 Sep 1995 17:24:23 GMT Organization: Protein Engineering Lines: 9 Message-ID: <42fco7$pj8@lyra.csx.cam.ac.uk> NNTP-Posting-Host: sonja.acad.cai.cam.ac.uk Mime-Version: 1.0 Content-Type: Text/Plain; charset=ISO-8859-1 X-Newsreader: WinVN 0.99.5 Dear Is there anybody who is working on Telomerase and its structure? Thanks, Jong From owner-proteins@net.bio.net Sun Sep 03 23:00:00 1995 Path: biosci!bloom-beacon.mit.edu!newsfeed.internetmci.com!news.sprintlink.net!dispatch.news.demon.net!demon!sunsite.doc.ic.ac.uk!daresbury!nntp-trd.UNINETT.no!news.uit.no!news From: allanh@petrus.uit.no (allan hey) Newsgroups: bionet.molbio.proteins Subject: NetOglyc ( was: removing o-glycosylation) Date: Mon, 04 Sep 95 14:41:21 WET Organization: University of Tromsø Lines: 97 Message-ID: <42esf3$nqb@news.uit.no> References: <421bvm$8ps@news.uit.no> <4234ph$3jv@ocean.silcom.com> <4241el$e5@news.uit.no> NNTP-Posting-Host: imb11.fagmed.uit.no Mime-Version: 1.0 X-Newsreader: WinVN 0.93.6 In article , afn26055@freenet.ufl.edu says... > >Alan, >You mentioned a NetOglyc prediction service. Could you follow up with >info for accessing this service. Thank you. >Ed >FERMCO NetOglyc is a free service for prediction of O-glycosylation, provided by the Center for Biological Sequence Analysis at The Technical University of Denmark in Lyngby, Denmark. I've taken the liberty of copying the following from them: *************** NetOglyc Mail Server V1.0 *************** Prediction of O-glycosylation of mammalian proteins Center for Biological Sequence Analysis The Technical University of Denmark DK-2800 Lyngby, Denmark DESCRIPTION: The NetOglyc mail server is a service producing neural network predictions of mucin type O-glycosylation sites in mammalian proteins as described in: J.E. Hansen, O. Lund, J. Engelbrecht, H. Bohr, J.O. Nielsen, J.E.S. Hansen and S. Brunak, Prediction of O-glycosylation of mammalian proteins: Specificity patterns of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase. The Biochemical Journal, 308, 801-813, 1995. ABSTRACT: The specificity of the enzyme(s) catalyzing the covalent link between the hydroxyl side-chains of serine or threonine and the sugar moiety GalNAc is unknown. Pattern recognition by artificial neural networks and weight matrix algorithms was performed to determine the exact position of in vivo O-linked GalNAc glycosylated serine and threonine residues from the primary sequence exclusively. The acceptor sequence context for O-glycosylation of serine was found to differ from that of threonine and the two types were therefore treated separately. The context of the sites showed a high abundance of proline, serine and threonine extending far beyond the previously reported region covering positions -4 through +4 relative to the glycosylated residue. The O-glycosylation sites were found to cluster and to have a high abundance in the amino-terminal part of the protein. The sites were also found to have an increased preference for three different classes of beta-turns. No simple consensus like rule could be deduced for the complex glycosylation sequence acceptor patterns. The neural networks were trained on the hitherto largest data material consisting of 48 carefully examined mammalian glycoproteins comprising 264 O-glycosylation sites. For detection neural network algorithms were much more reliable than weight matrices. The networks correctly found 60-95% of the O-glycosylated serine/threonine residues and 89-97% of the non-glycosylated residues in two independent test sets of known glycoproteins. A computer server using E-mail for prediction of O-glycosylation sites has been implemented and made publicly available. CURRENT NETWORK The network will be updated and predictions can alter due to different versions. The network is balanced to give optimal predictions whether you submit sequences with no homology to the known O-glycosylated proteins or not. If however the submitted sequence is very close to or identical to the sequences in our training dataset, wewill notify you by sending you both the assigment of the homologous (or identical) sequence in our data set and the prediction. FURTHER INFORMATION: The NetOglyc server returns a help file if the submitted file contains the word `help'. CONFIDENTIALITY Your submitted sequences will be deleted automatically immediately after processing by NetOglyc. ******************* The procedure is to send an E-mail to netOglyc@genome.cbs.dtu.dk. The first line should be a greater-then sign (>) followed by the name for the sequence. On the next line put the sequence (in single letter code), followed by an empty line to indicate the end of the sequence. Several sequences can be submitted in the same e-mail. You get the results back by e-mail - in my experience within a few hours of submitting the sequence. Hope this is of help to you! Allan From owner-proteins@net.bio.net Sun Sep 03 23:00:00 1995 Path: biosci!agate!lhom From: lhom@nature.Berkeley.EDU (Louis Hom) Newsgroups: bionet.molbio.proteins Subject: Books on Peptide Synthesis Date: 4 Sep 1995 19:56:01 GMT Organization: Agricultural and Resource Economics Lines: 7 Message-ID: <42flkh$83f@agate.berkeley.edu> References: <427mk9$1tr@agate.berkeley.edu> NNTP-Posting-Host: nature.berkeley.edu Can someone recommend a good introductory book on peptide synthesis? It looks like there is a long history of books by Miklos Bodanszky(?) in our library. Has anyone had experience with his writing? -- _______________________________________________________________________________ Lou Hom Pete Wilson & David Duke in '96. lhom@nature.berkeley.edu Be scared. Be very, very scared. From owner-proteins@net.bio.net Mon Sep 04 23:00:00 1995 Path: biosci!daresbury!hgmp.mrc.ac.uk!sanger.ac.uk!pmr From: pmr@sanger.ac.uk (Peter Rice) Newsgroups: bionet.molbio.proteins Subject: Re: Sequence Analysis Question Date: 05 Sep 1995 16:29:08 GMT Organization: The Sanger Centre Lines: 20 Message-ID: References: <42hpkm$ii0@news.duke.edu> NNTP-Posting-Host: unst.sanger.ac.uk In-reply-to: bussiere@PROBLEM_WITH_INEWS_GATEWAY_FILE's message of 5 Sep 1995 15:16:38 GMT In article <42hpkm$ii0@news.duke.edu> bussiere@PROBLEM_WITH_INEWS_GATEWAY_FILE (Dirksen Bussiere) writes: > Are there any programs out there that analyze and identify dna-binding > motifs in a protein sequence-particularly the helix turn helix? Thanks > for all of your help! There is one for helix-turn-helix in the EGCG package of extensions to EGCG. If yu have GCG 8.0, then EGCG is available from ftp.sanger.ac.uk in directory pub/pmr/egcg8 -- ------------------------------------------------------------------------ Peter Rice | Informatics Division E-mail: pmr@sanger.ac.uk | The Sanger Centre Tel: (44) 1223 494967 | Hinxton Hall, Hinxton, Fax: (44) 1223 494919 | Cambs, CB10 1RQ URL: http://www.sanger.ac.uk/~pmr/ | England From owner-proteins@net.bio.net Mon Sep 04 23:00:00 1995 Path: biosci!daresbury!nntp-trd.UNINETT.no!Norway.EU.net!EU.net!news.sprintlink.net!newsfeed.internetmci.com!howland.reston.ans.net!vixen.cso.uiuc.edu!ux1.cso.uiuc.edu!elarson From: elarson@ux1.cso.uiuc.edu (larson eric) Newsgroups: bionet.molbio.proteins,bionet.molbio.proteins Subject: Re: Protein Purification Date: 5 Sep 1995 21:24:12 GMT Organization: University of Illinois at Urbana Lines: 13 Message-ID: <42if5s$2eb@vixen.cso.uiuc.edu> References: NNTP-Posting-Host: ux1.cso.uiuc.edu Xref: biosci bionet.molbio.proteins:5557 sg@nwu.edu (Stephen Gately) writes: >Can anyone recommend a newsgroup that discusses protein purification by >ion exchange and affinity chromatography? ============== You're in a reasonable newsgroup for discussion. -- Eric Larson | University of Illinois at Urbana-Champaign USDA/Agronomy | 190 ERML; 1201 W. Gregory; Urbana, IL 61801 elarson@ux1.cso.uiuc.edu | Voice 217.244.3079 Fax 217.244.4419 Fidonet: 1:233/4.1 | My opinions are my own, but correct :-) From owner-proteins@net.bio.net Mon Sep 04 23:00:00 1995 Path: biosci!bloom-beacon.mit.edu!boulder!tali.UCHSC.edu!news From: Holers Lab Newsgroups: bionet.molbio.proteins Subject: Help trouble shooting expression and folding in bacterial system Date: 5 Sep 1995 19:21:46 GMT Organization: UCHSC, Div. of Rheumatology Lines: 127 Message-ID: <42i80a$76a@tali.UCHSC.edu> NNTP-Posting-Host: patroklos.uchsc.edu Mime-Version: 1.0 Content-Type: text/html Content-Transfer-Encoding: quoted-printable X-Mailer: Mozilla 1.1N (Windows; I; 16bit) From: guthridj@essex.hsc.colorado.edu (Joel Guthridge, Ph.D.) Newsgroups: bionet.molbio.methds-reagnts Subject: Help troubleshooting bacterial expression of prop. folded protein Date: Tue, 5 Sep 1995 13:52:03 Organization: Div. of Rheumatology, Univ. of Colorado Health Sci Ctr. Message-ID: I'm trying to produce protein to NMR analysis using IBI's FLAG epitope = tagged system. I'm using a vector which places the OmpA signal peptide = N-terminal to the N-terminal FLAG sequence, so the protein expression is = inducible by IPTG, and hopefully properly folded material is recovered in t= he = periplasmic fraction (detectible using the M1 antibody from IBI). In my = initial trials, I cloned this into the DH5a, and got detectible, but low = production. The affinity purified material appeared to be properly folded = (note my protein has 4 Cys. that are disulfide bonded) and proper disulfide= d = created. The problem was the level of production. Subsequently, I've put = this vector into the BL21 protease neg. strain, and got substantially bette= r = production, however now most of the material (even though soluble, from the= = periplasm and M1 (N-term) FLAG detectable) is found in higher molecular wei= ght = forms under non-reducing conditions, but reducable to a strong band of the = appropriate mol. wt. I think that the conditions we are now producing is n= ot = allowing appropriate disulfide bond formation. Does anyone have any = suggestions for how to overcome this problem? The production in the DH5a w= as = far too low to make that a cost effective option. = We've tried: Lower induction temp. Lower IPTG levels. Possible Altern. (We need advise here) -do expression in the presence of Glutatione, etc. = -? better periplasmic fractionation (how do others do this?) -do expression in presence or absence of Ca2+ (FLAG req. Ca2+= ) = -change epitope tag system ???? -change expression system (Pichia??) = -reduce and refold protein in vitro (methods ??) The protein we are producing is fairly hydrophobic and proper disulfide = bonding is required for function. Could the epitope tag be influencing the= = proper folding of the molecule. Any advise would be greatly appreciated, as would pointers to appropriat= e = reference materials on bacterial expression systems of eukaryotic proteins = for = structural analysis. Joel Guthridge email: Joel.Guthridge@UCHSC.edu From owner-proteins@net.bio.net Mon Sep 04 23:00:00 1995 Path: biosci!daresbury!nntp-trd.UNINETT.no!Norway.EU.net!EU.net!howland.reston.ans.net!agate!news.duke.edu!bussiere From: bussiere@PROBLEM_WITH_INEWS_GATEWAY_FILE (Dirksen Bussiere) Newsgroups: bionet.molbio.proteins Subject: Sequence Analysis Question Date: 5 Sep 1995 15:16:38 GMT Organization: Duke University, Durham, NC, USA Lines: 8 Message-ID: <42hpkm$ii0@news.duke.edu> NNTP-Posting-Host: abacus.mc.duke.edu X-Newsreader: TIN [version 1.2 PL2] Are there any programs out there that analyze and identify dna-binding motifs in a protein sequence-particularly the helix turn helix? Thanks for all of your help! -Dirk Bussiere 'bussiere@abacus.mc.duke.edu' From owner-proteins@net.bio.net Mon Sep 04 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!torn!nott!cunews!freenet.carleton.ca!FreeNet.Carleton.CA!at332 From: at332@FreeNet.Carleton.CA (Matt Parker) Subject: Re: circular dichroism of immunoglobulin like protein Message-ID: Sender: at332@freenet2.carleton.ca (Matt Parker) Reply-To: at332@FreeNet.Carleton.CA (Matt Parker) Organization: The National Capital FreeNet References: Date: Tue, 5 Sep 1995 21:42:15 GMT Lines: 23 Salvatore Sechi (SSechi@helix.nih.gov) writes: > Hello, everybody, > I am trying to do the thermal unfolding of a protein that has two > immunoglobulin like domain. I follow the denaturation using the CD. > I do 5 degrees steps and I equilibrate the sample for 10 min. I scan from > 200nm to 220nm from 22 degrees to 85. I would like to follow the > denaturation at 217nm (maximum for the beta-sheet negative peak). Supposly > the protein denature and the negative ellepticity at 217nm should decrease ( > less negative). Strangely the negative peak keeps encreasing. How would you > interprete this data? Does anybody know of any other protein that behaves > similarly? Should I go higher with the temperature? > Hope some body can help. > Thanks > Salvatore Sechi, Ph. D. > Laboratory of Molecular Allergy and Immunology > E-mail: ssechi@helix.nih.gov Is it possible that the protein might be changing from a predominantly beta-sheet to an alpha -helical conformation? I would try extending the range of your scans, and seeing if you get peaks at about 208 and 222 nm. From owner-proteins@net.bio.net Mon Sep 04 23:00:00 1995 Path: biosci!bloom-beacon.mit.edu!senator-bedfellow.mit.edu!usenet From: Newsgroups: bionet.molbio.proteins Subject: HPLCs Date: 5 Sep 1995 23:15:57 GMT Organization: Massachvsetts Institvte of Technology Lines: 10 Message-ID: <42ilnd$1aq@senator-bedfellow.MIT.EDU> NNTP-Posting-Host: ebirah.mit.