From owner-proteins@net.bio.net Thu Dec 01 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!gatech!howland.reston.ans.net!vixen.cso.uiuc.edu!uxa.cso.uiuc.edu!zemser From: zemser@uxa.cso.uiuc.edu (Rachel Zemser ) Newsgroups: bionet.molbio.proteins Subject: Anyone interested in a Food Science newsgroup Date: 3 Dec 1994 03:10:49 GMT Organization: University of Illinois at Urbana Lines: 10 Message-ID: <3bonjp$dje@vixen.cso.uiuc.edu> NNTP-Posting-Host: uxa.cso.uiuc.edu I was wondering if anyone is interested in a sci.foodscience newsgroup. Topics will include proteins and how they ar related to the food industry, as well as protein research being conducted at various food science departments around the world. Please let me know if you would be willing to vote on such a new group... I am currently writing the RFD and will post that in a few days. Rachel Zmeser zemser@uxa.cso.uiuc.edu From owner-proteins@net.bio.net Thu Dec 01 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!bloom-beacon.mit.edu!gatech!howland.reston.ans.net!vixen.cso.uiuc.edu!newsrelay.iastate.edu!gw1.phibred.com!dalmiabk.phibred.com!dalmiabk From: bipin dalmia Subject: Re: E.Coli Westerns Message-ID: X-Xxmessage-Id: X-Xxdate: Fri, 2 Dec 94 13:56:57 GMT Sender: news@phibred.com (USENET News System) Organization: pioneer hi-bred X-Useragent: Version 1.1.3 References: Date: Fri, 2 Dec 1994 20:03:03 GMT Lines: 11 In article Anne Spang, spang@vms.biochem.mpg.de writes: >via nitrocellulose strips on which you antigen is immobilized. The >advantage of this methode is, that you do not need pure antigen, because >you just cut out the band of your protein of interest. If you are >interested in this protocol,I can send it to you can you send me this protocol? (i had to post because of some mailer problems. it allows me to post but not reply directly, go figure) bip From owner-proteins@net.bio.net Thu Dec 01 22:00:00 1994 Path: biosci!SNFMA1.IF.USP.BR!szeinfel From: szeinfel@SNFMA1.IF.USP.BR (Rafael Iosef Najmanovich Szeinfeld {S) Newsgroups: bionet.molbio.proteins Subject: Re: protein-protein binding Date: 2 Dec 1994 12:19:34 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 43 Sender: daemon@net.bio.net Distribution: world Message-ID: References: <1994Dec2.092532.34320@cc.usu.edu> NNTP-Posting-Host: net.bio.net On 2 Dec 1994 sl16s@cc.usu.edu wrote: > I am trying to look for Protein binding sites in the protein I am working with. > I really appreciate iof any body can enlighten me on this matter. > 1. For a protein to bind naother protein should there bespecific DNA sequence > or a broad consensu sequence? There's no relation between DNA sequence and binding affinity because what enables an amino acid (a.a) to participate in binding is a property such as hydrophobicity which is present in a broad class of a.a's. > 2. Can I find the proein binding sites by the DNA sequence, translated by > computer? It's quite impossible on my point of view. Let me tell you why. Suppose the only interaction involved in binding were of hydrophobic nature (we're neglecting Hydrogen bonds and so on) then we can suppose that the binding site is composed mainly by apolar a.a's. Even in this case we wouldn't know what are the apolar residues in the sequence that are close together in the native structure, so we cannot predict the binding site, we would better predict the protein's interior. Now add to this the fact that you also don't know what exactly are the a.a's that can participate in the binding process. > I am working with a chaperone protein. I really apreciate if anybody can give > me any pointers on this matter. Supposing your chaperone protein is also a heat shock protein then would be interesting to look for portion of your protein that are heat resistant. Good luck, Rafael. *---------------------------------------------------------------------* * Rafael Iosef Najmanovich Szeinfeld | Depto. de Fisica Geral * * General Physics Department | Instituto de Fisica * * Physics Institute | Universidade Sao Paulo * * University of Sao Paulo - Brazil | Rua do Matao s/n CEP 01452-990 * *____________________________________| Caixa Postal 20516 * * E-mail: szeinfel@snfma1.if.usp.br | Sao Paulo - Brasil * *---------------------------------------------------------------------* From owner-proteins@net.bio.net Thu Dec 01 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!uhog.mit.edu!europa.eng.gtefsd.com!news.umbc.edu!haven.umd.edu!purdue!lerc.nasa.gov!magnus.acs.ohio-state.edu!bhomoell From: bhomoell@magnus.acs.ohio-state.edu (Bradley J Homoelle) Newsgroups: bionet.molbio.proteins Subject: Method to quantify number of cysteine residues Date: 2 Dec 1994 16:43:42 GMT Organization: The Ohio State University Lines: 8 Message-ID: <3bniru$8oe@charm.magnus.acs.ohio-state.edu> NNTP-Posting-Host: beauty.magnus.acs.ohio-state.edu Hi. I was wondering if anyone out there knew of a good method to determine the number of cysteine residues in a peptide. If so, could you kindly send me an e-mail or post to this newsgroup. Thank you in advance! -- Bradley J. Homoelle The Ohio State University From owner-proteins@net.bio.net Thu Dec 01 22:00:00 1994 Path: biosci!agate!howland.reston.ans.net!pipex!warwick!unicorn.nott.ac.uk!macfd.biochem.nottingham.ac.uk!user From: mbxfd@unicorn.nott.ac.uk (Fergus Doherty) Newsgroups: bionet.molbio.proteins Subject: Detecting hemoglobin on blots? Followup-To: bionet.molbio.proteins Date: 2 Dec 1994 14:10:07 GMT Organization: Nottingham University, UK. Lines: 18 Distribution: world Message-ID: NNTP-Posting-Host: macfd.biochem.nottingham.ac.uk Hello, First a probably silly question: does hb loose its heme when denatured in SDS PAGE sample buffer? I assume it does. Second: Any suggestions as to how I might detect rabbit hb on a Western? Of course an antibody would do it (have you got one I can have?). Or, if it retains the heme is there some kind of chemical detection method? I see a band on a gel which could easily be hb (polypeptides that bind and sediment when retic lysate incubated with mitos, especially in the presence of iron), similar size, but I'm not 100% sure....... Fergus Doherty, dept. Biochemistry, University Medical School, Queen's Medical Centre, Nottingham NG7 2UH Tel: 602 709366 FAX 602 422225 Internet: mbxfd@unicorn.nott.ac.uk From owner-proteins@net.bio.net Thu Dec 01 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!gatech!newsxfer.itd.umich.edu!zip.eecs.umich.edu!umn.edu!newsdist.tc.umn.edu!mayonews.mayo.edu!Mayo.EDU!eberhard From: eberhard@Mayo.EDU (norm eberhardt) Newsgroups: bionet.molbio.proteins Subject: Re: Minor Groove Binding TFs Date: 1 Dec 1994 23:42:15 GMT Organization: Mayo Foundation, Rochester, Minnesota Lines: 29 Distribution: world Message-ID: <3bln0n$3tr@fermat.mayo.edu> References: NNTP-Posting-Host: feynman.mayo.edu In article , Sinasac David Steven writes: |> Here is a little trivia question that I hope some one can answer. Can |> anyone give me the name of a specific cell specific transcription factor |> that binds to the minor groove of the DNA double helix. This has to be |> found in higher eukaryotes and it has to be true binding (though not |> necessarily sequence specific). The only thing that I could come up with |> is the IHF motif found in E. coli. Any help would be appreciated. |> |> |> |> Dave Sinasac |> sinasa2@server.uwindsor.ca |> University of Windsor 1. TBP (TATA Binding Protein)--Nikolov et al., Nature 360:40, 1992. 