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; PPC) X-URL: news:bionet.molbio.proteins Hi, I just wanted to thank everyone for their useful comments/suggestions on buying an HPLC. Dave Schaffer Chemical Engineering MIT From owner-proteins@net.bio.net Tue Sep 05 23:00:00 1995 Path: biosci!agate!howland.reston.ans.net!newsjunkie.ans.net!hermes.louisville.edu!news From: gharding@e-mail.kdp-baptist.louisville.edu (George Harding) Newsgroups: bionet.molbio.proteins Subject: 4..5 x 9.5 Immunodiffusion plates Date: Wed, 06 Sep 1995 14:00:30 GMT Organization: University of Louisville, Louisville KY USA Lines: 7 Message-ID: <42k9bg$sj4@hermes.louisville.edu> NNTP-Posting-Host: eklein2.kdp-mdr.louisville.edu X-Newsreader: Forte Free Agent 1.0.82 We lost our access to this newsgroup between August 24th and today, so I'd like to pose the same question again: does anyone know where we can buy empty 4.5 x 9.5 cm immunodiffusion plates in order to make our own assay plates ? They were previously sold by Miles Labs, but are not offered there any longer. Thanks. EKLEIN e0klei01@ULKYVM.LOUISVILLE.EDU From owner-proteins@net.bio.net Tue Sep 05 23:00:00 1995 Path: biosci!ACS.UCALGARY.CA!jbooth From: jbooth@ACS.UCALGARY.CA (Joseph Boothe) Newsgroups: bionet.molbio.proteins Subject: Re: Ion exchange, protein purification Date: 6 Sep 1995 17:32:20 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 35 Sender: daemon@net.bio.net Distribution: world Message-ID: References: <6SEP199511475077@watson.bms.com> NNTP-Posting-Host: net.bio.net On 6 Sep 1995, Michael S Curtis wrote: > I am looking for a very generic protocol to use as a starting point for some > FPLC work. I have some mono Q (Pharmacia) in the lab, as well as mono S. Does > anyone have a general buffer system that they commonly use. I hope this is not > too naive a question? I have done some research into different types of > resins, etc. but no one seems to give general running conditions etc. Any help > would be appreciated. > > Thnak you, > > Mike Curtis > curtis@bms.com > > > Purification on ion exchange resins is charge, and hence pH dependent. Therefore, it is extremely helpful if you know the pI of the protein you want to purify. For binding to an anion exchange resin (eg Mono Q) a pH above the pI of your protein would be chosen such that the protein would be negatively charged and vice versa for a cation exchanger. A buffer is selected which will have maximum buffering capacity at the chosen pH (pKa close to pH). While it is possible to elute proteins by altering the pH, this is more commonly accomplished by using the same buffer to which is added competing counterions in the form of salts. I think Pharmacia recommends initially trying to elute with a 1M solution of NaCl applied as a 0-35% gradient over 20 mls for the Mono Q HR 5/5 column. Pharmacia also supplies, free of charge (?), booklets describing principles and applications for different types of protein purification including ion exchange. I hope this helps. Joe From owner-proteins@net.bio.net Tue Sep 05 23:00:00 1995 Path: biosci!daresbury!nntp-trd.UNINETT.no!Norway.EU.net!EU.net!howland.reston.ans.net!vixen.cso.uiuc.edu!ux1.cso.uiuc.edu!elarson From: elarson@ux1.cso.uiuc.edu (larson eric) Newsgroups: bionet.molbio.proteins Subject: Re: Ion exchange, protein purification Date: 6 Sep 1995 18:32:09 GMT Organization: University of Illinois at Urbana Lines: 41 Message-ID: <42kpf9$mt@vixen.cso.uiuc.edu> References: <6SEP199511475077@watson.bms.com> NNTP-Posting-Host: ux1.cso.uiuc.edu curtis@watson.bms.com (Michael S Curtis) writes: >I am looking for a very generic protocol to use as a starting point for some >FPLC work. I have some mono Q (Pharmacia) in the lab, as well as mono S. Does >anyone have a general buffer system that they commonly use. I hope this is not >too naive a question? I have done some research into different types of >resins, etc. but no one seems to give general running conditions etc. Any help >would be appreciated. ================= It really depends a lot on the biological system you are using. In general, ion exchange using mono Q is used after some other column treatment. In purifying recombinant proteins from E. coli, we would commonly use a quick fast-flow column of QAE-Sepharose (same chemistry as MonoQ), desalt, then use MonoQ on the result. This prevented fouling of the very expensive MonoQ resin. Not too many enzyme/proteins will stick to MonoS, but if yours does, then purification can follow the same general scheme (i.e. pre-purify on bulk resin then clean up with MonoS). The buffers are often chosen for with an eye toward ensuring stability of the enzyme (pH 7 to 8, metals if needed, low levels of the enzymes' substrate if that helps stability, glycerol, etc.) and of ensuring a low enough ionic strength for the protein to bind (i.e., low divalent anions with MonoQ and low to moderate monoanions). In our case, we'd use Bicine at 20 mM (pH = 8), two substrates necessary for enzyme stability (10 mM MgCl2, 66 mM NaHCO3), 10 mM betamercaptoethanol (or DTT), with development of the column by increasing the NaCl concentration from 0 to 1 M (proteins eluted usually from 50 mM NaCl to around 600 mM NaCl). The key is having a good assay for the protein in question. Without this, progress really can't occur. -- Eric Larson | University of Illinois at Urbana-Champaign USDA/Agronomy | 190 ERML; 1201 W. Gregory; Urbana, IL 61801 elarson@ux1.cso.uiuc.edu | Voice 217.244.3079 Fax 217.244.4419 Fidonet: 1:233/4.1 | My opinions are my own, but correct :-) From owner-proteins@net.bio.net Tue Sep 05 23:00:00 1995 Path: biosci!daresbury!usenet From: "H.C. Whitaker" Newsgroups: bionet.molbio.proteins Subject: Re: Sequence Analysis Question Date: 6 Sep 1995 09:55:14 GMT Organization: SERC Daresbury Lab, Warrington, U.K. Lines: 7 Distribution: bionet Message-ID: <42jr62$o0j@mserv1.dl.ac.uk> References: <42hpkm$ii0@news.duke.edu> NNTP-Posting-Host: xserv1.dl.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (X11; I; SunOS 4.1.3 sun4m) To: bussiere@PROBLEM_WITH_INEWS_GATEWAY_FILE X-URL: news:42hpkm$ii0@news.duke.edu Try contacting Seqnet they have a WWW page (http://www.dl.ac.uk/Seqnet/home.html). They have HTH (a programme for finding helix-turn-helix motifs) and a multitude of motif finding and sequence analysis programmes. Hope this helps. From owner-proteins@net.bio.net Tue Sep 05 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!news.sprintlink.net!EU.net!Germany.EU.net!news.dfn.de!fu-berlin.de!zrz.TU-Berlin.DE!cs.tu-berlin.de!fauern!lrz-muenchen.de!news From: Wolfgang Schechinger Newsgroups: bionet.molbio.proteins Subject: Re: Books on Peptide Synthesis Date: 6 Sep 1995 08:32:10 GMT Organization: Institute for Diabetes Research Lines: 35 Distribution: world Message-ID: <42jmaa$3o0@sparcserver.lrz-muenchen.de> References: <427mk9$1tr@agate.berkeley.edu> <42flkh$83f@agate.berkeley.edu> NNTP-Posting-Host: u7k0201.ppp.lrz-muenchen.de Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Windows; I; 16bit) To: lhom@nature.Berkeley.EDU Hi Lou! I saw a book which I believe is very convenient on getteing started with this issue: don't remember the exact title and author, but it is published by IRL Press (Oxford University Press) and is a volume of the "chemistry primer" series. It cost about 20 Bucks or even less. Email me if this information is not sufficient to get it! Good luck! lhom@nature.Berkeley.EDU (Louis Hom) wrote: >Can someone recommend a good introductory book on peptide synthesis? It >looks like there is a long history of books by Miklos Bodanszky(?) in our >library. Has anyone had experience with his writing? >-- >_______________________________________________________________________________ >Lou Hom Pete Wilson & David Duke in '96. >lhom@nature.berkeley.edu Be scared. Be very, very scared. -- +++++++++++++++++++++++++++++++++++++++++++ Wolfgang Schechinger Institute for Diabetes Reserch Koelner Platz 1 80804 Munich Germany Phone +49 (89) 30 79 31 24 Fax +49 (89) 30 81 733 email u7k0201@sunmail.lrz-muenchen.de (Standard disclaimer applies) +++++++++++++++++++++++++++++++++++++++++++ From owner-proteins@net.bio.net Tue Sep 05 23:00:00 1995 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!news.sprintlink.net!in2.uu.net!senior.nectec.or.th!news.mahidol.ac.th!mucc!g3536468 From: g3536468@mucc.mahidol.ac.th (Nanthanit Chosungnoe - SCMI - 3536468 ) Newsgroups: bionet.molbio.proteins Subject: Cephalosporin C acylase Date: 6 Sep 1995 15:38:39 GMT Organization: Mahidol University, Thailand Lines: 33 Message-ID: <42kf9v$8sp@mars.mahidol.ac.th> NNTP-Posting-Host: 202.14.162.1 X-Newsreader: TIN [version 1.2 PL2] Hello Bionetters, I'm studying about Cephalosporin C acylase producing bacteria. Recently I asked for help from the library in my university to get the full paper. 1)TI : Simulaneous Determination of 7-Aminocephalosporanic acid by 2 nd Derivative spectrophotometry. AU : Murillo JA; Lemus JM; Garcia LF JN : Analytical Letters 24(4):683-699 PY : 1991 2)TI : Spectrophotometric Determination of 7-Aminocephalosporanic acid with Imidazole Reagent AU : Alwarnthan AA; Abdelfatth S; Zahran NM JN : Analytical Letters 24(2): 249-263 PY :1991 ---------------------------------------------------------------------------- Unforunately,I don't still receive that papers for over 4 months. So I asked for help from you who has them.If it's possible,please send the full paper by mail to me. " Nanthanit Choesongnern Faculty of Science Mahidol University Rama VI Rd.,Prayathai Bangkok, Thailand. " Thank you very much. **************************************************************************** From owner-proteins@net.bio.net Tue Sep 05 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!newsserver.jvnc.net!synapse.bms.com!watson.bms.com!curtis From: curtis@watson.bms.com (Michael S Curtis) Newsgroups: bionet.molbio.proteins Subject: Ion exchange, protein purification Date: 6 Sep 1995 11:47 EST Organization: Bristol Myers Squibb Pharmaceutical Research Institute Lines: 12 Distribution: world Message-ID: <6SEP199511475077@watson.bms.com> NNTP-Posting-Host: watson.bms.com News-Software: VAX/VMS VNEWS 1.41 I am looking for a very generic protocol to use as a starting point for some FPLC work. I have some mono Q (Pharmacia) in the lab, as well as mono S. Does anyone have a general buffer system that they commonly use. I hope this is not too naive a question? I have done some research into different types of resins, etc. but no one seems to give general running conditions etc. Any help would be appreciated. Thnak you, Mike Curtis curtis@bms.com From owner-proteins@net.bio.net Tue Sep 05 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!daresbury!nntp-trd.UNINETT.no!Norway.EU.net!EU.net!howland.reston.ans.net!swrinde!sgigate.sgi.com!uhog.mit.edu!rutgers!umdnj!greenfie From: greenfie@umdnj.edu (Norma Greenfield) Subject: Re: circular dichroism of immunoglobulin like protein Message-ID: Organization: Univ. of Medicine and Dentistry of NJ References: Date: Thu, 7 Sep 1995 00:58:22 GMT Lines: 35 at332@FreeNet.Carleton.CA (Matt Parker) writes: >Salvatore Sechi (SSechi@helix.nih.gov) writes: >> Hello, everybody, >> I am trying to do the thermal unfolding of a protein that has two >> immunoglobulin like domain. I follow the denaturation using the CD. >> I do 5 degrees steps and I equilibrate the sample for 10 min. I scan from >> 200nm to 220nm from 22 degrees to 85. I would like to follow the >> denaturation at 217nm (maximum for the beta-sheet negative peak). Supposly >> the protein denature and the negative ellepticity at 217nm should decrease ( >> less negative). Strangely the negative peak keeps encreasing. How would you >> interprete this data? Does anybody know of any other protein that behaves >> similarly? Should I go higher with the temperature? >> Hope some body can help. >> Thanks >> Salvatore Sechi, Ph. D. >> Laboratory of Molecular Allergy and Immunology >> E-mail: ssechi@helix.nih.gov > Is it possible that the protein might be changing from a predominantly >beta-sheet to an alpha -helical conformation? I would try extending the >range of your scans, and seeing if you get peaks at about 208 and 222 nm. It is not unusual for proteins with a large amount of beta structure to be fairly heat stable. Beta proteins are stabilized by hydrophobic forces which increase with increasing temperature. Often, instead of forming a random coil, upon denaturation beta proteins will form a large beta aggregate upon heating. This may eventually precipitate out of solution. You may want to try to denature the protein with guanidine HCL or urea rather than temperature. It would depend on what your goals are. If you want to study whether mutation change the stability, looking at the structure as a function of denaturant may work very well. From owner-proteins@net.bio.net Wed Sep 06 23:00:00 1995 Path: biosci!ucl.ac.uk!a.jackson From: a.jackson@ucl.ac.uk (Alison Jackson) Newsgroups: bionet.molbio.proteins Subject: recombinant luteinizing hormone Date: 7 Sep 1995 05:18:44 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 22 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Does anyone have any recombinant human LH that I could use in an ELISA I am trying to set up. I need approximately 75 ug. So if you have expressed it for instance in baculovirus then please get in touch. Thanks Jeremy Keusch Department of Immunology, Arthur Stanley House, U.C.L.S.M., 40-50 Tottenham Street, London, W1P 9PG. Tel: 44-171-636-8333 Ext.3145 Fax: 44-171-380-9357 Email: regfjjk@ucl.ac.uk From owner-proteins@net.bio.net Wed Sep 06 23:00:00 1995 Newsgroups: bionet.molbio.proteins,bionet.cellbiol,bionet.molec-model,bionet.structural-nmr,bionet.xtallography,bionet.general,sci.bio Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!howland.reston.ans.net!Germany.EU.net!news.dfn.de!gs.dfn.de!news.uni-bielefeld.de!techfak.uni-bielefeld.de!fuellen From: fuellen@techfak.uni-bielefeld.de (Georg Fuellen) Subject: Ribosomal Proteins <-> rRNA. How much is known ? Message-ID: Sender: fuellen@TechFak.Uni-Bielefeld.DE (Georg Fuellen) Date: Thu, 7 Sep 1995 13:50:07 GMT Nntp-Posting-Host: salbei.techfak.uni-bielefeld.de Organization: Universitaet Bielefeld, Technische Fakultaet. Followup-To: bionet.molbio.proteins Lines: 23 Xref: biosci bionet.molbio.proteins:5570 bionet.cellbiol:2859 bionet.molec-model:578 bionet.structural-nmr:746 bionet.xtallography:2010 bionet.general:16874 Hi there, How much is known about the interaction between Ribosomal Proteins and the ribosomal RNA ? Have points of contact between specific nucleotides and specific amino acids been identified ? In other words, how much detail does one know about the structure of the ribosome, beyond the standard textbook picture displaying the spatial arrangement of e.g. S1-S21, represented as spheres of different size ? [M.S.Capel et al, 1988, J.M.B. 200:65; Fig. 3-20 in Darnell, Lodish, Baltimore, Molecular Cell Biology, 2nd Ed., p.99] I eagerly await the education I'm about to get :) (Follow-Up set to bionet.molbio.proteins (so I hope)) best wishes, georg fuellen@dali.Mathematik.Uni-Bielefeld.DE, fuellen@MIT.EDU http://www.techfak.uni-bielefeld.de/techfak/persons/fuellen/georgF.html From owner-proteins@net.bio.net Wed Sep 06 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!vixen.cso.uiuc.edu!news.uoregon.edu!news.corpcomm.net!news.pepboys.com!netaxs.com!usenet From: "George