2. HSF (heat shock)--Fernandes, et al., Nucleic Acids Research 22: 167, 1994. 3. UBF--Copenhaver et al., Nucleic Acids Research 22(: 2651, 1994. 4. SRY--King et al., Sox-4Proc Natl Acad Sci USA 90(: 11990, 1993. 5. Sox-4--van de Wetering et al., EMBO J 12: 3847, 1993. 6. HNF-3--Clark et al., Nature 364: 412, 1993. Also interesing is GATA-1 which utilizes both major and minor groove binding. 7. GATA-1--Omichinski et al., Science 261: 438, 1993. This is not an exhaustive list, but should serve as a start. norman eberhardt eberhardt@mayo.edu From owner-proteins@net.bio.net Thu Dec 01 22:00:00 1994 Newsgroups: bionet.cellbiol,bionet.general,bionet.molbio.proteins,sci.bio,sci.bio.technology,comp.ai.neural-nets Path: biosci!newshost.lanl.gov!ncar!gatech!swrinde!pipex!uunet!zib-berlin.de!news.belwue.de!news.informatik.uni-stuttgart.de!msoberdo From: msoberdo@tick.informatik.uni-stuttgart.de (Matthias Oberdorfer) Subject: Q: protein prediction with neural networks Message-ID: Keywords: neural networks, water, denaturants, protein Sender: msoberdo@tick (Matthias Oberdorfer) Organization: University of Stuttgart, Germany Date: Thu, 1 Dec 1994 16:10:48 GMT Lines: 20 Xref: biosci bionet.cellbiol:1220 bionet.general:12229 bionet.molbio.proteins:3180 sci.bio:12608 sci.bio.technology:1910 comp.ai.neural-nets:11650 Hi, I'm interested in projects which involve using neural nets for prediction of protein function(s) and structure(s) from 1. Water Binding 2. Denaturants 2. Protein sequences Which network have been used (topology, type,...) ? Which result' s ? I would appreciate any information, or even references of where I can get information regarding this information. Please mail, I'll post a conclusion to the group. Thanks. Matthias Oberdorfer From owner-proteins@net.bio.net Thu Dec 01 22:00:00 1994 Path: biosci!agate!dog.ee.lbl.gov!news.cs.utah.edu!cc.usu.edu!sl16s From: sl16s@cc.usu.edu Newsgroups: bionet.molbio.proteins Subject: protein-protein binding Message-ID: <1994Dec2.092532.34320@cc.usu.edu> Date: 2 Dec 94 09:25:32 MDT Organization: Utah State University Lines: 8 I am trying to look for Protein binding sites in the protein I am working with. I really appreciate iof any body can enlighten me on this matter. 1. For a protein to bind naother protein should there be specific DNA sequences or a broad consensu sequence? 2. Can I find the proein binding sites by the DNA sequence, translated by computer? I am working with a chaperone protein. I really apreciate if anybody can give me any pointers on this matter. From owner-proteins@net.bio.net Thu Dec 01 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!uhog.mit.edu!europa.eng.gtefsd.com!gatech!swrinde!pipex!warwick!bham!usenet From: altabios@bham.ac.uk (John E. Fox) Newsgroups: bionet.molbio.proteins Subject: Re: Method to quantify number of cysteine residues Date: 2 Dec 1994 18:06:54 GMT Organization: The University of Birmingham, UK Lines: 13 Message-ID: <3bnnnu$fqe@sun4.bham.ac.uk> References: <3bniru$8oe@charm.magnus.acs.ohio-state.edu> NNTP-Posting-Host: bcs118.bham.ac.uk X-Newsreader: WinVN version 0.80 In article <3bniru$8oe@charm.magnus.acs.ohio-state.edu>, bhomoell@magnus.acs.ohio-state.edu (Bradley J Homoelle) says: > >Hi. I was wondering if anyone out there knew of a good method to determine the >number of cysteine residues in a peptide. If so, could you kindly send me an >e-mail or post to this newsgroup. Thank you in advance! >-- > > >Bradley J. Homoelle >The Ohio State University Try amino acid analysis after performic acid oxidation. From owner-proteins@net.bio.net Thu Dec 01 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!spool.mu.edu!uwm.edu!vixen.cso.uiuc.edu!howland.reston.ans.net!swrinde!pipex!uunet!zib-berlin.de!news.th-darmstadt.de!terra.wiwi.uni-frankfurt.de!zeus.rbi.informatik.uni-frankfurt.de!news.dfn.de!scsing.switch.ch!elna.ethz.ch!smac.ethz.ch!user From: tan@mol.biol.ethz.ch (Song Tan) Newsgroups: bionet.molbio.proteins Subject: Re: Minor Groove Binding TFs Date: Fri, 02 Dec 1994 13:39:53 +0100 Organization: ETH-Honggerberg (Swiss Federal Institute of Technology) Lines: 27 Message-ID: References: NNTP-Posting-Host: smac.ethz.ch In article , Sinasac David Steven wrote: > Here is a little trivia question that I hope some one can answer. Can > anyone give me the name of a specific cell specific transcription factor > that binds to the minor groove of the DNA double helix. This has to be > found in higher eukaryotes and it has to be true binding (though not > necessarily sequence specific). The only thing that I could come up with > is the IHF motif found in E. coli. Any help would be appreciated. I'm pretty sure I've seen several papers describing binding by transcription factors to the minor groove. The most obvious one that comes to mind is TBP (TATA binding protein). Cocrystal structures of Arabidopsis TBP/DNA from Burley's group and of yeast TBP/DNA from Sigler's group both show similar features including binding to the minor groove (back to back papers in Nature about 1 year ago). The primary sequence of this DNA binding region of TBP is so similar among eukaryotes that it's likely similar binding occurs in higher eukaryotes. Perhaps TBP won't meet your requirement of a "cell-specific" transcription factor, but it does meet the other requirements. -- Song Tan Institute for Molecular Biology and Biophysics ETH-Honggerberg (Swiss Federal Institute of Technology) 8093 Zurich, Switzerland email: tan@mol.biol.ethz.ch From owner-proteins@net.bio.net Thu Dec 01 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!spool.mu.edu!uwm.edu!psuvax1!news.ecn.bgu.edu!newspump.wustl.edu!crcnis3.unl.edu!news.mid.net!netserv.unmc.edu!netserv.unmc.edu!bghallor From: bghallor@unmc.edu (Brian Halloran) Newsgroups: bionet.molbio.proteins Subject: NEED: c-myb probe Date: 2 Dec 1994 21:18:21 GMT Organization: University of Nebraska Medical Center, Omaha, NE, USA Lines: 13 Message-ID: <3bo2ut$c56@netserv.unmc.edu> NNTP-Posting-Host: netserv.unmc.edu X-Newsreader: TIN [version 1.2 PL2] Hello Folks: I'm planning to do a Northern blot on human RNA and need a probe for c-myb. Can anyone direct me to where I can find/buy this? Thanks in advance. Reply to: bghallor@unmcvm.unmc.edu Brian Halloran University of Nebraska Medical Center From owner-proteins@net.bio.net Thu Dec 01 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!gatech!howland.reston.ans.net!news.moneng.mei.com!uwm.edu!lll-winken.llnl.gov!enews.sgi.com!decwrl!tribune.usask.ca!canopus.cc.umanitoba.ca!loewen5.microbio.umanitoba.ca!ahillar From: ahillar@your-servername.Lan1.UManitoba.CA Newsgroups: bionet.molbio.proteins Subject: Horseradish Peroxidase in E. coli??? Date: Fri, 2 Dec 1994 20:12:04 GMT Organization: University of Manitoba Lines: 6 Message-ID: NNTP-Posting-Host: loewen5.microbio.umanitoba.ca X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] Anybody know if a plasmid encoded version of the gene for horseradish peroxidase has been constructed that has been used to express the holoenzyme in E. coli? I am aware that the inactive apoenzyme has been successfully expressed from a synthetic gene constructed by A.T. Smith's group at Brighton, but wonder if anybody has had (even marginal) success at getting any active holoenzyme expressed in E. coli. From owner-proteins@net.bio.net Fri Dec 02 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!gatech!newsxfer.itd.umich.edu!news.itd.umich.edu!usenet From: Rick Neubig Newsgroups: bionet.molbio.proteins Subject: Re: Method to quantify number of cysteine residues Date: 3 Dec 1994 13:57:01 GMT Organization: University of Michigan Lines: 21 Message-ID: <3bptfd$mre@lastactionhero.rs.itd.umich.edu> References: <3bniru$8oe@charm.magnus.acs.ohio-state.edu> NNTP-Posting-Host: pm002-13.dialip.mich.net bhomoell@magnus.acs.ohio-state.edu (Bradley J Homoelle) wrote: > > Hi. I was wondering if anyone out there knew of a good method to determine the > number of cysteine residues in a peptide. If so, could you kindly send me an > e-mail or post to this newsgroup. Thank you in advance! > -- A very simple and inexpensive way to detect reduced cysteines is with Ellman's reagent, DTNB (it is dithionitrobis phenol - or some thing like that). I don't have a detailed protocol right here but could try to track down the reference. You just incubate your thiol-containing peptide with an excess of DTNB and read the absorbance. It will only detect reduced cysteines so if there is a chance that some have oxidized during storage (which was a problem for us) you will have to reduce the peptide with DTT or BME then get rid of the reducing agent before using the DTNB. RIck Neubig University of Michigan From owner-proteins@net.bio.net Fri Dec 02 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!uhog.mit.edu!europa.eng.gtefsd.com!howland.reston.ans.net!newsserver.jvnc.net!raffles.technet.sg!nuscc.nus.sg!eeserver.ee.nus.sg!guoyi From: guoyi@eeserver.ee.nus.sg (Guo Yi) Newsgroups: bionet.molbio.proteins Subject: ~{zoW^~} Date: 29 Nov 1994 06:58:27 GMT Organization: National University of Singapore Lines: 1 Message-ID: <3bejek$lf0@nuscc.nus.sg> NNTP-Posting-Host: guoyi%@eeserver-gw.ee.nus.sg X-Newsreader: TIN [version 1.2 PL2] From owner-proteins@net.bio.net Fri Dec 02 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!spool.mu.edu!howland.reston.ans.net!newsserver.jvnc.net!raffles.technet.sg!nuscc.nus.sg!eeserver.ee.nus.sg!guoyi From: guoyi@eeserver.ee.nus.sg (Guo Yi) Newsgroups: bionet.molbio.proteins Subject: ~{9y~} Date: 29 Nov 1994 06:55:00 GMT Organization: National University of Singapore Lines: 1 Message-ID: <3bej84$lf0@nuscc.nus.sg> NNTP-Posting-Host: guoyi%@eeserver-gw.ee.nus.sg X-Newsreader: TIN [version 1.2 PL2] From owner-proteins@net.bio.net Fri Dec 02 22:00:00 1994 Path: biosci!daresbury!news!ajb From: ajb@s-crim1.dl.ac.uk (Alan Bleasby) Newsgroups: bionet.molbio.proteins Subject: [ANNOUNCE] Principles of Protein Structure Date: 03 Dec 1994 23:23:09 GMT Organization: SERC Daresbury Lab, Warrington, U.K. Lines: 64 Distribution: world Message-ID: NNTP-Posting-Host: s-crim1.dl.ac.uk A Virtual Course PRINCIPLES OF PROTEIN STRUCTURE INVITATIONS for STUDENT REGISTRATION This is an interactive multimedia Internet course covering the basic principles of protein structure and related function. It is sponsored by: Birkbeck College, University of London and The Virtual School of Natural Sciences (Globewide Network Academy). Since the initial announcement 6 weeks ago, about 30 consultants worldwide have volunteered to help in various ways, and have already contributed extremely impressive material. There are many completely novel methods of learning in this course and it has already received much interest. WE ARE NOW INVITING APPLICATIONS FOR STUDENTS ON THE COURSE. There are no fees or formal qualifications required but there ARE a number of other requirements and all students MUST READ THE INSTRUCTIONS CAREFULLY before applying. Registration will ONLY BE ACCEPTED (OR EVEN ACKNOWLEDGED) VIA THE FORMS PROTOCOL UNDER HTML/CGI. (Most browsers such as Netscape, Mosaic and lynx (text-only) support this). ALL MATERIAL RELATING TO THE COURSE IS UNDER THE URL: http://www.cryst.bbk.ac.uk/PPS/index.html Please read this before mailing us with additional questions. There are also HTML/CGI forms for adding your comments, and this is a more efficient method than e-mail. All correspondence should go to the addresses given under the course URL and no longer to me. In this way we can record and manage your mail more efficiently. I am sorry that there has been a slight delay in registration, (due to technical server problems), but we don't expect it to delay the start of the course. We expect a heavy application, but it is NOT first-come-first-served, so please make sure you read the material carefully. Peter Murray-Rust December 3 1994 BTW The WWW pages at Daresbury (http://seqnet.dl.ac.uk:8000/vsns-pps) will remain as a mirror of the Birkbeck pages, but BBK is now the primary site. I believe that all the static documents link correctly, and we also believe that the forms and scripts work - use the bugs form for any problems. I am extremely grateful to several people for their technical help with the servers (Alan Bleasby, Dave Houldershaw, Alan Mills, Marcus Speh, and Richard Westlake). If there are technical problems with the DL material, mail me. P. Peter Murray-Rust (pmr1716@ggr.co.uk) Glaxo Research & Development, Greenford, UK mbglx@seqnet.dl.ac.uk, http://www.dl.ac.uk/CBMT/pmr.html (Thanks to Alan Bleasby) From owner-proteins@net.bio.net Fri Dec 02 22:00:00 1994 Path: biosci!daresbury!trane.uninett.no!sunic!pipex!uunet!news.inhouse.compuserve.com!news.compuserve.com!news From: John A. Newitt <73043.1773@CompuServe.COM> Newsgroups: bionet.molbio.proteins Subject: Re: Detecting hemoglobin on blots? Date: 3 Dec 1994 22:10:57 GMT Organization: NIH Lines: 4 Message-ID: <3bqqdh$kbr$1@mhade.production.compuserve.com> References: (null) In my experience hemoglobin does lose its heme when denatured in SDS-sample buffer (while I think cytochrome C may not). Sorry, but I can't help with any Ab's. - John A. Newitt From owner-proteins@net.bio.net Fri Dec 02 22:00:00 1994 Path: biosci!agate!howland.reston.ans.net!Germany.EU.net!netmbx.de!zib-berlin.de!informatik.tu-muenchen.de!lrz-muenchen.de!ipp-garching.mpg.de!alf.biochem.mpg.de!krasel From: krasel@alf.biochem.mpg.de (Cornelius Krasel) Newsgroups: bionet.molbio.proteins Subject: Re: E.Coli Westerns Date: 3 Dec 1994 11:54:28 GMT Organization: Rechenzentrum der Max-Planck-Gesellschaft in Garching Lines: 19 Message-ID: <3bpm9k$1ia4@sat.ipp-garching.mpg.de> References: NNTP-Posting-Host: alf.biochem.mpg.de X-Newsreader: TIN [version 1.2 PL2] bipin dalmia (dalmiabk@phibred.com) wrote: > In article Anne Spang, > spang@vms.biochem.mpg.de writes: > >via nitrocellulose strips on which you antigen is immobilized. The > >advantage of this methode is, that you do not need pure antigen, because > >you just cut out the band of your protein of interest. If you are > >interested in this protocol,I can send it to you > can you send me this protocol? (i had to post because of some mailer > problems. it allows me to post but not reply directly, go figure) Better yet, post it (others might be interested as well)! --Cornelius. -- /* Cornelius Krasel, Abt. Lohse, Genzentrum, D-82152 Martinsried, Germany */ /* email: krasel@alf.biochem.mpg.de fax: +49 89 8578 3795 */ /* "Science is the game you play with God to find out what His rules are." */ From owner-proteins@net.bio.net Fri Dec 02 22:00:00 1994 Path: biosci!news.cs.umb.edu!hsdndev!nmr-z.mgh.harvard.edu!132.183.200.17!lfk From: lfk@receptor.mgh.harvard.edu (Lee F. (Frank) Kolakowski) Newsgroups: bionet.molbio.proteins Subject: Re: Method to quantify number of cysteine residues Date: 03 Dec 1994 20:05:42 GMT Organization: Mass. General Hospital, Renal Unit Lines: 38 Message-ID: References: <3bniru$8oe@charm.magnus.acs.ohio-state.edu> <3bptfd$mre@lastactionhero.rs.itd.umich.edu> NNTP-Posting-Host: receptor.mgh.harvard.edu In-reply-to: Rick Neubig's message of 3 Dec 1994 13:57:01 GMT On 3 Dec 1994 13:57:01 GMT, Rick Neubig said: > bhomoell@magnus.acs.ohio-state.edu (Bradley J Homoelle) wrote: >> Hi. I was wondering if anyone out there knew of a good method to >> determine the number of cysteine residues in a peptide. > A very simple and inexpensive way to detect reduced cysteines is > with Ellman's reagent, DTNB (it is dithionitrobis phenol - or some > thing like that). That is Di Thio Nitro Benzoic acid. > I don't have a detailed protocol right here > but could try to track down the reference. You just incubate > your thiol-containing peptide with an excess of DTNB and read > the absorbance. By this from Pierce and they give you a little book with protocols. The main thing is that it has to be in 0.1 M NaPO4 ph 8.0. Also you can make a standard curve with some free Cysteine. > It will only detect reduced cysteines so if there is a chance > that some have oxidized during storage (which was a problem for us) > you will have to reduce the peptide with DTT or BME then get > rid of the reducing agent before using the DTNB. Of course Rick is right here. -- Frank Kolakowski Email: lfk@receptor.mgh.harvard.edu 617-735-7515 (LAB) GCRDb-WWW From owner-proteins@net.bio.net Sat Dec 03 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!spool.mu.edu!howland.reston.ans.net!Germany.EU.net!news.dfn.de!zeus.rbi.informatik.uni-frankfurt.de!terra.wiwi.uni-frankfurt.de!news.th-darmstadt.de!zib-berlin.de!informatik.tu-muenchen.de!lrz-muenchen.de!ipp-garching.mpg.de!genmac4.biochem.mpg.de!user From: spang@vms.biochem.mpg.de (Anne Spang) Newsgroups: bionet.molbio.proteins Subject: antibody purification protocol (nitrocellulose) Date: 4 Dec 1994 18:20:48 GMT Organization: Rechenzentrum der Max-Planck-Gesellschaft in Garching Lines: 32 Message-ID: NNTP-Posting-Host: genmac4.biochem.mpg.de Hi everybody! A very detailed protocol with a lot of references can be found in Methods in Enzymology Vol. 194, Guide to Yeast Genetics and Molecular Biology, pp.581. Short version: -run a preperative SDS-PAGE with your protein or a crude extract -transfer the proteins onto nitrocellulose paper -stain the membrane with Ponceau S -cut out your protein band and mark the protein side! -destain and block the nitrocellulose strip (5% milk in PBS) for at least 1 h RT -wash the strip in PBS -place a piece of parafilm on the bottom of a Petri dish and lay the nitrocellulose strip on top (drained but wet; proteins side up) -put 0.2-0.5 ml serum on the strip -put a wet kimwipe in the dish (wet chamber, evaporation!) -place the closed Petri dish on a shaker (shake fast enough that the serum moves on the strip, but not to fast) 2-3h RT -remove the serum with a pipetman -wash the strip 1xPBST, 3xPBS -place the strip on a fresh piece of parafilm in a Petri dish -elute the Antibodies with 0.2 ml 200mM glycine 1mM EDTA pH 2.5 for 10-30 min -remove the eluate and adjust the pH immediately with 0.01 ml 100 mM Tris pH 8 -the elution step can be repeated, if necessary -wash the strip and store it in PBS at -20C or after drying for reuse Hope this helps! Anne PS: For Cornelius only! If you need further information, you know where I am! Just come downstairs! From owner-proteins@net.bio.net Sun Dec 04 22:00:00 1994 Path: biosci!JASON.UTHCT.EDU!SHAUN From: SHAUN@JASON.UTHCT.EDU ("Shaun D. Black") Newsgroups: bionet.molbio.proteins Subject: Re: Method to quantify number of cysteine residues Date: 5 Dec 1994 10:22:45 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 151 Sender: daemon@net.bio.net Distribution: world Message-ID: <941205122917.dc9@jason.uthct.edu> NNTP-Posting-Host: net.bio.net Hi All, I though I'd paste my "everything you wanted to know about DTNB" blurb in to complement the current thread. Perhaps it will prove useful to some. Best regards, Shaun =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= = Use of Ellman's Reagent for Determination of Protein Thiols = =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= =-=-=-=-=-=-=-= = Background = =-=-=-=-=-=-=-= Ellman's reagent or 5,5'-dithiobis(2-nitrobenzoate) (DTNB) is a symmetrical aryl disulfide which readily undergoes the thiol-disulfide interchange reaction in the presence of a free thiol: - - - COO COO COO \___ ___/ \___ / \ / \ | - / \ | O N- O -S-S- O -N O + Cys-S ----> O N- O -S-S-Cys (Mixed 2 \___/ \___/ 2 | 2 \___/ | disulfide) (Excess) + - COO \___ / \ - (2-nitro-5- O N- O -S thiobenzoate) 2 \___/ (TNB) The TNB dianion has a relatively intense absorbance at 412nm compared to both disulfides. Because the stoichiometry of protein thiol to TNB formed is 1:1, TNB formation can be used to assess the number of thiols present. In the absence of denaturants, only accessible thiols will react, whereas in the presence of chaotropic agents the total number of reduced Cys residues present can be measured. Reduction of the protein followed by treatment with chaotropes and DTNB can yield the total number of cysteines (Cys-SH plus Cys-S-S-Cys). - - The reaction is sensitive to alkaline pH ( OH) competes with R-S ), acidic pH (disulfides can be broken), oxygen (reoxidation of R-SH), and temperature (thermochromism). Consequently, the reaction is usually carried out with an excess of DTNB to protein, at neutral pH, fixed temp- erature, and sometimes under anaerobic conditions. Furthermore, TNB is sensitive to various buffer ions, so the extinction coefficient used to calculate number of thiols must be properly matched to the reaction conditions. This is also true of reactions carried out in the presence of the denaturants urea or guanidine (GuHCl). The following table summarizes some useful data on DTNB and TNB under various conditions: -------------------------------------------------------------------------- Compound lambda max (pH~7) Extinction: (in PO4) (in Tris) (in GuHCl) --------- ----------------- -------------------- ---------- ---------- DTNB 324 nm 17,780 16,600 ---- TNB 412 nm 14,150 -??