A. Heavner" Newsgroups: bionet.molbio.proteins Subject: Re: Books on Peptide Synthesis Date: 5 Sep 1995 19:52:35 GMT Organization: Net Access - Philadelphia's Internet Connection Lines: 10 Message-ID: <42i9q3$8ua@netaxs.com> References: <427mk9$1tr@agate.berkeley.edu> <42flkh$83f@agate.berkeley.edu> NNTP-Posting-Host: 198.69.189.46 Bodansky's Peptide Synthesis is a good primer in this area as is Stewart and Young's book on Solid Phase Peptide Synthesis. For something after that there is a series of books entitled THE PEPTIDES, ANALYSIS, SYNTHESIS AND BIOLOGY published by Academic Press. George A. Heavner Senior Director, Peptide R&D Centocor, Inc. From owner-proteins@net.bio.net Wed Sep 06 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!newsjunkie.ans.net!newstf01.news.aol.com!newsbf02.news.aol.com!not-for-mail From: jonthn@aol.com (JONTHN) Newsgroups: bionet.molbio.proteins Subject: Sept 17-20, Mol. Biol. Conf. San Diego Date: 8 Sep 1995 00:01:59 -0400 Organization: America Online, Inc. (1-800-827-6364) Lines: 61 Sender: root@newsbf02.news.aol.com Message-ID: <42of7n$5ug@newsbf02.news.aol.com> Reply-To: jonthn@aol.com (JONTHN) NNTP-Posting-Host: newsbf02.mail.aol.com The Molecular Biology Conference, San Diego 1995 September 17-20 Held at the Lake San Marcos Resort, San Marcos, California U.S.A. ----------------------------------------- DAY PASSES AVAILABLE ----------------------------------------- Meeting organized and supported by: The Salk Institute, La Jolla, Calif. Richard Dana, Project Green Gene, San Diego, Calif. Harold Koopowitz, University of California, Irvine Calif State Univ, San Marcos J. Miksche, H. Shands, S. Heller, USDA/ARS, Beltsville, MD E.Roos, USDA/ARS, Ft Collins, Colorado Charles Simon, Germplasm Preservation Unit, USDA, Pullman, Color. Jonathan Sherman, Jason Frank, MicroForest, Inc., San Marcos, Calif Pyraponic Industries, Inc. II, San Marcos, Calif CDN Isotopes Inc., Pointe-Claire, Quebec, Canada A SAMPLE SESSION: Monday, September 18 7-10:30 pm SESSION IVb. STRUCTURAL BIOLOGY, MOLECULAR IMMUNOLOGY The Basic Structural Biology of Calcium Signalling, Walter Chazin, The Scripps Research Institute, La Jolla, CA Molecular Immunology of Self-Reactive Autoanti- bodies and Autoimmune Disease, Azad Kaushik, University of Guelph Designer Genes for Protein NMR Spectroscopy Danilo Casimiro, The Scripps Research Institute, La Jolla, CA Analysis with x-ray crystallography and yeast genetics, C. Brenner, Brandeis Univ NMR Studies of Sidechain Titration Behavior and Histidine Tautomeric States of Bacillus Circulans Xylanase, Leigh Plesniak, ACUSD, CA DNA Binding Determinants of the alpha Subunit of RNA Polymerase: A Novel DNA Binding Domain Architecture, Nuria Assa-Munt, La Jolla Cancer Research Foundation, La Jolla, CA Structural basis for DNA bending by the architec- tural transcription factor LEF-1, Li Xiang, The Scripps Research Institute, La Jolla, CA Contact MBC'95 for more information at: MBC'95 145 Vallecitos de Oro, Suite E San Marcos, California 92069 Attention: Jonathan Sherman Phone: 619-736-3065 FAX: 619-736-3193 E-Mail: jonthn@aol.com From owner-proteins@net.bio.net Wed Sep 06 23:00:00 1995 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!howland.reston.ans.net!swrinde!sgigate.sgi.com!sgiblab!cgl!picasso.ucsf.edu!smiller From: smiller@picasso.ucsf.edu (Stephen Miller) Newsgroups: bionet.molbio.proteins Subject: Re: Books on Peptide Synthesis Date: 7 Sep 95 22:31:39 GMT Organization: UCSF Computer Graphics Lab Lines: 35 Message-ID: References: <427mk9$1tr@agate.berkeley.edu> <42flkh$83f@agate.berkeley.edu> NNTP-Posting-Host: picasso-yp.ucsf.edu lhom@nature.Berkeley.EDU (Louis Hom) writes: >Can someone recommend a good introductory book on peptide synthesis? It >looks like there is a long history of books by Miklos Bodanszky(?) in our >library. Has anyone had experience with his writing? >-- >_______________________________________________________________________________ >Lou Hom Pete Wilson & David Duke in '96. >lhom@nature.berkeley.edu Be scared. Be very, very scared. Here's a short list of useful books/articles: Bodanszky, Principles of Peptide Synthesis, Second Edition (1993) Atherton and Sheppard, Solid Phase Peptide Synthesis (1989) Fields and Noble, Int'l J Pep Prot Res 35 p161 (1990) NovaBiochem Catalog (1994-95) Bodanszky is probably the best general reference for understanding the principles involved in peptide synthesis, but it isn't very up-to-date on current methodology. The NovaBiochem catalog gives a fairly good guide on the practical aspects, and is more current. Fields and Noble give a good description of Fmoc SPPS and its limitations, but again it doesn't represent the state-of-the-art. Regarding your other question about N->C vs C->N synthesis, virtually all non-enzymatic peptide syntheses are conducted in a C->N direction, since activation of the C-terminus of a growing peptide chain can lead to oxazolone formation and concomitant racemization. Bodanszky discusses some special cases where N->C syntheses have been conducted, but these are not broadly applicable. Stephen Miller Department of Pharmaceutical Chemistry University of California, San Francisco From owner-proteins@net.bio.net Wed Sep 06 23:00:00 1995 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!howland.reston.ans.net!newsserver.jvnc.net!synapse.bms.com!news-admin From: "Patricia W. Cash" Newsgroups: bionet.molbio.proteins Subject: Re: Books on Peptide Synthesis Date: 7 Sep 1995 17:55:05 GMT Organization: Bristol-Myers Squibb Company Lines: 21 Message-ID: <42nblp$d66@synapse.bms.com> References: <427mk9$1tr@agate.berkeley.edu> <42flkh$83f@agate.berkeley.edu> <42jmaa$3o0@sparcserver.lrz-muenchen.de> NNTP-Posting-Host: silvinj1.wf.bms.com > lhom@nature.Berkeley.EDU (Louis Hom) wrote: > >Can someone recommend a good introductory book on peptide synthesis? It > >looks like there is a long history of books by Miklos Bodanszky(?) in our > >library. Has anyone had experience with his writing? > >-- > >_______________________________________________________________________________ > >Lou Hom Pete Wilson & David Duke in '96. > >lhom@nature.berkeley.edu Be scared. Be very, very scared. > Hi, A good introductory book on peptide synthesis is Stewart and Young's "Solid-Phase Peptide Synthesis". It's available from Pierce. Bodansky's books are invaluable to the peptide scientist, but may contain more detail than you are interested in. He did come outwith a small paperback about 3 years ago which is more of an overview of peptide synthesis. I believe it's called something like "Peptide Chemistry". Hope this helps. Pat Cash From owner-proteins@net.bio.net Wed Sep 06 23:00:00 1995 Path: biosci!daresbury!nntp-trd.UNINETT.no!Norway.EU.net!EU.net!howland.reston.ans.net!vixen.cso.uiuc.edu!news.uoregon.edu!cisco-ts5-line11.uoregon.edu!user From: noone@nowhere.org (Dr. Ted) Newsgroups: bionet.molbio.proteins Subject: Re: Help trouble shooting expression and folding in bacterial system Date: 8 Sep 1995 06:32:32 GMT Organization: Bedlam Lines: 82 Message-ID: References: <42i80a$76a@tali.UCHSC.edu> NNTP-Posting-Host: cisco-ts5-line11.uoregon.edu In article <42i80a$76a@tali.UCHSC.edu>, Holers Lab wrote: > > > From: guthridj@essex.hsc.colorado.edu (Joel Guthridge, Ph.D.) > We've tried: Lower induction temp. > > Lower IPTG levels. > > > > Possible Altern. (We need advise here) > > > > -do expression in the presence of Glutatione, etc. = > > > -? better periplasmic fractionation (how do others do this?) > > -do expression in presence or absence of Ca2+ (FLAG req. Ca2+= > ) = > > > -change epitope tag system ???? > > -change expression system (Pichia??) = > > > -reduce and refold protein in vitro (methods ??) Joel, Your problem is a common one, if that makes you feel better. It is very easy to get your typical disulfide bonded eukaryotic protein into insoluble inclusion bodies by overexpressing it in E. coli. Note that these inclusion bodies can be formed in the cytoplasm OR the periplasm. You have probably checked to see if the signal peptide is cleaved in the solubilized monomers from the BL21, so you can see if the processing/ transport is working. Typical periplasmic fractionation is by osmotic shock, although this is awkward to scale up. Also note that the DH5alpha can be grown to about a kilogram of wet cell mass per 10L fermentation with the correct feed protocol, so low level expression is not the end. The stable isotope label/minimal media will be tough, though. As for the changes you mention: glutathione- E. coli is pretty particular about the redox potential it maintains inside, this will most likely not work periplasmic fractionation-Check the lit. to see if your up to speed, but this will not make the protein behave, simply make it more pure. Ca2+, epitope tag- your problem is the overexpression, and the cysteines so the tag is probably not going to help, you could omit it to see if it helps, but the purification will be harder. Invitrogen(Pichia) markets a thioredoxin fusion system and NEB has the maltose binding protein, these may help but probably not. Pichia- works great for some things but its only advantage over Sacc. cerve is the huge cell mass, I'd try reg. yeast first, then there is the odd glycosylation and stable isotope label problems... best to stick with E. coli. Renature/reduce the protein- YES TRY THIS. Inclusion bodies can be a shortcut to purification, they pellet in the centrifuge almost pure, and there are currently several large articles discussing renaturation/refolding, some using disulfide isomerase. You have only 4 disulfides= two possible bond combination; one right one wrong, worth a shot. Check medline, Bio/technlogy etc. I've seen this work with over 5 S-S bridges.These papers should be easy to find. Methods in Enzymology?.. > Lower IPTG levels. low level expression only occurs with the TAC promoter at IPTG levels below 1µM. The promoter is leaky though, sometimes uninduced cells give more active protein than induced cells...check it out. Good Luck. regards, Ted Michelini Institute of Molecular Biology University of Oregon tedm@darkwing.uoregon.edu From owner-proteins@net.bio.net Wed Sep 06 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!vixen.cso.uiuc.edu!news.uoregon.edu!news.corpcomm.net!news.pepboys.com!netaxs.com!usenet From: "George A. Heavner" Newsgroups: bionet.molbio.proteins Subject: Re: Peptide Synthesis Date: 5 Sep 1995 19:48:20 GMT Organization: Net Access - Philadelphia's Internet Connection Lines: 21 Message-ID: <42i9i4$8qi@netaxs.com> References: <427mk9$1tr@agate.berkeley.edu> <42d8u4$o74@netaxs.com> <42frri$4ch@gap.cco.caltech.edu> NNTP-Posting-Host: 198.69.189.46 One reference to N to C solid phase peptide synthesis is: A Novel Method of Solid Phase Peptide Synthesis, R.P. Sharma, D.A. Jones,D.L. Corina and M. Akhtar in Proceedings of the Thirteenth American Peptide Symposium, pp 127-129,R.S. Hodges and J.A. Smith, Eds. ESCOM, Leiden, 1994. Some of the earlier work in this area was done in the 60s by Robert Letsinger. The problems with this approach is attachment to the resin, activation of the resin-bound carboxyl group and forcing the reaction to completion. With carboxylate activation, you cannot use an excess of the activated agent. If amino group activation becomes practical, and some work has been done in this area, then the N to C solid phase method could be competitive with C to N synthesis. George A. Heavner From owner-proteins@net.bio.net Wed Sep 06 23:00:00 1995 Path: biosci!celtrix.com!yvonne_thorstenson From: yvonne_thorstenson@celtrix.com ("Yvonne Thorstenson") Newsgroups: bionet.molbio.proteins Subject: RE: Help trouble shooting expression and folding in bacterial system Date: 7 Sep 1995 11:02:08 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 166 Sender: daemon@net.bio.net Distribution: world Message-ID: <199509071744.KAA13329@netcomsv.netcom.com> NNTP-Posting-Host: net.bio.net Dear Joel, It seems like your protein is able to fold correctly in the bacterial periplasm since you saw it (even in low quantities) in DH5a. When you changed to BL21, is it possible that your protein went to inclusion bodies? If that is the case, then it probably won't help to adjust the fractionation protocol. On the other hand, if your protein is still being transported to the periplasm in BL21, then my bet is that the increased production of your protein has overwhelmed the oxidizing capability of the periplasm. The most recent issue of Bio/Technology has an article that may be of interest to you: Lisa A. Collins-Racie et al. Sept 1995. "Production of recombinant bovine enterokinase catalytic subunit in E. coli using the novel secretory fusion partner DsbA." Bio/Technology 13: 982-87. Hope that helps. I would be interested in knowing what you end up doing. Cheers, Yvonne _______________________________________________________________________________ To: proteins@net.bio.net From: Holers Lab on Tue, Sep 5, 1995 7:08 PM Subject: Help trouble shooting expression and folding in bacterial system RFC Header:From BIOSCI-REQUEST@net.bio.net Tue Sep 5 18:57:14 1995 Received: from net.bio.net by netcomsv.netcom.com with ESMTP (8.6.12/SMI-4.1) id SAA16054; Tue, 5 Sep 1995 18:57:14 -0700 Received: (from daemon@localhost) by net.bio.net (8.6.12/8.6.6) id SAA08164 for protein-analysis-list; Tue, 5 Sep 1995 18:38:22 -0700 Received: (from news@localhost) by net.bio.net (8.6.12/8.6.6) id SAA08160 for bionet-protein-analysis-arpanet; Tue, 5 Sep 1995 18:38:20 -0700 To: proteins@net.bio.net From: Holers Lab Subject: Help trouble shooting expression and folding in bacterial system Date: 5 Sep 1995 19:21:46 GMT Message-ID: <42i80a$76a@tali.UCHSC.edu> NNTP-Posting-Host: patroklos.uchsc.edu Mime-Version: 1.0 Content-Type: text/html Content-Transfer-Encoding: quoted-printable X-Mailer: Mozilla 1.1N (Windows; I; 16bit) Message Body: From: guthridj@essex.hsc.colorado.edu (Joel Guthridge, Ph.D.) Subject: Help troubleshooting bacterial expression of prop. folded protein Date: Tue, 5 Sep 1995 13:52:03 Message-ID: I'm trying to produce protein to NMR analysis using IBI's FLAG epitope = tagged system. I'm using a vector which places the OmpA signal peptide = N-terminal to the N-terminal FLAG sequence, so the protein expression is = inducible by IPTG, and hopefully properly folded material is recovered in t= he = periplasmic fraction (detectible using the M1 antibody from IBI). In