- 13,700 -------------------------------------------------------------------------- Extinction coefficients are in units of per Molar per cm path length, 30degC Decreased TNB extinction in the presence of GuHCl is due to a shift in the lambda max from 412 to 422 nm. The extinction of DTNB at 412 nm is 212/M/cm, small but measurable. =-=-=-=-=-=-=-= = Protocol = =-=-=-=-=-=-=-= Stock Solutions: Protein solution (native or previously reduced and desalted) (~5-40uM in 0.1M potassium phosphate buffer, pH 7.4) DTNB stock (20 mM in 0.1M KPO4, pH 7.4; MW 396.35; 7.93 mg/ml) (DTNB is very slow to go into solutions; vortex periodically over the period of 2-3 hours to achieve complete solution, which will be very light yellow in color) Urea or Guanidine-HCl (high purity, crystalline solids) Major Equipment: Dual beam spectrophotometer set in kinetic mode at 412nm Thermostated cells, 30degreesC Procedure: Sample cell 1 = 1.0 mL protein solution + 50 uL 20mM DTNB Reference cell 1 = 1.0 mL buffer + 50 uL DTNB Record the absorbance before addition of the DTNB as "zero". Follow the reaction at 412 nm at 30degC continuously. Record past the time when the reaction reaches a maximum. If any significant down slope is seen after the maximum absorbance is reached, this will need to be corrected for in order to get the correct number of accessible thiols. In the second experiment, add crystalline Guanidine-HCl to your protein solution to achieve a final concentration of about 4M. Note the volume change and the resulting significant dilution of your protein solution! Sample cell 2 = 1.0 mL protein solution (+4M GuHCl) + 50 uL 20 mM DTNB Reference cell 2 = 1.0 mL buffer (+4M GuHCl) + 50 uL 20mM DTNB Again, record OD 412 nm at 30 degC up to and beyond peak absorbance. This experiment will yield the total number of reduced thiols. Sample cell 3 = 1.0 mL pre-denatured,reduced, and dialyzed protein + 50 uL 20mM DTNB Reference cell 3 = 1.0 mL buffer (+4M GuHCl) + 50 uL 20 mM DTNB Again, record OD 412 nm at 30degC vs time to peak absorbance and beyond. This experiment will yield the total number of Cys residues in the protein. =-=-=-=-=-=-=-=-=-= = Data Analysis = =-=-=-=-=-=-=-=-=-= Calculate the number of thiols modified (or concentration thereof) using the maximum absorbance at 412 nm measured (or the corrected value thereof), Beer's Law, and the appropriate extinction coefficient; divide this value by the accurately known molar protein concentration in the reaction cuvette for each of the three experiments. This will give you accessible thiols, total Cys-SH, and total Cys-SH plus Cys-SS-Cys. Do each experiment in duplicate or triplicate and average the respective replicates. If your numbers are statistically integers, you're done. If not, consider repeating the protocol, but using anaerobic (degassed) buffers for all reactions. =-=-=-=-=-=-=-=-=-=-= = Some References = =-=-=-=-=-=-=-=-=-=-= "A colorimetric method for determining low concentrations of mercaptans" Ellman, G.L. (1958) Arch. Biochem. Biophys. 74, 443-450. "Reassessment of Ellman's reagent" Riddles, P.W., Blakeley, R.L., and Zerner, B. (1983) Methods Enzymol. 91, 49-60. "Studies on the identity of the heme-binding cysteinyl residue in rabbit liver microsomal cytochrome P-450 isozyme 2" Black, S.D. and Coon, M.J. (1985) Biochem, Biophys. Res. Commun. 128, 82-89. =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= = Shaun D. Black, PhD | Internet address: shaun@jason.uthct.edu = = Dept. of Biochemistry | University of Texas Health Center, at Tyler = =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= From owner-proteins@net.bio.net Sun Dec 04 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!spool.mu.edu!howland.reston.ans.net!pipex!sunsite.doc.ic.ac.uk!daresbury!not-for-mail From: Mohammed Saad Newsgroups: bionet.molbio.proteins Subject: fibres Date: 5 Dec 1994 17:00:41 -0000 Lines: 20 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <3bvgvp$s3n@mserv1.dl.ac.uk> Original-To: proteins@dl.ac.uk Hi, everybody. Is there anyone who is interested in or knows how to create a newgroup specific for fibrous proteins (X-ray/neutron structure analysis, modelisation,data analysis etc... ) ? \\|// (o o) --------------------------ooO---(_)---Ooo-------------------------- Protein Crystallography ----------------------- SAAD Mohamed Ph.D. | Experimental Division European Synchrotron Radiation | Structural Biology Group Facility | B.P. 220 | Tel: 0033 76 88 22 13 38043 GRENOBLE CEDEX 9 | Fax: 0033 76 88 25 42 FRANCE | E-Mail: saad@esrf.fr ------------------------------------------------------------------- From owner-proteins@net.bio.net Sun Dec 04 22:00:00 1994 Path: biosci!agate!overload.lbl.gov!xpat.postech.ac.kr!news.kreonet.re.kr!worak.kaist.ac.kr!chiak.kaist.ac.kr!jylee From: jylee@chiak.kaist.ac.kr (Lee jang young) Newsgroups: bionet.molbio.proteins Subject: biological demethylation ? Date: 5 Dec 1994 14:35:43 GMT Organization: Korea Advanced Institute of Science and Technology Lines: 10 Message-ID: <3bv8fv$8kj@worak.kaist.ac.kr> NNTP-Posting-Host: chiak.kaist.ac.kr X-Newsreader: TIN [version 1.1 PL9] Hi bionetters Does anyone have a knowledge about the biological demethylation occurring in vivo ? Any suggestion would be welcomed at jylee@chiak.kaist.ac.kr Thanks in advance J.Y.Lee From owner-proteins@net.bio.net Sun Dec 04 22:00:00 1994 Path: biosci!JASON.UTHCT.EDU!SHAUN From: SHAUN@JASON.UTHCT.EDU ("Shaun D. Black") Newsgroups: bionet.molbio.proteins Subject: RE: biological demethylation ? Date: 5 Dec 1994 09:51:02 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 36 Sender: daemon@net.bio.net Distribution: world Message-ID: <941205115747.c69@jason.uthct.edu> NNTP-Posting-Host: net.bio.net Lee Jang Young asked if anyone had knowledge of in vivo biological demethylation. I'm not sure what the actual orientation of the question is, but wanted to note that there is an extensive body of research on in vivo demethylation reactions carried out by cytochrome P450. This microsomal enzyme system can oxidatively catalyze N-, O-, S-, and C- demethylation reactions on a wide variety of substrates, deemed by some to number in the 1000's. If there is any interest to follow this lead up further, you might want to consult one of the books or reviews below: "Cytochrome P450" in the Handbook of Experimental Pharmacology, vol. 105, (1993) H. Greim and J.B. Schenkman, eds., Springer Verlag, Berlin. Methods in Enzymology, vol. 206 (1991) M.R. Waterman and E.F. Johnson, eds. Academic Press, New York. Cytochrome P450: Structure, Mechanism, and Biochemistry (1986) P. Ortiz de Montellano, ed., Plenum Press, New York. Human Hepatic Cytochromees P450 Involved in Drug Medabolism. Wrighton, S.A. and Stevens, J.C. (1992) Crit. Rev. Toxicol. 22(1): 1-22. Enzymatic Activation of Chemicals to Toxic Metabolites. Guengerich, F.P., and Liebler, D.C. (1985) CRC Crit. Rev. Toxicol. 14: 259-307. P-450 Cytochromes: Structure and Function. Black, S.D., and Coon, M.J. (1987) Adv. Enzymol. Relat. Areas Mol. Biol. 60: 35-87. I hope this helps you a bit. Best regards, =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= = Shaun D. Black, PhD | Internet: shaun@jason.uthct.edu = = Dept. of Biochemistry | Old: shaun%jason.decnet@relay.the.net = = UT Health Center, Tyler | Phone: (903)877-2806 FAX: (903)877-7558 = = Tyler, TX 75710-2003 | B-) (Start every day with a smile...) = =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= From owner-proteins@net.bio.net Sun Dec 04 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!gatech!howland.reston.ans.net!newsserver.jvnc.net!synapse.bms.com!lanrover2.bms.com!user From: roberts_d@msmail.bms.com (dan roberts) Newsgroups: bionet.molbio.proteins Subject: Endogenous Biotinylated Proteins in SF9 Cells? Followup-To: bionet.molbio.proteins Date: 6 Dec 1994 02:43:36 GMT Organization: Bristol-Myers Squibb Company Lines: 3 Distribution: world Message-ID: NNTP-Posting-Host: lanrover2.bms.com Anyone one have any idea if there are any endogenous biotinylated proteins in SF9 cells? Also does streptavidin have any known or bothersome cross reactivities?.. Thanks.>Dan Roberts From owner-proteins@net.bio.net Sun Dec 04 22:00:00 1994 Path: biosci!ZOOL.UMD.EDU!GOODE From: GOODE@ZOOL.UMD.EDU ("Dennis Goode") Newsgroups: bionet.molbio.proteins Subject: Re: fibres Date: 5 Dec 1994 10:47:03 -0800 Organization: University of Maryland Zoology Lines: 45 Sender: daemon@net.bio.net Distribution: world Message-ID: <1AB6342E08@zool.umd.edu> NNTP-Posting-Host: net.bio.net Mohammed Saad ON THE Subject: fibres said: (5 Dec 1994 17:00:41) 0000 > Hi, everybody. > > Is there anyone who is interested in or knows how to create a newgroup specific > for fibrous proteins (X-ray/neutron structure analysis, modelisation,data > analysis etc... ) ? > > \\|// > (o o) > --------------------------ooO---(_)---Ooo-------------------------- > > Protein Crystallography > ----------------------- > SAAD Mohamed Ph.D. | Experimental Division > European Synchrotron Radiation | Structural Biology Group > Facility | > B.P. 220 | Tel: 0033 76 88 22 13 > 38043 GRENOBLE CEDEX 9 | Fax: 0033 76 88 25 42 > FRANCE | E-Mail: saad@esrf.fr > ------------------------------------------------------------------- > > Mohammed: I think there would be sufficient interest to "subclone" a fibrous protein group. Why don't you contact the bionet coordinator. Of course two different meanings of "fibrous" proteins are used: 1. Long, thin proteins (with a high axial ratio). 2. Proteins that polymerize to form the fibers, filaments, and tubules so important in biological systems (many of which are small and globular like actin and tubulin). Perhaps both should be included, since the problems and techniques are similar in many cases. Dennis "The greatest shortcoming of the human race is our inability to understand the exponential function." A.A. Bartlet From owner-proteins@net.bio.net Mon Dec 05 22:00:00 1994 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!network.ucsd.edu!tpillay.extern.ucsd.edu!tpillay From: T. S. Pillay Newsgroups: bionet.molbio.proteins Subject: Re: Amino Acid Homology Date: 6 Dec 1994 15:04:39 GMT Organization: UCSD Lines: 7 Distribution: world Message-ID: <3c1ui7$176@network.ucsd.edu> References: <37234.ewright@fox.nstn.ns.ca> NNTP-Posting-Host: tpillay.extern.ucsd.edu X-UserAgent: Version 1.1.3 X-XXMessage-ID: X-XXDate: Tue, 6 Dec 94 07:05:50 GMT In article <37234.ewright@fox.nstn.ns.ca> Edwin Wright, ewright@FOX.NSTN.NS.CA writes: >What is the latest word on amino acid homology with respect to conservative >versus moderate or radical amino acid substitutions in protein sequences? > Yeah, I don't understand your question either? From owner-proteins@net.bio.net Mon Dec 05 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!bloom-beacon.mit.edu!spool.mu.edu!howland.reston.ans.net!EU.net!sun4nl!cs.vu.nl!balaena!mac239.bio.vu.nl!user From: luirink@bio.vu.nl (J.Luirink) Subject: colocalisation of proteins with ribosomes Message-ID: Followup-To: bionet.molbio.proteins Sender: news@bio.vu.nl Organization: Molecular Microbioly, Vrije Universiteit Amsterdam, NL Date: Tue, 6 Dec 1994 14:19:55 GMT Lines: 18 Does anyone know about a reliable protocol to separate coli ribosomes from membranes. I would like to localise a protein which I think is associated with both fractions. Thanks. -- Joen Luirink Dept. Mol. Microbiol. Vrije Universiteit De Boelelaan 1087 1081 HV Amsterdam The Netherlands Tel: (20) 4447175 Fax: (20) 4447123 E-mail: luirink@bio.vu.nl From owner-proteins@net.bio.net Mon Dec 05 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!gatech!swrinde!pipex!lyra.csx.cam.ac.uk!warwick!unicorn.nott.ac.uk!macfd.biochem.nottingham.ac.uk!user From: mbxfd@unicorn.nott.ac.uk (Fergus Doherty) Newsgroups: bionet.molbio.proteins Subject: Re: Endogenous Biotinylated Proteins in SF9 Cells? Followup-To: bionet.molbio.proteins Date: 6 Dec 1994 16:28:11 GMT Organization: Nottingham University, UK. Lines: 26 Distribution: world Message-ID: References: NNTP-Posting-Host: macfd.biochem.nottingham.ac.uk In article , roberts_d@msmail.bms.com (dan roberts) wrote: > > Anyone one have any idea if there are any endogenous biotinylated proteins > in SF9 cells? Also does streptavidin have any known or bothersome cross > reactivities?.. Thanks.>Dan Roberts A friend of mine in Hungary had some problems with 'odd' bands in Sf9 cells using a biotinylated antibody/avidin system, and I certainly have seen endogenous biotinylated proteins in mitochondrial fractions, especially with 125-streptavidin to detect bound antibody. Carboxylases have biotin, and I would expect Sf9 to contain these, why not? You can overcome this by blocking firt with avidin to bind endogenous biotin, then wash and incubate with biotin. Biotin can only be bound by one avidin/streptavidin so when you add your avidin-peroxidase or whatever, it will only bind to the biotinylated antibody. Check out Vector Labs info to make sure I have this right! The streptavidin or avidin from the likes of Vector Labs is claimed to be free of 'cross reactivities'. They modify their avidin in some way to cut down background. Fergus Doherty, dept. Biochemistry, University Medical School, Queen's Medical Centre, Nottingham NG7 2UH Tel: 602 709366 FAX 602 422225 Internet: mbxfd@unicorn.nott.ac.uk From owner-proteins@net.bio.net Tue Dec 06 22:00:00 1994 Path: biosci!MAILGATE.GLAXO.COM!agostino~mj%a1.usa.umc From: agostino~mj%a1.usa.umc@MAILGATE.GLAXO.COM ("Mike Agostino") Newsgroups: bionet.molbio.proteins Subject: how many receptors are there? Date: 7 Dec 1994 05:27:27 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 27 Sender: daemon@net.bio.net Distribution: world Message-ID: <75522170214991.326975@USA> NNTP-Posting-Host: net.bio.net [This message is converted from WPS-PLUS to ASCII] There have been a lot of numbers kicked around, based on various techniques for estimating gene frequencies, that put the total number of G-protein coupled receptors encoded by the human genome at anywhere from a couple of hundred to several thousand. Does anyone out there in Netland have what they are comfortable with as a number for GPCR's? Or, do you have any idea where some of these estimates came from? If so, could you post or email me with it and please include where the estimate came from and how it was derived (no astrology, please). I realize that all of these estimates have an element of fantasy to them, so I promise not to publish or otherwise attribute an estimate to you unless you beg appropriately. It would probably be most useful to break it down into total and non-odorant/sensory receptors. I will post back to the net a summary of replies (with names omitted unless otherwise requested). Thanks, Rich Buckholz RGB12955@glaxo.com From owner-proteins@net.bio.net Tue Dec 06 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!gatech!swrinde!pipex!sunic!news.uni-c.dk!exon!janhan From: janhan@exon (Jan Hansen) Newsgroups: bionet.molbio.proteins Subject: secondary structure prediction Date: 7 Dec 1994 10:56:22 GMT Organization: News Server at UNI-C, Danish Computing Centre for Research and Education. Lines: 18 Message-ID: <3c44cm$k64@news.uni-c.dk> NNTP-Posting-Host: exon.cbs.fki.dth.dk X-Newsreader: TIN [version 1.2 PL1] Do anyone know of availability of secondary structure prediction from sequence using E-mail or FTP other than the PHD method at EMBL?? -- Jan Hansen Center for Biological Sequence Analysis Laboratory for Infectious Diseases Department of Physical Chemistry The Technical University of Denmark Building 206 DK-2800 Lyngby Denmark Phone: +45 4593 1222 ext. 2485 (tone) Fax: +45 4593 4808 E-mail: janhan@cbs.dth.dk or janhan@exon.cbs.fki.dth.dk From owner-proteins@net.bio.net Tue Dec 06 22:00:00 1994 Path: biosci!rutgers!gatech!swrinde!pipex!uunet!zib-berlin.de!informatik.tu-muenchen.de!lrz-muenchen.de!ipp-garching.mpg.de!usenet From: kweber@vms.biochem.mpg.de (Klaus Weber) Newsgroups: bionet.molbio.proteins Subject: phosphoprotein enrichment Date: 7 Dec 1994 13:31:54 GMT Organization: Your Organization Lines: 10 Message-ID: <3c4dga$13cd@sat.ipp-garching.mpg.de> NNTP-Posting-Host: pcge10.biochem.mpg.de X-Newsreader: WinVN 0.92.5 Hi netters! There is a method for enrichment of phosphoproteins by phenolic extraction of triton lysates. The method was published in 1980 by Hunter and Sefton in PNAS. Does anybody know WHY it works and what the factor of enrichment is? Please mail to: jungbluth@genmic.biochem.mpg.de A.Jungbluth From owner-proteins@net.bio.net Tue Dec 06 22:00:00 1994 Path: biosci!rutgers!gatech!howland.reston.ans.net!vixen.cso.uiuc.edu!portisa1.agn.uiuc.edu!arportis From: arportis@vmd.cso.uiuc.edu (Archie Portis) Newsgroups: bionet.molbio.proteins Subject: Re: secondary structure prediction Date: Wed, 7 Dec 1994 22:16:19 GMT Organization: USDA Lines: 15 Message-ID: References: <3c44cm$k64@news.uni-c.dk> NNTP-Posting-Host: portisa1.agn.uiuc.edu X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] In article <3c44cm$k64@news.uni-c.dk> janhan@exon (Jan Hansen) writes: >Do anyone know of availability of secondary structure prediction >from sequence using E-mail or FTP other than the PHD method at EMBL?? Try psa-request@darwin.bu.edu. For help I believe you email without any message. _________________________________________________________________ Archie R. Portis arportis@vmd.cso.uiuc.edu USDA/ARS & Agronomy/UIUC 217-244-4419 [FAX] 193 PABL 217-244-3083 [Voice] 1201 W. Gregory Urbana, IL 61801 _________________________________________________________________ From owner-proteins@net.bio.net Tue Dec 06 22:00:00 1994 Path: biosci!rutgers!gatech!howland.reston.ans.net!vixen.cso.uiuc.edu!news.uoregon.edu!ntuix.ntu.ac.sg!raffles.technet.sg!nuscc.nus.sg!mcblab43 From: mcblab43@leonis.nus.sg (Mr. Loh Chang Shiung) Newsgroups: bionet.molbio.proteins Subject: ENCAD availability Date: 5 Dec 1994 03:23:11 GMT Organization: Paris Opera House Lines: 10 Message-ID: <3bu12v$f2r@nuscc.nus.sg> NNTP-Posting-Host: mcblab43%@leonis.nus.sg X-Newsreader: TIN [version 1.2 PL2] Hello everyone, Can anybody advise me on the availability of Energy Calculation and Dynamics (ENCAD) software (Levitt et al)? I hope this is the correct newsgroup to post this! Thanks. Loh Chang Shiung, Internet e-mail address: mcblab43@leonis.nus.sg Centre for Natural Products Research, Institute of Molecular and Cell Biology From owner-proteins@net.bio.net Tue Dec 06 22:00:00 1994 Path: biosci!daresbury!sunsite.doc.ic.ac.uk!uknet!pipex!swrinde!gatech!rutgers!csn!borcore.usbr.gov!sacsa3.mp.usbr.gov!fws.gov!ash.lab.r1.fws.gov!bob.lab.r1.fws.gov!HoeschB From: HoeschB@fws.gov (Bob Hoesch) Newsgroups: bionet.molbio.proteins Subject: Re: Detecting hemoglobin on blots? Date: Wed, 7 Dec 1994 12:01:54 Organization: National Fish and Wildlife Forensics Lab Lines: 18 Message-ID: References: (null) <3bqqdh$kbr$1@mhade.production.compuserve.com> NNTP-Posting-Host: bob.lab.r1.fws.gov X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] >In my experience hemoglobin does lose its heme when denatured >in SDS-sample buffer (while I think cytochrome C may not). >Sorry, but I can't help with any Ab's. > - John A. Newitt I didn't see the original post, but if someone is looking for anti-HB, it is avalable from Sigma. Polyclonal rabbit anti-human Hb, catalog # H 4890 (p.1245 in the 1994 catalog). Regards, Bob Hoesch U.S. Fish and Wildlife Forensics Laboratory Ashland, OR HoeschB@fws.gov From owner-proteins@net.bio.net Wed Dec 07 22:00:00 1994 Path: biosci!CS.Arizona.EDU!news.Arizona.EDU!hamblin.math.byu.edu!sol.ctr.columbia.edu!spool.mu.edu!uwm.edu!psuvax1!news.cc.swarthmore.edu!mac04.parrishd.swarthmore.edu!user From: ehom1@cc.swarthmore.edu (Erik Forbes Y. Hom '95) Newsgroups: bionet.molbio.proteins Subject: Q: searching for phosphorylation sites Followup-To: bionet.molbio.proteins Date: Thu, 08 Dec 1994 00:13:55 +0000 Organization: Laboratory of Neurobiology, Swarthmore College Lines: 7 Message-ID: NNTP-Posting-Host: mac04.parrishd.swarthmore.edu Mime-Version: 1.0 Content-Type: text/plain; charset=US-ASCII Content-Transfer-Encoding: 7bit Hi netters, Could anyone here tell the best way to search for potential phosphorylation sites or sequence homologies to phosphorylated regions. Are there programs that do this for you? What would be the best way to proceed? Thanks in advanced for your help! erik From owner-proteins@net.bio.net Wed Dec 07 22:00:00 1994 Path: biosci!CS.Arizona.EDU!news.Arizona.EDU!hamblin.math.byu.edu!sol.ctr.columbia.edu!news.moneng.mei.com!howland.reston.ans.net!swrinde!elroy.jpl.nasa.gov!lll-winken.llnl.gov!overload.lbl.gov!xpat.postech.ac.kr!news.kreonet.re.kr!sorak.kaist.ac.kr!cojoe From: cojoe@sorak.kaist.ac.kr (Doo Yeon Kim) Newsgroups: bionet.molbio.proteins Subject: Determination of protein concentration? Date: 8 Dec 1994 01:26:02 GMT Organization: Korea Research Environment Open Network (KREONet) Lines: 8 Message-ID: <3c5nba$867@news.kreonet.re.kr> NNTP-Posting-Host: sorak.kaist.ac.kr X-Newsreader: TIN [version 1.2 PL2] I have problems in determine the protein concentration because of the high detergent concentraion of my protein sample. Is anyone knows the detergent free protein assay? -- ////////Doo Yeon Kim////// From owner-proteins@net.bio.net Wed Dec 07 22:00:00 1994 Path: biosci!agate!news.ucdavis.edu!library.ucla.edu!csulb.edu!nic-nac.CSU.net!usc!howland.reston.ans.net!gatech!newsxfer.itd.umich.edu!news.itd.umich.edu!usenet From: Rick Neubig Newsgroups: bionet.molbio.proteins Subject: Re: Determination of protein concentration? Date: 8 Dec 1994 18:01:54 GMT Organization: University of Michigan Lines: 17 Message-ID: <3c7hmi$t88@lastactionhero.rs.itd.umich.edu> References: <3c5nba$867@news.kreonet.re.kr> NNTP-Posting-Host: warbler.med.umich.edu cojoe@sorak.kaist.ac.kr (Doo Yeon Kim) wrote: > > I have problems in determine the protein concentration because of the high detergent concentraion of my protein sample. Is anyone knows the detergent free protein assay? A commonly used protein assay for dilute proteins in detergent solution is by Schaffner and Weisman 1. Schaffner, W. and Weissmann, C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal.Biochem. 