my = initial trials, I cloned this into the DH5a, and got detectible, but low = production. The affinity purified material appeared to be properly folded = (note my protein has 4 Cys. that are disulfide bonded) and proper disulfide= d = created. The problem was the level of production. Subsequently, I've put = this vector into the BL21 protease neg. strain, and got substantially bette= r = production, however now most of the material (even though soluble, from the= = periplasm and M1 (N-term) FLAG detectable) is found in higher molecular wei= ght = forms under non-reducing conditions, but reducable to a strong band of the = appropriate mol. wt. I think that the conditions we are now producing is n= ot = allowing appropriate disulfide bond formation. Does anyone have any = suggestions for how to overcome this problem? The production in the DH5a w= as = far too low to make that a cost effective option. = We've tried: Lower induction temp. Lower IPTG levels. Possible Altern. (We need advise here) -do expression in the presence of Glutatione, etc. = -? better periplasmic fractionation (how do others do this?) -do expression in presence or absence of Ca2+ (FLAG req. Ca2+= ) = -change epitope tag system ???? -change expression system (Pichia??) = -reduce and refold protein in vitro (methods ??) The protein we are producing is fairly hydrophobic and proper disulfide = bonding is required for function. Could the epitope tag be influencing the= = proper folding of the molecule. Any advise would be greatly appreciated, as would pointers to appropriat= e = reference materials on bacterial expression systems of eukaryotic proteins = for = structural analysis. Joel Guthridge email: Joel.Guthridge@UCHSC.edu From owner-proteins@net.bio.net Thu Sep 07 23:00:00 1995 Path: biosci!agate!spool.mu.edu!howland.reston.ans.net!tank.news.pipex.net!pipex!oleane!jussieu.fr!univ-lyon1.fr!ws41.cnusc.fr!ciril.fr!u-strasbg.fr!news From: nussbaum@neurochem.u-strasbg.fr Newsgroups: bionet.molbio.proteins Subject: ECM proteins Date: 8 Sep 1995 13:29:38 GMT Organization: CNRS Lines: 7 Message-ID: <42pgg2$eat@apopi.u-strasbg.fr> NNTP-Posting-Host: noiset.u-strasbg.fr X-Newsreader: Could someone tell me how to proceed to get rapidely and easily the bulk of the ECM proteins of brain; I want to test if my protein binds to one of these ECM proteins by using the Western blot method. or where I can buy such a total extract. Many thanks From owner-proteins@net.bio.net Thu Sep 07 23:00:00 1995 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!tank.news.pipex.net!pipex!howland.reston.ans.net!spool.mu.edu!caen!kuhub.cc.ukans.edu!xiaokezh From: xiaokezh@kuhub.cc.ukans.edu Newsgroups: bionet.molbio.proteins Subject: arachidonic acid poster Message-ID: <1995Sep8.133416.102998@kuhub.cc.ukans.edu> Date: 8 Sep 95 13:34:16 CDT Organization: University of Kansas Academic Computing Services Lines: 7 Several years ago, I got a color poster about arachidonic acid pathway, including all of the enzymes and inhibitors involved in each step. I would like to obtain such a poster now, but I forget which company it came from. I would appreciate if anyone could let me know where I can find such thing.. thank you. tole and e oorr From owner-proteins@net.bio.net Thu Sep 07 23:00:00 1995 Path: biosci!ns1.faseb.org!lamarck.sura.net!newsfeed.internetmci.com!tank.news.pipex.net!pipex!lade.news.pipex.net!pipex!sunic!sunic.sunet.se!news.uni-c.dk!eagle.novo.dk!usenet From: "" <\@novo.dk> Newsgroups: bionet.molbio.proteins Subject: Re: CD secondary structure Date: 8 Sep 1995 11:16:38 GMT Organization: Novo Nordisk A/S Lines: 11 Message-ID: <42p8mm$p42@eagle.novo.dk> References: <31AUG95.24226600.0073@VM1.MCGILL.CA> NNTP-Posting-Host: pof.novo.dk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Windows; I; 16bit) To: bgzw@musicb.mcgill.ca Hi, Laura The are two good programs that you can use which introduce the far-UV CD spectrum down to 178 nm. As far as I know, I think you have to write to the authors to ask them to send you the programs. 1)CCA. Perczel et al. Analytical Biochemistry 203,83-93 (1992) 2)VSM. Parthasarathy et al. Analytical Biochemistry 167,76-85 (1987) Best gregards Per-Ola pof@novo.dk From owner-proteins@net.bio.net Thu Sep 07 23:00:00 1995 Path: biosci!NMSU.EDU!hroychow From: hroychow@NMSU.EDU (Hiranya Roychowdhury) Newsgroups: bionet.molbio.proteins Subject: Re: Protein Purification Date: 8 Sep 1995 09:06:42 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 19 Sender: daemon@net.bio.net Distribution: world Message-ID: References: NNTP-Posting-Host: net.bio.net On Tue, 5 Sep 1995, Stephen Gately wrote: > Can anyone recommend a newsgroup that discusses protein purification by > ion exchange and affinity chromatography? > > Thank you > > What other group than this very one!? >>>>>>>>>>>>>>>>>>>>>>>>>>> Hiranya S. Roychowdhury Plant Genetic Engineering Lab. Box 3GL, NM State Univ. Las Cruces, NM 88003 Phone: (505) 646-5785 hroychow@nmsu.edu <<<<<<<<<<<<<<<<<<<<<<<<<<< From owner-proteins@net.bio.net Fri Sep 08 23:00:00 1995 Path: biosci!agate!howland.reston.ans.net!news.sprintlink.net!in1.uu.net!nntp.hk.super.net!tst.hk.super.net!techcomp From: techcomp@hk.super.net (Mr Eric Chan) Newsgroups: bionet.molbio.proteins Subject: www from Techcomp- agarose Date: 9 Sep 1995 08:12:35 GMT Organization: Hong Kong Supernet Lines: 1 Message-ID: <42ri9j$ds1@tst.hk.super.net> NNTP-Posting-Host: is1.hk.super.net X-Newsreader: TIN [version 1.2 PL2] From owner-proteins@net.bio.net Fri Sep 08 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!agate!howland.reston.ans.net!news.sprintlink.net!in1.uu.net!newsflash.concordia.ca!CC.UMontreal.CA!news.uqam.ca!merlin!tm100429 From: tm100429@merlin.si.uqam.ca (TREMBLAY GUY) Subject: Re: Ribosomal Proteins <-> rRNA. How much is known ? Message-ID: Sender: news@news.uqam.ca Nntp-Posting-Host: merlin.si.uqam.ca Organization: Universite du Quebec a Montreal X-Newsreader: TIN [version 1.2 PL2] References: Date: Sat, 9 Sep 1995 21:31:39 GMT Lines: 34 Georg Fuellen (fuellen@techfak.uni-bielefeld.de) wrote: ... > How much is known about the interaction between Ribosomal Proteins > and the ribosomal RNA ? Have points of contact between specific nucleotides > and specific amino acids been identified ? In other words, how much > detail does one know about the structure of the ribosome, beyond > the standard textbook picture displaying the spatial arrangement of ... The following articles may interest you: Probing the phosphates of the E.coli ribosomal 16S rRNA in its naked form in the 30S subunit and in the 70S ribosome, F. Baudin et al. Biochemistry, 1989(28) 5847-5855 Model for the 3D folding of 16S ribosomal RNA. Stern, S. et al. J. mol. biol. 1988(204) 447-481. As far as I know it is too hard to obtain a good crystal structure with ribosomes. The maximal resolution reached for X-ray is around 15-20 Angstrom so it is very poor. The modelling of the ribosome is made with softwares like mcsym. It is getting obvious that "there is no such thing" as ribosomal RNA and ribosomal proteins because the complex may be very different than its components. So good luck trying to solve the structure. --------------------------------------------------------------------- | /\-/\ | Guy Tremblay | | ((O O)) | (514) 521-2195 | | \\\ \\>// /// | tm100429@merlin.si.uqam.ca | | \\\\///\\//// | Universite du Quebec a Montreal | --------------------------------------------------------------------- From owner-proteins@net.bio.net Fri Sep 08 23:00:00 1995 Path: biosci!agate!newsxfer.itd.umich.edu!newsfeed.internetmci.com!news.sprintlink.net!in2.uu.net!cis.ohio-state.edu!magnus.acs.ohio-state.edu!lerc.nasa.gov!purdue!news.bu.edu!taco.cc.ncsu.edu!news From: dtoroser@unity.ncsu.edu (Dikran Toroser) Newsgroups: bionet.molbio.proteins Subject: Re: Ion exchange, protein purification Date: 8 Sep 1995 23:36:20 GMT Organization: North Carolina State University Lines: 34 Distribution: world Message-ID: <42qk1k$f0l@taco.cc.ncsu.edu> References: <6SEP199511475077@watson.bms.com> NNTP-Posting-Host: leachr11.usda.ncsu.edu Mime-Version: 1.0 X-Newsreader: WinVN 0.93.14 In article <6SEP199511475077@watson.bms.com>, curtis@watson.bms.com says... > >I am looking for a very generic protocol to use as a starting point for some >FPLC work. I have some mono Q (Pharmacia) in the lab, as well as mono S. Does >anyone have a general buffer system that they commonly use. I hope this is not >too naive a question? I have done some research into different types of >resins, etc. but no one seems to give general running conditions etc. Any help >would be appreciated. > >Thnak you, > >Mike Curtis >curtis@bms.com > We use a linear gradient to separate PEG precipitated proteins; buffer A= 50 mM MOPS/NaOH (Ph 7.5), 10 mM MgCl2, 2.5 mM DTT; buffer B= as previous but with 1M NaCl. Limit the Separation from ) to O.5 mM NaCl over the desired period of time and then jum to 100 %B for cleaning. Works well with Mono-Q and Res-Q. Hope i goes well. Dikran Toroser USDA-ARS Plant Science Research 3127 Ligon Street, Raleigh, NC 27607, USA. From owner-proteins@net.bio.net Sat Sep 09 23:00:00 1995 Path: biosci!agate!spool.mu.edu!howland.reston.ans.net!news.sprintlink.net!in1.uu.net!EU.net!sun4nl!news.euro.net!usenet From: "Andre W. Schram" Newsgroups: bionet.molbio.proteins Subject: ANNOUNCEMENT: PROTEIN BIOTECHNOLOGY COURSE Date: 10 Sep 1995 16:43:24 GMT Organization: BioUpdate Foundation Lines: 26 Message-ID: <42v4jc$1l5@news.euro.net> NNTP-Posting-Host: dial6.nedernet.nl Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) X-URL: news://news.nedernet.nl/bionet.molbio.proteins Protein Biotechnology Course held in the Netherlands October 30 - November 3 1995 Organised by the BioUpdate Foundation, the Netherlands Studies of the biochemistry of disease, coupled with advances in recombinant DNA technology have led to massive commercial interest in the production of highly purified, stable proteins. Apart from protein therapeutics, expanding markets also exist in diagnostics, industrial enzymes as biocatalysts, veterinary products and molecular biology reagents. This protein biotechnology course addresses developments in protein technology from protein extraction, through the various protein purification stages, to their presentation as stable products. The respective merits of various methods for protein characterisation and quality control are compared, issues of protein stability are treated in detail, and topics of current interest, such as protein engineering and protein glycosylation are reviewed. More information available on the BioUpdate homepage: http://www.nedernet.nl/bioupdate/index.html From owner-proteins@net.bio.net Sat Sep 09 23:00:00 1995 Path: biosci!agate!howland.reston.ans.net!news.sprintlink.net!in1.uu.net!news.compuserve.com!news.production.compuserve.com!news From: Sherilyn Bell <72650.1760@CompuServe.COM> Newsgroups: bionet.molbio.proteins Subject: Messy Western blots Date: 11 Sep 1995 02:18:25 GMT Organization: CompuServe, Inc. (1-800-689-0736) Lines: 17 Message-ID: <43069h$19j$1@mhade.production.compuserve.com> For some months I have been trying to examine the protein product of a DNA construct by transfecting cos-1 cells, lysing it in SDS sample buffer, running it on 8% acrylamide, blotting on nitrocellulose and incubating in anitbody against a portion of the construct. I have been detecting with ECL but my blots have many nonspecific bands. I have tried preclearing with untransfected cos-1 lysate, varying incubation times, washing for longer times and more changes (6x5min). I have been seeing the product I want, but its hard to interpret with so much background. The antibody is know to be specific against larger constructs containing the same sequence with less background. Does anyone have suggestions? -- Sherilyn Bell slbell@sickkids.on.ca From owner-proteins@net.bio.net Sat Sep 09 23:00:00 1995 Path: biosci!bloom-beacon.mit.edu!newsfeed.internetmci.com!tank.news.pipex.net!pipex!in1.uu.net!news.compuserve.com!news.production.compuserve.com!news From: Patrick R. Jones <102570.2734@CompuServe.COM> Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.molluscs,bionet.molbio.proteins,bionet.molbio.rapd,bionet.molbio.yeast Subject: Need Help!! Date: 10 Sep 1995 20:13:45 GMT Organization: Personal Lines: 19 Message-ID: <42vgtp$9h8$1@mhafm.production.compuserve.com> Xref: biosci bionet.molbio.methds-reagnts:33310 bionet.molbio.molluscs:143 bionet.molbio.proteins:5587 bionet.molbio.rapd:1280 bionet.molbio.yeast:3709 I am a undergraduate student in biology. Some day I hope to go to graduate school in biochemistry. My grades are good enough but I am afraid that this may not be enough to get in. I am looking for something to set myself apart from the other students. At this point I am at a loss on what to do. I have no real resources to work on projects in the areas that interest me. Does anybody have any ideas? I do have one useful skill. I am a professional computer programmer. I don’t mean someone who has taken a FORTRAN class and is working as a work-study student, but a 10 year application programming veteran in the computing industry. If I could use this skill great if not, I don’t care. I need something to further my career; something good. Please help!!! I know that somebody out there knows of something I could do. Thank you for your time!!! Patrick R. Jones -- <> From owner-proteins@net.bio.net Sat Sep 09 23:00:00 1995 Path: biosci!bloom-beacon.mit.edu!spool.mu.edu!howland.reston.ans.net!vixen.cso.uiuc.edu!news.uoregon.edu!news.u.washington.edu!verlinde From: verlinde@u.washington.edu (Christophe Verlinde) Newsgroups: bionet.molbio.proteins Subject: SN2 reaction and transition state inhibitors Date: 11 Sep 1995 04:12:23 GMT Organization: University of Washington Lines: 6 Message-ID: <430cv7$ptm@nntp3.u.washington.edu> NNTP-Posting-Host: saul3.u.washington.edu NNTP-Posting-User: verlinde I want to design transition state inhibitors of an enzyme that has an SN2 catalytic mechanism. Since modelers are much better at mimicking than designing from scratch I would like to hear about a few enzymes that operate via SN2 and for which transition-state inhibtiors are known. Thanks, Christophe Verlinde From owner-proteins@net.bio.net Sun Sep 10 23:00:00 1995 Path: biosci!daresbury!nntp-trd.UNINETT.no!Norway.EU.net!EU.net!howland.reston.ans.net!tank.news.pipex.net!pipex!sunsite.doc.ic.ac.uk!lyra.csx.cam.ac.uk!swhbmac9.bioc.cam.ac.uk!user From: Trumpington (LAGER) Newsgroups: bionet.molbio.proteins Subject: Re: Help with CD data analysis. Date: Mon, 11 Sep 1995 18:24:47 -0200 Organization: shrubbery Lines: 43 Message-ID: References: <43159f$rod@news.ox.ac.uk> NNTP-Posting-Host: swhbmac9.bioc.cam.ac.uk In article <43159f$rod@news.ox.ac.uk>, njh@mail.nerc-oxford.ac.uk wrote: > Hi, > > I have just made my first tentative steps into the world of circular dichroism and I am now > looking for any information on interpreting those squiggly lines!! Does anyone know of any > article or review or basic introduction to the interpretation of CD data? What are the > significant wavelengths for both proteins and RNA? I am investigating an RNA-binding protein > and looking for changes on binding in both the protein and the RNA. > > Any help appreciated. Thanks. > > > Nigel > > njh@mail.nerc-oxford.ac.uk Pages 190 -191 of Creighton, second ed. give a simple outline of CD. The following references give descriptions of three different means by which the secondary structure of proteins can be estimated from a CD spectra (i.e. FAR UV CD spectra which are, in practice, from around 178 to 260nm; NEAR UV CD spectra however, are from around 260 to +350nm, and are sensitive to chromophores and disulpide bonds in chiral environments in the tertiary structure of proteins: averaging in the random coil state of a protein, will result in no Near UV CD signal, hence these spectra are sensitive to the tertiary structure of proteins) Provencher et al; (1981) Biochemistry V.20 P.33-38 Johnson et al; (1987) Anal.Biochem. V.167 P.76-85 Fasman et al; (1992)PROTEINS: Struct. Funct. Gen. V.13 p.57-69 However, as a word of warning about over-interpreting CD spectra, I'd strongly advise reading : Vuilleumier et al; (1993) Biochemistry V.32 P.10303 -10313; Finally, there should be lots of people in the group of Chris Dobson, in Oxford who could tell you about CD. Hope this helps Laurence Tisi lct11@uk.ac.cam.bio.mole From owner-proteins@net.bio.net Sun Sep 10 23:00:00 1995 Path: biosci!agate!dharma.hip.berkeley.edu!user From: genecutl@mendel.berkeley.edu (gc) Newsgroups: bionet.molbio.proteins Subject: Re: Help with CD data analysis. Date: Mon, 11 Sep 1995 10:20:53 -0800 Organization: world domination Lines: 37 Message-ID: References: <43159f$rod@news.ox.ac.uk> NNTP-Posting-Host: dharma.hip.berkeley.edu X-Newsreader: Value-Added NewsWatcher 2.0b27.1+ In article <43159f$rod@news.ox.ac.uk>, njh@mail.nerc-oxford.ac.uk wrote: > Hi, > > I have just made my first tentative steps into the world of circular dichroism and I am now > looking for any information on interpreting those squiggly lines!! Does anyone know of any > article or review or basic introduction to the interpretation of CD data? What are the > significant wavelengths for both proteins and RNA? I am investigating an RNA-binding protein > and looking for changes on binding in both the protein and the RNA. > > Any help appreciated. Thanks. > > > Nigel > > njh@mail.nerc-oxford.ac.uk The generic answer to that question is look in any basic protein textbook and you can find that info. As a followup to that, does anybody know of any software to deconvolute CD spectra to get percent helix and sheet? Thanks. -- gc. --gc ...doom is like the handle of a pot, it's there, know it, have ice in your tea, marry, have children, visit your dentist, do not scream at night even genecutl@mendel.berkeley.edu if you feel like screaming... -cb http://sp1.berkeley.edu/gc.html From owner-proteins@net.bio.net Sun Sep 10 23:00:00 1995 Path: biosci!agate!news.duke.edu!godot.cc.duq.edu!newsfeed.pitt.edu!dsinc!netnews.upenn.edu!cronkite.ocis.temple.edu!astro.ocis.temple.edu!driska From: driska@astro.ocis.temple.edu (Stephen P. Driska PhD) Newsgroups: bionet.molbio.proteins Subject: Protein standards for DENATURING (9M urea) IEF Date: 11 Sep 1995 15:18:19 GMT Organization: Temple University, Academic Computer Services Lines: 22 Message-ID: <431jvr$sr1@cronkite.ocis.temple.edu> NNTP-Posting-Host: astro.ocis.temple.edu X-Newsreader: TIN [version 1.2 PL2] Many companies sell protein standards for isoelectric focusing (IEF), but it seems that nearly all of the products are intended for NON-DENATURING IEF of native proteins. I'm looking for a standard mixture for IEF under denaturing conditions, e.g. 9M urea, Nonidet P-40, dithioerythritol. The only product I have seen that seems like it would be appropriate is the Sigma kit for 2-Dimensional electrophoresis. This kit only has 4 proteins, though, so it might not be complete enough for what I want to do. There are lots of mixtures for 1D-SDS electrophoresis, but I wouldn't necessarily expect these standards to have nice banding patterns in IEF gels. My question is, can any one tell me where I might buy a standard mixture for denaturing IEF, or perhaps recommend a selection of known proteins which could provide nice bands in 9 M urea ? Thanks very much for your help, Steve Driska From owner-proteins@net.bio.net Sun Sep 10 23:00:00 1995 Path: biosci!bloom-beacon.mit.edu!newsfeed.internetmci.com!tank.news.pipex.net!pipex!uknet!bhamcs!news.ox.ac.uk!news From: njh@mail.nerc-oxford.ac.uk Newsgroups: bionet.molbio.proteins Subject: Help with CD data analysis. Date: 11 Sep 1995 11:07:27 GMT Organization: Oxford University Lines: 15 Message-ID: <43159f$rod@news.ox.ac.uk> NNTP-Posting-Host: ivpc038.nerc-oxford.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Windows; I; 16bit) Hi, I have just made my first tentative steps into the world of circular dichroism and I am now looking for any information on interpreting those squiggly lines!! Does anyone know of any article or review or basic introduction to the interpretation of CD data? What are the significant wavelengths for both proteins and RNA? I am investigating an RNA-binding protein and looking for changes on binding in both the protein and the RNA. Any help appreciated. Thanks. Nigel njh@mail.nerc-oxford.ac.uk From owner-proteins@net.bio.net Sun Sep 10 23:00:00 1995 Path: biosci!cc.kmitt.ac.th!s781201 From: s781201@cc.kmitt.ac.th (Operator) Newsgroups: bionet.molbio.proteins Subject: Re: IMPORTANT - BIOSCI MOVE IS TODAY!! Date: 11 Sep 1995 00:37:51 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 3 Sender: daemon@net.bio.net Distribution: world Message-ID: References: <3set13$a7m@net.bio.net> NNTP-Posting-Host: net.bio.net m From owner-proteins@net.bio.net Sun Sep 10 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!vixen.cso.uiuc.edu!news.ecn.bgu.edu!willis.cis.uab.edu!nntp.msstate.edu!fiona.umsmed.edu!fiona.umsmed.edu!hutchins From: hutchins@fiona.umsmed.edu (Jim Hutchins) Newsgroups: bionet.molbio.proteins Subject: Re: Messy Western blots Date: 11 Sep 1995 15:47:53 GMT Organization: University of Mississippi Medical Center Lines: 42 Message-ID: <431ln9$c3b@fiona.umsmed.edu> References: <43069h$19j$1@mhade.production.compuserve.com> NNTP-Posting-Host: fiona.umsmed.edu X-Newsreader: TIN [version 1.2 PL1] Sherilyn Bell (72650.1760@CompuServe.COM) wrote: : For some months I have been trying to examine the protein product : of a DNA construct by transfecting cos-1 cells, lysing it in SDS [stuff deleted] : the construct. I have been detecting with ECL but my blots have : many nonspecific bands. I have tried preclearing with : untransfected cos-1 lysate, varying incubation times, washing for : longer times and more changes (6x5min). [more stuff deleted] : Does anyone have suggestions? Yes. You might try: 1. A blocking step; I'm not sure this is compatible with ECL but I think it is. Depending on your protein, you might use 1-2% BSA or 1-5% Blotto(tm) [Carnation non-fat dry milk in buffer] or 1-2% fish gelatin or any number of other blocking agents. 0.05% Tween-20 also seems to work sometimes. 2. Reduce your protein loading. For most purposes, 10-50 ug per lane seems to be optimal. No one seems to know why. If your cells are really pumping the stuff out, you may need to go even lower in terms of total protein. 3. Reduce your primary antibody concentration 4. Reduce your secondary antibody concentration I would try these steps in the order listed until you get satisfactory results. You may have to play with all of these parameters to get an optimal blot. Have you tried PVDF or nylon instead of nitrocellulose? That might be #5 on the list. :-) Hope this helps -- Jim Hutchins Assoc Prof Anatomy, Asst Prof Neurology (Research), Univ Mississippi Med Ctr http://fiona.umsmed.edu/~hutchins/ *** E-mail: hutchins@umsmed.edu ``It became necessary to destroy the town in order to save it.''--American infantry officer firing on Ben Tre, Vietnam, 2/8/68 From owner-proteins@net.bio.net Sun Sep 10 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!ihnp4.ucsd.edu!agate!howland.reston.ans.net!news.sprintlink.net!in2.uu.net!nih-csl!loglady.ninds.nih.gov!johnk From: johnk@spasm.niddk.nih.gov (John Kuszewski) Subject: Re: Ribosomal Proteins <-> rRNA. How much is known ? Message-ID: <1995Sep11.160545.13543@alw.nih.gov> Sender: postman@alw.nih.gov (AMDS Postmaster) Nntp-Posting-Host: spasm.niddk.nih.gov Reply-To: johnk@spasm.niddk.nih.gov (John Kuszewski) Organization: National Insts. of Health References: Date: Mon, 11 Sep 1995 16:05:45 GMT Lines: 63 In article , tm100429@merlin.si.uqam.ca (TREMBLAY GUY) writes: |> Georg Fuellen (fuellen@techfak.uni-bielefeld.de) wrote: |> ... |> > How much is known about the interaction between Ribosomal Proteins |> > and the ribosomal RNA ? Have points of contact between specific nucleotides |> > and specific amino acids been identified ? In other words, how much |> > detail does one know about the structure of the ribosome, beyond |> > the standard textbook picture displaying the spatial arrangement of |> ... |> |> The following articles may interest you: |> |> Probing the phosphates of the E.coli ribosomal 16S rRNA in its naked form |> in the 30S subunit and in the 70S ribosome, F. Baudin et al. |> Biochemistry, 1989(28) 5847-5855 |> |> Model for the 3D folding of 16S ribosomal RNA. Stern, S. et al. J. mol. |> biol. 1988(204) 447-481. |> |> As far as I know it is too hard to obtain a good crystal structure with |> ribosomes. The maximal resolution reached for X-ray is around |> 15-20 Angstrom so it is very poor. The modelling of the ribosome is made |> with softwares like mcsym. It is getting obvious that "there is no such |> thing" as ribosomal RNA and ribosomal proteins because the complex may be |> very different than its components. So good luck trying to solve the |> structure. Yes, but that's not quite the question that was asked. I have heard of some footprinting studies on intact or partial ribosomes that point out the specific rRNA sequences that are bound by specific ribosomal proteins, but I don't have the reference in front of me. There was also a paper in Science last year (I think) that showed which rRNA sequences were involved in the peptidyl transferase reaction center. Finally, there are a few high-resolution structures of pieces of ribosomes. Steve White at Duke got one ribosomal protein to give good crystals, and Peter Moore at Yale did the structure of the sarcin/ricin RNA loop by NMR. So I think a more accurate answer is that quite a bit more has become known about the structure of ribosomes since the late 1980s, but that we still don't have a great picture of the overall "machine," or even of all of the interesting parts of it. -- _____________ | ___/_ | |/ / -- /\ // /-- || || / /|| || || / / || || ||/ / || John Kuszewski || |/ /| || johnk@spasm.niddk.nih.gov || / /|| || \/ / / || \/ that's MISTER protein G to you! |/__/| | /_________| "Biophysics has driven me to an attitude of apocalyptic doom" --Frank Delaglio From owner-proteins@net.bio.net Sun Sep 10 23:00:00 1995 Path: biosci!SCRIPPS.EDU!ryang From: ryang@SCRIPPS.EDU (Ri-Ya Yang/SBR4/4-9980) Newsgroups: bionet.molbio.proteins Subject: Re: Messy Western blots Date: 11 Sep 1995 08:55:10 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 26 Sender: daemon@net.bio.net Distribution: world Message-ID: References: <43069h$19j$1@mhade.production.compuserve.com> NNTP-Posting-Host: net.bio.net Maybe the the antibody concentrations were too high. Ri-Yao On 11 Sep 1995, Sherilyn Bell wrote: > For some months I have been trying to examine the protein product > of a DNA construct by transfecting cos-1 cells, lysing it in SDS > sample buffer, running it on 8% acrylamide, blotting on > nitrocellulose and incubating in anitbody against a portion of > the construct. I have been detecting with ECL but my blots have > many nonspecific bands. I have tried preclearing with > untransfected cos-1 lysate, varying incubation times, washing for > longer times and more changes (6x5min). I have been seeing the > product I want, but its hard to interpret with so much > background. The antibody is know to be specific against larger > constructs containing the same sequence with less background. > > Does anyone have suggestions? > > -- > Sherilyn Bell > slbell@sickkids.on.ca > > From owner-proteins@net.bio.net Sun Sep 10 23:00:00 1995 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!tank.news.pipex.net!pipex!dispatch.news.demon.net!demon!sunsite.doc.ic.ac.uk!lyra.csx.cam.ac.uk!mole.bio.cam.ac.uk!cdw1000 From: cdw1000@mole.bio.cam.ac.uk (Christopher Walentas (Bioc)) Newsgroups: bionet.molbio.proteins Subject: His tags & best second codons Date: 11 Sep 1995 18:42:17 GMT Organization: University of Cambridge, England Lines: 31 Message-ID: <431vu9$6ol@lyra.csx.cam.ac.uk> NNTP-Posting-Host: mole.bio.cam.ac.uk Hi. I've been trying to express a tagged version of glutathione reductase but to no avail. The wt expresses very well (100mg/L) from a Ptac (pKK223-3) vector (the tag isn't for straightforward purification purposes, but has a "higher" task). At first I tried MetHis6... but that didn't work (no protein could be seen on a gel, or by activity). I was then going to try sticking a few more neutral residues between the Met and the tag itself. pET seems to fancy MetGlySerSerHis6, while QIAGEN likes MetArgGlySerHis6. Does anyone have any personal favorites, or any comments upon the realtive levels of expression between these two? GR is a fairly large (100kDa homodimer) and is very stable, so I can only guess that the tag is somehow disturbed things at either the transcriptional or translational level-- rather than not allow the protein to fold and degrading. I guess this is obvious, but any suggestions wou be appreciated. Thanks. ------------------------------------------------------------------------------- C. D. Walentas Cambridge Centre for Molecular Recognition, University of Cambridge, Cambridge, England CB2 1QW Phone: 0223 333 662 Fax: 0223 333 345 email: c.d.walentas@bioc.cam.ac.uk From owner-proteins@net.bio.net Sun Sep 10 23:00:00 1995 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!howland.reston.ans.net!math.ohio-state.edu!newsfeed.acns.nwu.edu!news.cc.uic.edu!F017.DEN.UIC.EDU!pbm From: pbm@uic.edu (phillip messersmith) Newsgroups: bionet.molbio.proteins,bionet.molbio.proteins,sci.chem Subject: Transient Heating Effects on Proteins Date: Mon, 11 Sep 1995 13:27:52 Organization: University of Illinois at Chicago Lines: 19 Message-ID: NNTP-Posting-Host: f017.den.uic.edu X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] Xref: biosci bionet.molbio.proteins:5598 sci.chem:38732 I am trying to find out what is known about transient thermal degradation/denaturation of proteins. I am generally aware that thermal denaturation of some proteins can occur at just a few degrees above physiological temperature. However, I am interested more in transient heating effects, such as what happens to proteins when they are heated and cooled quickly. For example, I want to heat up a protein-containing matrix rapidly (100-1000 deg/sec) and quench (same rate) immediately after attaining a final temperature of 100-500C, hopefully preserving the structure/bioactivity of the protein. Is anything known about the effect of such rapid and short-term heating on structure and bioactivity of proteins? If anybody has experience in this area or can point me to key references on this topic, please send a response by e-mail to PBM@UIC.EDU. Thanks in advance. Phillip B. Messersmith pbm@uic.edu From owner-proteins@net.bio.net Sun Sep 10 23:00:00 1995 Path: biosci!ccmail.monsanto.com!MSABAD From: MSABAD@ccmail.monsanto.com ("MARK S ABAD") Newsgroups: bionet.molbio.proteins Subject: Re[2]: Messy Western blots Date: 11 Sep 1995 16:15:32 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 38 Sender: daemon@net.bio.net Distribution: world Message-ID: <9508118108.AA810868093@ccmail.monsanto.com> Dear Sherilyn Bell, It may help to block your membrane in 5% nonfat dry milk overnite after electrophoretic transfer and then do all incubations of primary, secondary and conjugate in milk also, but wash away the milk before you do the detection step. Mark Abad _________ Maybe the the antibody concentrations were too high. Ri-Yao On 11 Sep 1995, Sherilyn Bell wrote: > For some months I have been trying to examine the protein product > of a DNA construct by transfecting cos-1 cells, lysing it in SDS > sample buffer, running it on 8% acrylamide, blotting on > nitrocellulose and incubating in anitbody against a portion of > the construct. I have been detecting with ECL but my blots have > many nonspecific bands. I have tried preclearing with > untransfected cos-1 lysate, varying incubation times, washing for > longer times and more changes (6x5min). I have been seeing the > product I want, but its hard to interpret with so much > background. The antibody is know to be specific against larger > constructs containing the same sequence with less background. > > Does anyone have suggestions? > > -- > Sherilyn Bell > slbell@sickkids.on.ca > > From owner-proteins@net.bio.net Sun Sep 10 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!tank.news.pipex.net!pipex!in2.uu.net!nih-csl!fp5-9n.domain!user From: tomasd@bdg10.niddk.nih.gov (Tomas Drgon) Subject: protein microseqencing (summary) Message-ID: Sender: postman@alw.nih.gov (AMDS Postmaster) Nntp-Posting-Host: fp5-9n.domain Organization: NIH Date: Mon, 11 Sep 1995 21:35:07 GMT Lines: 75 Hi. Thanks everybody who responded. This is the summary: ==================================================== ------------------------------------------------------------------ From: pelpw@helix.nih.gov My lab would be willing to help you out (free)if you only have a limited number of samples. Further, I do not want to commit to long term projects etc. But, if you only have a few samples we will be willing to help you out. Paul Wingfield National Institutes of Health Building 6B, Room 1B 130 Bethesda MD-20892-2775 pelpw@helix.nih.gov ------------------------------------------------------------------ From: rah@mrc-lmb.cam.ac.uk Tomas, We operate a protein sequencing service, primarily for MRC staff, but will perform runs for outside customers as and when capacity allows. The person to contact is Kate Sparks, MIP Unit, MRC Centre, Hills Road, Cambridge CB2 2QH, UK. Tel: +1223-402001. Dick Harrison ------------------------------------------------------------- From altabios@bham.ac.uk Dear Tomas, Have just seen your posting on the bionet newsgroup concerning protein sequencing. I run, amongst other things, a protein sequencing operation. We use an ABI 473A, can cope with either free proteins or blots and have a wide customer base in the UK. The charge is 18 pounds per amino acid with a minimum of 5 cycles. We are normally able to start work within 2 or 3 days. If you are interested, I can send you our handbook which describes in a bit more detail what we do and how we do it. John Fox Alta Bioscience School of Biochemistry The University of Birmingham Edgbaston tel 0121 414 5450 Birmingham. U.K. fax 0121 414 3376 B15 2TT email altabios@bham.ac.uk ---------------------------------------------------------------- From: LESZYK@SCI.WFEB.EDU Hi Tomas, My name is John Leszyk and I am the director of the Worcester Foundation Microsequencing Facility. We perform microsequencing services fora large number of investigators throughout the country on a commercial basis. We alsohave extensive experience in sequencing samples on PVDF membranes for both N-terminal sequencing and internal sequencing. If you are interested in finding out more please contact me at (508)-842-8921 or FAX (508)-842-9632. Sincerely, John Leszyk ------------------------------------------------------------------- -- I speak for nobody but myself and my pet frog. From owner-proteins@net.bio.net Mon Sep 11 23:00:00 1995 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!elroy.jpl.nasa.gov!lll-winken.llnl.gov!fnnews.fnal.gov!unixhub!news.Stanford.EDU!b151-mac.stanford.edu!user From: tsunehis@cmgm.stanford.edu (Nam) Newsgroups: bionet.molbio.proteins Subject: Suspension CHO culture Date: Tue, 12 Sep 1995 13:53:16 +0000 Organization: Stanford University Lines: 9 Message-ID: Reply-To: tsunehis@cmgm.stanford.edu NNTP-Posting-Host: b151-mac.stanford.edu I am interested in making a secretable protein in large scale. To achieve this goal, I am planning to transfect the cDNA construct into CHO cell and adapt it in suspension culture. Does anybody know CHO cell line which is already suspension-adapted? Or is there easy method to adapt by myself? Thanks Nam tsunehis@cmgm.stanford.edu From owner-proteins@net.bio.net Mon Sep 11 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!usc!nic-nac.CSU.net!csulb.edu!drivel.ics.uci.edu!paris.ics.uci.edu!news.service.uci.edu!rablab.biochem.uci.edu!user From: user@host.uci.edu (Enter Your Name Here) Newsgroups: bionet.molbio.proteins Subject: What mammalian expression vector to choose? Date: Tue, 12 Sep 1995 19:38:21 +1000 Organization: UC Irvine Lines: 7 Message-ID: Reply-To: dmthomas@uci.edu NNTP-Posting-Host: rablab.biochem.uci.edu I want to express high level of a protein in mammalian cell lines (PC12 and fibroblasts) with a vector containg an inducible promoter. Also I'd like to add a tag to this protein. Any advices comcerning the choice of such a vector would be much appreciated. Please, address your answer to DMTHOMAS@uci.edu DT From owner-proteins@net.bio.net Mon Sep 11 23:00:00 1995 Path: biosci!botany.uq.edu.au!J.Marcus From: J.Marcus@botany.uq.edu.au ("Marcus, Dr J.") Newsgroups: bionet.molbio.proteins Subject: Re: Desalting Date: 12 Sep 1995 19:58:51 -0700 Organization: Dept of Botany, Univ of Qld Lines: 34 Sender: daemon@net.bio.net Distribution: world Message-ID: <5B646A6728C@botany.uq.edu.au> NNTP-Posting-Host: net.bio.net > We wish to purify an enzyme using ion exchange from serum-free conditioned > medium. The NaCl concentration is approximately 100 mM. We would like to > desalt this medium prior to binding to DEAE-sephadex. We have tried to > desalt using buffer exchange (Centri-Prep), however, the enzyme loses its > activity after desalting by this approach, even if NaCl concentrations are > raised back to physiological levels! > > Are there any other techniques available that may be used to desalt the > conditioned medium? > What about straight forward dialysis? Another thought is to try loading your material directly on to your ion-exchange column; it just might stick with 100mM NaCl. If it does stick, then you no longer have to worry about desalting. If your volumes are not too large you might also be able to dilute your sample with an equal volume of buffer with no NaCl, thus lowering the salt concentration and allowing your protein to bind to the column. Hoping that helps, John _________________________________________________________ John Marcus Marcus@tpp.uq.edu.au (Dr J.Marcus) Cooperative Research Centre for Tropical Plant Pathology 5th Level John Hines Building University of Queensland St. Lucia, QLD 4072 AUSTRALIA Fax: 61-7-365-4771 Phone: 61-7-365-4764 From owner-proteins@net.bio.net Mon Sep 11 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!tank.news.pipex.net!pipex!dispatch.news.demon.net!demon!sunsite.doc.ic.ac.uk!news.cc.ic.ac.uk!usenet From: mpalmer Newsgroups: bionet.molbio.proteins Subject: Re: Messy Western blots Date: 12 Sep 1995 15:44:40 GMT Organization: sm.ic.ac.uk Lines: 31 Message-ID: <4349t8$2cv@oban.cc.ic.ac.uk> References: <43069h$19j$1@mhade.production.compuserve.com> NNTP-Posting-Host: bc-43.sm.ic.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) X-URL: news:43069h$19j$1@mhade.production.compuserve.com Sherilyn Bell <72650.1760@CompuServe.COM> wrote: >For some months I have been trying to examine the protein product >of a DNA construct by transfecting cos-1 cells, lysing it in SDS >sample buffer, running it on 8% acrylamide, blotting on >nitrocellulose and incubating in anitbody against a portion of >the construct. I have been detecting with ECL but my blots have >many nonspecific bands. I have tried preclearing with >untransfected cos-1 lysate, varying incubation times, washing for >longer times and more changes (6x5min). I have been seeing the >product I want, but its hard to interpret with so much >background. The antibody is know to be specific against larger >constructs containing the same sequence with less background. > >Does anyone have suggestions? You should repeat the Western with secondary antibody alone to determine whether the extra bands are due to binding of the primary or secondary AB. If secondary you could preclear with non-conjugated Ig of the same type together with protein A-sepharose beads. Block the blots with 5% non fat dried milk in TBS-Tween and do all washes and hybes in TBS-Tween. Titre out both antibodies, especially the secondary AB. You can also dilute the ECL reagents if the required band is coming up quite quickly and optimise the autorad exposure time. Mark Palmer Dept. of Biochemistry, St Mary's Hospital Medical School, London, UK From owner-proteins@net.bio.net Mon Sep 11 23:00:00 1995 Path: biosci!agate!newsxfer.itd.umich.edu!newsfeed.internetmci.com!howland.reston.ans.net!Germany.EU.net!news.dfn.de!fu-berlin.de!zrz.TU-Berlin.DE!ppc6100.lb.tu-berlin.de!user From: muel1534@mailszrz.zrz.tu-berlin.de (Dirk Mueller) Newsgroups: bionet.molbio.proteins Subject: Aspergillus catalase Followup-To: bionet.molbio.proteins Date: Tue, 12 Sep 1995 16:35:41 +0200 Organization: Mikrobiologie und Genetik Lines: 5 Message-ID: NNTP-Posting-Host: ppc6100.lb.tu-berlin.de Is there anyone working on aspergillus catalase?? Bye Dirk. E-Mail: muel1534@mailszrz.zrz.tu-berlin.de From owner-proteins@net.bio.net Mon Sep 11 23:00:00 1995 Path: biosci!daresbury!nntp-trd.UNINETT.no!Norway.EU.net!EU.net!howland.reston.ans.net!news.cac.psu.edu!news.math.psu.edu!psuvax1!news.eecs.nwu.edu!newsfeed.acns.nwu.edu!news.acns.nwu.edu!med048050.medicine.nwu.edu!user From: sg@nwu.edu (Stephen Gately) Newsgroups: bionet.molbio.proteins Subject: Desalting Date: Mon, 11 Sep 1995 16:35:31 -0500 Organization: Northwestern University Lines: 9 Message-ID: NNTP-Posting-Host: med048050.medicine.nwu.edu We wish to purify an enzyme using ion exchange from serum-free conditioned medium. The NaCl concentration is approximately 100 mM. We would like to desalt this medium prior to binding to DEAE-sephadex. We have tried to desalt using buffer exchange (Centri-Prep), however, the enzyme loses its activity after desalting by this approach, even if NaCl concentrations are raised back to physiological levels! Are there any other techniques available that may be used to desalt the conditioned medium? From owner-proteins@net.bio.net Mon Sep 11 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!EU.net!uknet!yama.mcc.ac.uk!usenet From: abashein@fs1.ho.man.ac.uk (Abdulla Bashein) Newsgroups: bionet.molbio.proteins Subject: test do not read Date: 12 Sep 1995 10:29:06 GMT Organization: Clinical Sciences - Univeristy Of Manchester, England Lines: 2 Message-ID: <433ndi$e1t@yama.mcc.ac.uk> NNTP-Posting-Host: pc96.hop.man.ac.uk Mime-Version: 1.0 X-Newsreader: WinVN 0.93.11 this is just a test. From owner-proteins@net.bio.net Mon Sep 11 23:00:00 1995 Path: biosci!daresbury!sunsite.doc.ic.ac.uk!dispatch.news.demon.net!demon!btnet!tank.news.pipex.net!pipex!howland.reston.ans.net!vixen.cso.uiuc.edu!ux1.cso.uiuc.edu!elarson From: elarson@ux1.cso.uiuc.edu (larson eric) Newsgroups: bionet.molbio.proteins Subject: Re: Desalting Date: 12 Sep 1995 17:50:06 GMT Organization: University of Illinois at Urbana Lines: 87 Message-ID: <434h8e$s56@vixen.cso.uiuc.edu> References: NNTP-Posting-Host: ux1.cso.uiuc.edu sg@nwu.edu (Stephen Gately) writes: >We wish to purify an enzyme using ion exchange from serum-free conditioned >medium. The NaCl concentration is approximately 100 mM. We would like to >desalt this medium prior to binding to DEAE-sephadex. We have tried to >desalt using buffer exchange (Centri-Prep), however, the enzyme loses its >activity after desalting by this approach, even if NaCl concentrations are >raised back to physiological levels! ========== You can desalt by dialysis, desalting gel filtration, protein ppt., or remove the ions by Sterogene Ionclear Bigbead resin. Dialysis can be combined with protein concentration by putting polyethylene glycol 10,000 (sigma) in the dialysis buffer (not in the bag) and by using a dialysis bag that will exclude the PEG 10K (standard 10,000 to 12,000 cut-off works fine). Gel Filtration requires a properly-sized column (generally 5 to 10X the volume being desalted). Sephadex G25-300 (approx 2,500 mol. wt. exclusion with 300 micrometer beads) is generally used (but there are many others available). Occasionally, precipitation of the protein can be used to advantage. I routinely ppt. proteins using PEG 10K (with 10 mM Mg++), to "reprecipitate" proteins that were already ppt. by ammonium sulfate. Ammonium sulfate ppts. tend to have a large fraction of membranes for the initial cut (35% for our plant protein). However, the resuspended AS pellets often contain enough ammonium sulfate to cause binding problems on ion exchange resins. We've found that many (not all), of our proteins will reprecipitate out of the now dilute AS solution using 14 to 17% PEG 10,000 (same as used above for dialysis/concentration). The advantage is the supernatent of the PEG ppt. contains the dilute AS. Another distinct advantage is once the PEG pellet has been resolubilized, it is found that the membranes are now *not* soluble and can be spun out (I believe dilute AS helps keep the membrane fragments in solution). Essentially, doing a PEG ppt. on the heels of an AS ppt. removes both the AS and contaminating membranes (but maybe in your case the membranes are the wanted fragment?, fine, they could spin out now with far fewer contaminating soluble proteins). The Ionclear Bigbeads is a new technique I'm itching to try. Product literature claims that ions can be clear from solution without significant protein loss. I tend to believe product literature claims after I've tried the technqiue (1-800-535-2284). >Are there any other techniques available that may be used to desalt the >conditioned medium? It is often the case that simply diluting the protein sample containing monovalents allows for protein to bind to the column in question. DEAE is a relatively weak binder, with some proteins eluting as low as 30 mM NaCl. This would probably be a lower limit for diluting. By altering the binding chemistry to a slightly tigher binding group, the need for diluting can be reduced. I suggest trying QAE Sepharose (fast flow is usually best when the sample is possibly contaminated or if this is the first column in the series). Sizing of the column can be a bit tricky, but for first trys, I usually load about 3 to 5 mg of protein per mL of resin. If the sample contains significant divalents (sulfate from AS the principal problem), then some significant increase in col. size is warranted. Divalent anions tend to stick to DEAE and QAE resins very strongly, to the point they almost need to be considered as "permanent" binders for the loading phase. This can mean uneasily large columns for relatively small amounts of protein. I have had occasion to use a bulk-column method for catching a rare protein from dilute solutions. Usually, I'll dilute the sample to monovalents to around 50 mM (i.e. 50 mM NaCl), then add washed QAE resin and mix. The weak slurry is spun in a prepartive centrifuge (equal volumns in opposing tubes as when the resin is spun, the balance of the tube is markedly altered), the supernatent is aspirated, and the resin is resuspended with minimal buffer and poured into a column that contains 25% of QAE already settled (with excess buffer already aspirated). This "column" is then subjected to a rapid gradient (usually NaCl increases). The goal with any protein that shows lability is to move quickly to get it onto a column and away from possible degradative agents (i.e. proteases). If it were me? I'd dilute the sample to 50 mM NaCl, load it onto an oversized QAE column, and do a quick gradient with deliberate speed at all stages (especially the analysis phase, don't let the samples in the fraction collector "just sit" as you do assays). Good luck. -- Eric Larson | University of Illinois at Urbana-Champaign USDA/Agronomy | 190 ERML; 1201 W. Gregory; Urbana, IL 61801 elarson@ux1.cso.uiuc.edu | Voice 217.244.3079 Fax 217.244.4419 Fidonet: 1:233/4.1 | My opinions are my own, but correct :-) From owner-proteins@net.bio.net Mon Sep 11 23:00:00 1995 Path: biosci!agate!news.ucdavis.edu!library.ucla.edu!newsfeed.internetmci.com!tank.news.pipex.net!pipex!oleane!jussieu.fr!univ-lyon1.fr!swidir.switch.ch!scsing.switch.ch!elna.ethz.ch!usenet From: michael kertesz Newsgroups: bionet.molbio.proteins Subject: Re: Desalting Date: 12 Sep 1995 15:46:51 GMT Organization: Microbiology ETHZ Lines: 18 Message-ID: <434a1b$1ki@elna.ethz.ch> References: NNTP-Posting-Host: b22-pro486-1.ethz.ch Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.2b5 (Windows; I; 16bit) We had a similar problem with Centripreps, losing >50% of our enzyme activity every time we concentrated (a bit of a nuisance during a multistep purification!). The problem was solved with a trick we learnt from the producers - when we pretreated the Centripreps overnight with a 5% Tween solution, then washed them thoroughly before use, we retained all our enzyme activity (a sulfatase, in this case). We decided that our activity loss was probably due to binding of our quite hydrophobic enzyme to the plastic of the Centriprep unit. How hydrophobic is your enzyme? Might be worth a try, in any case. good luck, Michael Kertesz ETH-Microbiology e-mail kertesz@micro.biol.ethz.ch tel: +41-1-632 3357 From owner-proteins@net.bio.net Tue Sep 12 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!bcm.tmc.edu!cs.utexas.edu!swrinde!tank.news.pipex.net!pipex!uknet!newsfeed.ed.ac.uk!festival!festival.ed.ac.uk!pnb01 From: pnb01@festival.ed.ac.uk (Paul Barlow) Subject: Senior Physical/Biological Chemist Message-ID: Sender: news@festival.ed.ac.uk (remote news read deamon) Organization: Edinburgh University Date: Wed, 13 Sep 1995 12:44:58 GMT Keywords: Post-doc, PrP, Prions Lines: 63 Job description Institution : BBSRC Institute for Animal Health, Compton Laboratory, Compton (Near Newbury), Berkshire RG20 7NN Principal Investigator : Dr J. Hope (E-Mail : james.hope@bbsrc.ac.uk) Title of project : Physical structure, folding and stability of PrP protein forms Start date : 01 December 1995 End date : 30 November 1998 Grade of post : Band 6 The conversion of the normal form of the prion protein (PrPC) to its protease-resistant isoform (PrPSc) is a key process in the development of scrapie and related prion diseases. PrPSc is enriched in preparations of infectious particles and, either by itself or in association with other molecules, is regarded by many as the infectious agent. Spectral changes monitored (by circular dichroism or fluorescence) during the thermal or chemical denaturation of PrP27-30 (an active fragment of PrPSc) indicate a complex "melt" of this fibrillar form of the protein, possibly involving a transition state with similar properties to a "molten globule". Circular dichroism (CD), Fourier-transform infra-red spectroscopy (FT-IR) and fluoresecence measurements on PrPC and PrPSc give estimates of a high alpha-helix secondary structure in PrPC, less in PrPSc, and least in PrP27-30 . This spectrocopic data and computer simulations of the secondary and tertiary structure of PrP have led to a four-helix bundle model of PrPC and a mechanism involving the switch of one or more of these helices to beta-sheet structure when this normal isoform changes to its protease-resistant forms (PrPSc and PrP27-30). Knowledge of PrP structure and an understanding of the mechanism of PrPSc formation and prion replication are needed for rationale drug design, therapy, prevention of intra-and inter-species transmission and the development of more effective methods of disinfection of the agents of BSE, scrapie and Creutzfeldt-Jakob disease. To compliment studies on the natural forms of PrPC and PrPSc and our transgenic programme of research on the biological aspects of PrP in the spongiform encephalopathies, we are investigating the structure, stability and folding of the wild-type and mutant, recPrP proteins. To support this programe of work, we are seeking to appoint a senior Biological/Physical Chemist (Grade 6) at IAH Compton to direct the characterisation of recPrP by a variety of biophysical techniques including mass spectrometry, Fourier Transform-infra red spectroscopy, circular dichroism, fluorescence, surface plasmon resonance, laser light scattering, ultra-centrifugation and ultrafiltration. The preferred candidate will have extensive experience in at least two of these areas of protein analysis. He/she will also take responsibility for the setting up the different permutations of solutions, protein concentrations, etc need to crystallise the PrP protein and the preliminary screening by light and electron microscopy of these liquors for crystals. If you require more details about this post, please call 0131-667-5204), fax (0131-668-3892) or mail (above) Jim Hope, or contact the Personnel Officer, Institute for Animal Health, Compton, Berkshire RG20 7NN (phone, 01635-578411; fax, 01635-577131; e-mail ; hughesj@bbsrc.ac.uk). The Institute for Animal Health is an Equal Opportunities Employer. From owner-proteins@net.bio.net Tue Sep 12 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!swrinde!tank.news.pipex.net!pipex!oleane!jussieu.fr!univ-lyon1.fr!cri.ens-lyon.fr!news From: Patrice Boissy Newsgroups: bionet.molbio.proteins Subject: help ! Looking for antibodies against chicken Integrins Date: 13 Sep 1995 13:16:59 GMT Organization: Ecole Normale Superieure de Lyon, France Lines: 12 Message-ID: <436lkb$6vr@cri.ens-lyon.fr> NNTP-Posting-Host: mac-lr5-143b.ens-lyon.fr Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) X-URL: news:bionet.molbio.proteins Hello! I¹m looking for a source of monoclonal or polyclonal antibodies against chicken integrins for immunohistology, immunoprecipitation and functional effects studies, Any information will be greatly appreciated! Many thanks!! From owner-proteins@net.bio.net Tue Sep 12 23:00:00 1995 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!news.sprintlink.net!in1.uu.net!mail.llu.edu!usenet From: bLangridge@ccmail.llu.edu (Bill Langridge) Newsgroups: bionet.molbio.proteins Subject: GLYCOPROTEINS Date: 13 Sep 1995 20:15:24 GMT Organization: Loma Linda University Lines: 8 Message-ID: <437e4s$eg0@mail.llu.edu> NNTP-Posting-Host: 151.112.27.53 Mime-Version: 1.0 Content-Type: Text/Plain; charset=ISO-8859-1 X-Newsreader: WinVN 0.99.5 Can anyone tell me if Plant, Pichia, and Baculovirus protein synthesizing systems will properly glycosylate a viral (HIV coat protein)? Thanks for your help! Bill Langridge From owner-proteins@net.bio.net Tue Sep 12 23:00:00 1995 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!howland.reston.ans.net!Germany.EU.net!news.dfn.de!news.rwth-aachen.de!newsserver.rrzn.uni-hannover.de!tubsibr!neumann From: neumann@ibr.cs.tu-bs.de (Tilman Neumann) Newsgroups: bionet.molbio.proteins Subject: calculation of sequence characteristics from 3D structure Date: 13 Sep 1995 17:57:46 GMT Organization: TU Braunschweig, Informatik (Bueltenweg), Germany Lines: 23 Distribution: world Message-ID: <43762q$ij4@ra.ibr.cs.tu-bs.de> NNTP-Posting-Host: eos.ibr.cs.tu-bs.de Hello, I'm developing an exemplar based protein secondary structure prediction system. I think that the classification accuracy will be the better the more information I'll put in. Therefore I'm looking for any kind of programs that calculate characteristics for protein sequences, as well global characteristics like molecular weight as amino acid residue wise characteristics like alpha-, beta-propensities or accesible surface... One example for such a program is DSSP, that defines secondary structures and accessible surface area for single residues. If you know something about similar programs that are available, it would be very kind of you to send me a note about it. Thank you very much, Tilman Neumann neumann@ibr.cs.tu-bs.de From owner-proteins@net.bio.net Tue Sep 12 23:00:00 1995 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!howland.reston.ans.net!news.sprintlink.net!EU.net!sun4nl!news.iaf.nl!biochem!gertjan From: gertjan@biochem.iaf.nl (Gert-Jan Euverink) Newsgroups: bionet.molbio.proteins Subject: Re: Desalting Distribution: world Message-ID: <810988857.36snx@biochem.iaf.nl> References: Date: Wed, 13 Sep 95 10:40:57 GMT Reply-To: gertjan@biochem.iaf.nl Lines: 25 In article sg@nwu.edu writes: > >We wish to purify an enzyme using ion exchange from serum-free conditioned >medium. The NaCl concentration is approximately 100 mM. We would like to >desalt this medium prior to binding to DEAE-sephadex. We have tried to >desalt using buffer exchange (Centri-Prep), however, the enzyme loses its >activity after desalting by this approach, even if NaCl concentrations are >raised back to physiological levels! > >Are there any other techniques available that may be used to desalt the >conditioned medium? > You can try dialysis. If the volume is to large you might consider to dilute your sample untill the salt concentration is low enough to allow protein binding to DEAE sephadex. Maybe you can do this in combination of raising the pH. -- Gert-Jan Euverink gertjan@biochem.iaf.nl Groningen The Netherlands. From owner-proteins@net.bio.net Tue Sep 12 23:00:00 1995 Path: biosci!agate!boulder!tali.UCHSC.edu!Guy.Oshiro From: oshiro_G@defiance.hsc.colorado.edu (Guy Oshiro) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.molluscs,bionet.molbio.proteins,bionet.molbio.rapd,bionet.molbio.yeast Subject: Re: Need Help!! Date: 13 Sep 1995 19:25:57 GMT Organization: University of Colorado Health Sciences Center Lines: 31 Sender: -Not-Authenticated-[6503] Message-ID: <437b85$s49@tali.UCHSC.edu> References: <42vgtp$9h8$1@mhafm.production.compuserve.com> NNTP-Posting-Host: brbmt8.uchsc.edu X-Posted-From: InterNews 7.2@tali.uchsc.edu. Xdisclaimer: No attempt was made to authenticate the sender's name. Xref: biosci bionet.molbio.methds-reagnts:33508 bionet.molbio.molluscs:146 bionet.molbio.proteins:5623 bionet.molbio.rapd:1295 bionet.molbio.yeast:3738 Patrick, My a 10 year computer programmer ventran and you want to pursue a career in biological sciences. Hmmm...are you sure this is what you want to do with your life? I hope that you realize that science is not a money-making carreer. I hope that you are prepared to make less than a fast-food worker - for the next 7-10 years that it takes you to finish your PhD and then a post-doc. Then of course the next step would be to find a position. But enough lamenting about the current state of affairs and things may change in the next few years. You can definitely set yourself apart with your programming skill if you want to continue with it in your biology-computer science career. The first thought that comes to mind is computer modeling of protein structures. The dream of a lot of biologists would be able to enter the primary DNA sequence and have a computer algorithm convert it into its functional tertiary structure. There are many other possibilities but you should look into groups near your area that are doing computer modeling to get some hands on experience. Good Luck. Guy Oshiro EMAIL: Oshiro_G@defiance.hsc.colorado.edu /\ /\ /\ / \/ \/\/ \ Science in the Rockies. / \ \/ \ From owner-proteins@net.bio.net Tue Sep 12 23:00:00 1995 Path: biosci!agate!howland.reston.ans.net!vixen.cso.uiuc.edu!news.ecn.bgu.edu!psuvax1!news.eecs.nwu.edu!newsfeed.acns.nwu.edu!news.acns.nwu.edu!med048050.medicine.nwu.edu!user From: sg@nwu.edu (Stephen Gately) Newsgroups: bionet.molbio.proteins Subject: Affinity Purification Date: Wed, 13 Sep 1995 16:08:22 -0500 Organization: Northwestern University Lines: 15 Message-ID: NNTP-Posting-Host: med048050.medicine.nwu.edu We are trying to purify an enzyme from serum-free conditioned medium. We have tested a variety of exogenous inhibitors to block the enzyme activity, and have found a potent inhibitor that is also available bound to cyanogen bromide activiated agarose. Despite blocking 100% of activity when the inhibitor is added exogenously, passing the conditioned medium through a column containing the inhibitor agarose, or batch adsorption does not block the enzyme activity. We would appreciate any useful guidelines for affinity purification, ie. salt, pH etc., or specific comments on why we observe the difference between exogenous inhibitor and bound inhibitor ability to block activity. Thank you. From owner-proteins@net.bio.net Tue Sep 12 23:00:00 1995 Path: biosci!CSHL.ORG!boyce From: boyce@CSHL.ORG (Joan Boyce) Newsgroups: bionet.molbio.proteins Subject: New WWW Resource - an on-line buyer's guide from Cold Spring Harbor Lab Press Date: 13 Sep 1995 13:48:58 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 28 Sender: daemon@net.bio.net Distribution: world Message-ID: <9509132010.AA22041@phage.cshl.org> NNTP-Posting-Host: net.bio.net Cold Spring Harbor Lab Press introduces BIOSUPPLYNET-An online directory of biotech research products and services. This new WWW site offers free searching in the CSH Lab Manual Source Book database of 15,000 laboratory products (an entire section is devoted to protein chemostry reagents) and 1,400 suppliers. BIOSUPPLYNET features "Interactive Product Listings" (IPLs), which provide users with instant access to details on many of the products contained within its extensive database. BIOSUPPLYNET is updated weekly and provides: 1.) Information on new products and special offers 2.) Access to 15,000 laboratory product listings from over 1,400 suppliers 3.) An opportunity for users to share expertise through product user groups 4.) Immediate access to suppliers via email for technical queries and ordering information 5.) Links to more than 100 biology-relevant Web servers BSN is accessible at: http://www.biosupplynet.com/bsn/ A FREE copy of the CSH Lab Manual Source Book can be obtained through Cold Spring Harbor Web Site, CLIO, http://www.cshl.org/ Hope you all find it useful! Joan Boyce boyce@cshl.org From owner-proteins@net.bio.net Tue Sep 12 23:00:00 1995 Path: biosci!bcm.tmc.edu!pendragon.jsc.nasa.gov!news.msfc.nasa.gov!newsfeed.internetmci.com!news.sprintlink.net!in1.uu.net!zib-berlin.de!news.rrz.uni-hamburg.de!rzaix05!pz4a004 From: pz4a004@rzaix05.uni-hamburg.de (Aquiles Luna-Rodriguez) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.molluscs,bionet.molbio.proteins,bionet.molbio.rapd,bionet.molbio.yeast Subject: Re: Need Help!! Followup-To: bionet.molbio.methds-reagnts,bionet.molbio.molluscs,bionet.molbio.proteins,bionet.molbio.rapd,bionet.molbio.yeast Date: 13 Sep 1995 13:43:30 GMT Organization: University of Hamburg -- Germany Lines: 29 Message-ID: <436n62$8dk@rzsun02.rrz.uni-hamburg.de> References: <42vgtp$9h8$1@mhafm.production.compuserve.com> NNTP-Posting-Host: rzaix05.rrz.uni-hamburg.de X-Newsreader: TIN [version 1.2 PL2] Xref: biosci bionet.molbio.methds-reagnts:33462 bionet.molbio.molluscs:145 bionet.molbio.proteins:5616 bionet.molbio.rapd:1293 bionet.molbio.yeast:3732 In article <42vgtp$9h8$1@mhafm.production.compuserve.com> you wrote: : I am a undergraduate student in biology. Some day I hope to go to : graduate school in biochemistry. : I am a professional computer programmer. Hi Patrick! I may have just what your4re looking for. I4m a psycho- logy student, and I write neural simulation programs since many years. Now, I4m developing a tool-box to simulate neural behavior, a kind of formal lenguage to describe brain theories. It can be used to simulate small subsystems, maybe even the "learning" part of animal models like Aplysia, Hermissenda or C. elegans. The idea is using a "chemical" learning algorithm, and test the model against empirical data, either neural or behavioral. I need contact with people interested in molecular biology, neurophisio- logy/anatomy, computer programming, behavior science, etc. Don4t worry if you4re less than a specialist in all these fields, modesty is a disadvantage if you4re shooting at a Nobel Prize. If you4re interested, I can send you C source for Turbo C 3.0, just email me. *********************************** * Aquiles Luna-Rodriguez * * Universitaet Hamburg, Germany * * pz4a004@rrz.uni-hamburg.de * *********************************** From owner-proteins@net.bio.net Tue Sep 12 23:00:00 1995 Path: biosci!bcm.tmc.edu!pendragon.jsc.nasa.gov!news.msfc.nasa.gov!newsfeed.internetmci.com!newsxfer.itd.umich.edu!nntp.cs.ubc.ca!cs.ubc.ca!unixg.ubc.ca!news.bc.net!rover.ucs.ualberta.ca!tribune.usask.ca!news.sasknet.sk.ca!canopus.cc.umanitoba.ca!zahradka2.sbrc.umanitoba.ca!davidw From: davidw@sbrc.umanitoba.ca (David Wilson) Newsgroups: bionet.molbio.proteins Subject: cox-2 antibody Date: Wed, 13 Sep 1995 08:48:24 Organization: St. Boniface Hospital Research Centre Lines: 12 Message-ID: NNTP-Posting-Host: zahradka2.sbrc.umanitoba.ca Keywords: antibody, cox-2, porcine X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] Hello, I am looking for a cox-2 antibody that crossreacts with porcine tissue or cells. I am aware of a number of suppliers of antibody to Cox-2, but none of them have any information regarding cross reactivity with porcine tissue. If anyone has any experience specifially with Cox-2 antibody and porcine tissue I would be greatful if you could provide me with the source. Thank you. Peter Zahradka St. Boniface General Hospital Research Ctr. Winnipeg, Manitoba, Canada From owner-proteins@net.bio.net Tue Sep 12 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!tank.news.pipex.net!pipex!in2.uu.net!nih-csl!NewsWatcher!user From: newitt@nih.gov (John A. Newitt) Subject: Re: Desalting Message-ID: Sender: postman@alw.nih.gov (AMDS Postmaster) Nntp-Posting-Host: 128.231.113.35 Organization: National Institutes of Health X-Newsreader: Yet Another NewsWatcher 2.0.1 References: Date: Wed, 13 Sep 1995 17:50:26 GMT Lines: 20 In article , sg@nwu.edu (Stephen Gately) wrote: > Are there any other techniques available that may be used to desalt the > conditioned medium? I think straightforward dilution of a sample into low ionic strength buffer is the easiest and best way to prepare a protein solution prior to binding to an ion-exchange column. The column will re-concentrate the protein for you. Of course, if you know that dilution leads to loss of activity, this method isn't for you. Dilution should be practical in your case because your salt is only 100 mM. Depending upon the affinity of your protein for the DEAE-Sephadex (Sepharose ?), you may be able to get away with only a 2-fold dilution. Regards, John A. Newitt, Ph.D. | National Institutes of Health | FAX: 301-402-0387 Bethesda, Maryland USA | From owner-proteins@net.bio.net Tue Sep 12 23:00:00 1995 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!news.sprintlink.net!in2.uu.net!noc.near.net!news3.near.net!yale!yale.edu!news.ycc.yale.edu!news From: Danny Chamovitz Newsgroups: bionet.molbio.proteins Subject: Re: Messy Western blots Date: 13 Sep 1995 13:59:59 GMT Organization: Yale University Lines: 7 Message-ID: <436o4v$j2g@news.ycc.yale.edu> References: <43069h$19j$1@mhade.production.compuserve.com> NNTP-Posting-Host: mustelid.biology.yale.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (X11; I; HP-UX A.09.03 9000/735) X-URL: news:43069h$19j$1@mhade.production.compuserve.com Is your antibody affinity purified? This may get rid of most of your nonspecific bands. Also, we have much better luck in reducing background using PVDF rather than nitrocellulose. Danny Chamovitz chamo@peaplant.biology.yale.edu From owner-proteins@net.bio.net Wed Sep 13 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!news.sprintlink.net!EU.net!Germany.EU.net!news.dfn.de!gs.dfn.de!fauern!lrz-muenchen.de!news From: Wolfgang Schechinger Newsgroups: bionet.molbio.proteins Subject: Re: Help for a rookie? (peptide sequences) Date: 14 Sep 1995 09:58:14 GMT Organization: Institute for Diabetes Research Lines: 43 Distribution: world Message-ID: <438ubm$40b@sparcserver.lrz-muenchen.de> References: <437uaf$o3j@ixnews2.ix.netcom.com> NNTP-Posting-Host: u7k0201.ppp.lrz-muenchen.de Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Windows; I; 16bit) To: MHAYTHOR@ix.netcom.com Try HYPERCHEM. Ireqiures Windows 3.1. The best I've seen in this field. Don't know an URL for getting the demo, but maybe someone else knows? (Try bionet.software or bionet.software.www) Wolfgang (not connected with hyperchem) MHAYTHOR@ix.netcom.com (Mark Haythorn) wrote: >Anyone know of a drawing program that will allow to to draw a peptide >by clicking in an amino-acid name and having the structure appear on >the screen? > > >Forgive my lack of expertise, but any info would be much appreciated!! > > > >Mark Haythorn >MHAYTHOR@IX.NETCOM.COM > > -- +++++++++++++++++++++++++++++++++++++++++++ Wolfgang Schechinger Institute for Diabetes Reserch Koelner Platz 1 80804 Munich Germany Phone +49 (89) 30 79 31 24 Fax +49 (89) 30 81 733 email u7k0201@sunmail.lrz-muenchen.de (Standard disclaimer applies) +++++++++++++++++++++++++++++++++++++++++++ From owner-proteins@net.bio.net Wed Sep 13 23:00:00 1995 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!EU.net!Germany.EU.net!news.dfn.de!gs.dfn.de!fauern!lrz-muenchen.de!news From: Wolfgang Schechinger Newsgroups: bionet.molbio.proteins Subject: Re: Affinity Purification Date: 14 Sep 1995 09:53:40 GMT Organization: Institute for Diabetes Research Lines: 66 Distribution: world Message-ID: <438u34$40b@sparcserver.lrz-muenchen.de> References: NNTP-Posting-Host: u7k0201.ppp.lrz-muenchen.de Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Windows; I; 16bit) To: sg@nwu.edu,diabetes@sunmail.lrz-muenchen.de Dear Stephen. Suppose that 1) Your inhibitor is maybe attached with the "wrong" sinde, i.e. the binding site for your enzyme to the agarose 2) or your enzyme runs through the coulumn despite tha fact that the column is appropriate (because there's too much salt in the solution or the pH is "wrong"). Try to 1) desalt your your conditioned medium, eg. by dialysis or by using a desalting column, then try if you are able to precipitate your enzyme with the modified agarose. If not, try to change pH, buffer, ... (The Pharmacia company has a nice small booklet they give upon request on this topic) 2) if nothing of 1) will work, try another acivated agarose that will bind other functional groups of your inhibitor. Maybe NHSactivated sepharose (small prepacked cheap 1 ml-columns!) from Pharmacia or similar products from BioRad will do) 2a)if everything failes, try other media like heparin-sepharose. I made quite good experience with this (heparin is used as an affinity reagent for growth factors, but IMHO, it might act as a cation exchanger). Try ANYTHING you can imagine: Phosphotyrosine or phosphoserine might be interesting as well. 3) If you finally succeed in getting stuck your protein to your columns, try to elute from low salt (e.g. 10mM tris buffer) to high salt buffer (0.5 or 1M NaCl e.g.) Hope this helps! Wolfgang (not connected with any company mentioned) sg@nwu.edu (Stephen Gately) wrote: >We are trying to purify an enzyme from serum-free conditioned medium. We >have tested a variety of exogenous inhibitors to block the enzyme >activity, and have found a potent inhibitor that is also available bound >to cyanogen bromide activiated agarose. > >Despite blocking 100% of activity when the inhibitor is added exogenously, >passing the conditioned medium through a column containing the inhibitor >agarose, or batch adsorption does not block the enzyme activity. > >We would appreciate any useful guidelines for affinity purification, ie. >salt, pH etc., or specific comments on why we observe the difference >between exogenous inhibitor and bound inhibitor ability to block >activity. > >Thank you. -- +++++++++++++++++++++++++++++++++++++++++++ Wolfgang Schechinger Institute for Diabetes Reserch Koelner Platz 1 80804 Munich Germany Phone +49 (89) 30 79 31 24 Fax +49 (89) 30 81 733 email u7k0201@sunmail.lrz-muenchen.de (Standard disclaimer applies) +++++++++++++++++++++++++++++++++++++++++++ From owner-proteins@net.bio.net Wed Sep 13 23:00:00 1995 Path: biosci!newshost.lanl.gov!ncar!newsxfer.itd.umich.edu!newsfeed.internetmci.com!news.compuserve.com!news.production.compuserve.com!news From: Alan Gamble <74443.2760@CompuServe.COM> Newsgroups: bionet.jobs.wanted,bionet.molbio.gdb,bionet.molbio.proteins,comp.databases.ms-access,comp.sys.mac.databases Subject: **Position Available- 4D Programmer Date: 14 Sep 1995 12:29:51 GMT Organizatio