56:502-514, 1973. The detection limit is about 1-2 ug. You precipitate the protein with TCA then collect on nitrocellulose and stain with Coomassie blue. You can elute the Coomassie blue with solvent and quantitate or just eyeball it compared to standards. Rick Neubig University of Michigan Pharmacology From owner-proteins@net.bio.net Wed Dec 07 22:00:00 1994 Path: biosci!ns1.faseb.org!darwin.sura.net!mother.usf.edu!chuma!mulholla From: "Dale Mulholland (BIO)" Newsgroups: bionet.molbio.proteins Subject: polyproline help Date: Thu, 8 Dec 1994 15:51:22 -0500 Organization: University of South Florida Lines: 13 Message-ID: NNTP-Posting-Host: chuma.cas.usf.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII X-Sender: mulholla@chuma Anyone out there know the chemical properties of a polyproline a-helix? I'm talking about a string of ~25 P's that form an a-helix, then three helices make a triple-helix a la collagen triple-helices. Possibly thermostability or possible chain-to-chain bonds giving extra stability? Thanks heaps! Dale S. Mulholland mulholla@chuma.cas.usf.edu University of South Florida Department of Biology Tampa, Florida 33620-5150 From owner-proteins@net.bio.net Wed Dec 07 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!daresbury!hgmp.mrc.ac.uk!sunsite.doc.ic.ac.uk!uknet!bcc.ac.uk!link-1.ts.bcc.ac.uk!ucbcplw From: ucbcplw@ucl.ac.uk (Mr Paul Wells) Subject: hplc Summary: help wanted Message-ID: <1994Dec8.151835.46573@ucl.ac.uk> Date: Thu, 8 Dec 1994 15:18:35 GMT Organization: University College London Keywords: Wanted inject valve Lines: 12 Newsgroups: bionet.molbio.proteins Subject: hplc end Keywords: wanted inject valve Hi, We are a metabolic group at UCL desperately seeking an inject valve for an old HPLC machine. Any help? post reply to ucbcmss@ucl.ac.uk From owner-proteins@net.bio.net Wed Dec 07 22:00:00 1994 Path: biosci!rutgers!gatech!howland.reston.ans.net!vixen.cso.uiuc.edu!news.uoregon.edu!netnews.nwnet.net!mule.fhcrc.org!news From: Tim Buss Newsgroups: bionet.molbio.proteins Subject: Getting Proteins out of the Periplasm Date: 8 Dec 1994 20:59:12 GMT Organization: Fred Hutchinson Cancer Research Center Lines: 9 Distribution: world Message-ID: <3c7s30$kjl@mule.fhcrc.org> NNTP-Posting-Host: 140.107.14.25 X-UserAgent: Nuntius v1.1.1d24 X-XXDate: Thu, 8 Dec 94 21:02:50 GMT I'm expressing humanized antibody fragments in E.coli. The proteins are concentrated into the periplasm and I'm looking for a good technique to get them out, with minimal cell disruption. Many protocols make use of lysozyme, but I cannot use this (it's an anti-lysozyme Fv). I am also trying to avoid low pH as I think this may well disrupt the fragment. Any tried and tested methods you folks would be willing to share???? Tim. From owner-proteins@net.bio.net Wed Dec 07 22:00:00 1994 Path: biosci!rutgers!uwm.edu!vixen.cso.uiuc.edu!howland.reston.ans.net!pipex!uunet!zib-berlin.de!informatik.tu-muenchen.de!lrz-muenchen.de!ipp-garching.mpg.de!alf.biochem.mpg.de!krasel From: krasel@alf.biochem.mpg.de (Cornelius Krasel) Newsgroups: bionet.molbio.proteins Subject: Re: Determination of protein concentration? Date: 8 Dec 1994 21:19:30 GMT Organization: Rechenzentrum der Max-Planck-Gesellschaft in Garching Lines: 19 Message-ID: <3c7t92$2f3m@sat.ipp-garching.mpg.de> References: <3c5nba$867@news.kreonet.re.kr> NNTP-Posting-Host: alf.biochem.mpg.de X-Newsreader: TIN [version 1.2 PL2] Doo Yeon Kim (cojoe@sorak.kaist.ac.kr) wrote: > I have problems in determine the protein concentration because of the > high detergent concentraion of my protein sample. Is anyone knows the > detergent free protein assay? You have several possibilities. One is to precipitate your protein before assaying it (e.g. with TCA). You could also try the BCA assay from Pierce which is reported to be quite insensitive against up to 1% detergent whilst having a sensitivity for proteins comparable with the Lowry assay, however, it does not like reducing agents. There are other assays described which involve TCA precipitation to get rid of unwanted substances in your protein solution. --Cornelius. -- /* Cornelius Krasel, Abt. Lohse, Genzentrum, D-82152 Martinsried, Germany */ /* email: krasel@alf.biochem.mpg.de fax: +49 89 8578 3795 */ /* "Science is the game you play with God to find out what His rules are." */ From owner-proteins@net.bio.net Thu Dec 08 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!bloom-beacon.mit.edu!gatech!howland.reston.ans.net!pipex!uunet!nih-csl!postman From: "Malcolm Moos Jr." Subject: Re: Determination of protein concentration? Message-ID: <1994Dec9.172826.4200@alw.nih.gov> Sender: postman@alw.nih.gov (AMDS Postmaster) Organization: National Institutes of Health References: <3c5nba$867@news.kreonet.re.kr> Date: Fri, 9 Dec 1994 17:28:26 GMT Lines: 11 cojoe@sorak.kaist.ac.kr (Doo Yeon Kim) wrote: > > I have problems in determine the protein concentration because of the high detergent concentraion of my protein sample. Is anyone knows the detergent free protein assay? > > > -- > > ////////Doo Yeon Kim////// > > Try the methanol chloroform procedure of Wessel and Pflugge; it was in Analytical Biochemistry several years ago. From owner-proteins@net.bio.net Thu Dec 08 22:00:00 1994 Path: biosci!rutgers!gatech!swrinde!pipex!uunet!news.inhouse.compuserve.com!news.compuserve.com!news From: John A. Newitt <73043.1773@CompuServe.COM> Newsgroups: bionet.molbio.proteins Subject: Re: HPLC Capability Question Date: 9 Dec 1994 23:40:18 GMT Organization: NIH Lines: 6 Message-ID: <3capt2$aa9$1@mhadg.production.compuserve.com> References: Particulates and HPLC don't mix. Also using HPLC would probably be slower, since you sacrifice the parallel nature of doing multiple ELISA's simultaneously versus having to run one sample at a time on an HPLC (unless, of course, you have 96 separate HPLC's). John From owner-proteins@net.bio.net Thu Dec 08 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!gatech!swrinde!pipex!bnr.co.uk!corpgate!bcarh189.bnr.ca!nott!nrcnet0.nrc.ca!nrcbsa!phi From: phi@nrcbsa.bio.nrc.ca (Dr. Barry Phipps) Newsgroups: bionet.molbio.proteins Subject: Re: Getting Proteins out of the Periplasm Date: 9 Dec 1994 14:04:20 GMT Organization: National Research Council, Canada Lines: 20 Distribution: world Message-ID: <3c9o54$neh@nrcnet0.nrc.ca> References: <3c7s30$kjl@mule.fhcrc.org> NNTP-Posting-Host: nrcbsa.bio.nrc.ca X-Newsreader: TIN [version 1.1 PL8] Tim, Have you tried osmotic shock? It uses EDTA to compromise outer membrane integrity and a sudden change in osmotic pressure (addition of 20% sucrose) to release periplasmic contents. Quite effective. If interested, I can FAX you a protocol and some refs. Barry *************************************************************************** Barry M. Phipps Institute for Biological Sciences National Research Council Building M-54, Montreal Road Ottawa, Ontario CANADA K1A 0R6 E-mail: phi@nrcbsa.bio.nrc.ca (132.246.240.13) Phone: (613) 990-0889 Fax: (613) 941-4475 **************************************************************************** From owner-proteins@net.bio.net Thu Dec 08 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!gatech!swrinde!pipex!uunet!news.fdn.fr!jussieu.fr!univ-lyon1.fr!pasteur.fr!usenet From: Hassan Badrane Newsgroups: bionet.molbio.proteins,bionet.molbio.genome-program Subject: Marrakech CONGRES Cancer & Therapie Date: 9 Dec 1994 14:05:01 GMT Organization: Institut Pasteur, Paris, France Lines: 60 Message-ID: <3c9o6d$s10@montespan.pasteur.fr> NNTP-Posting-Host: mendel.sis.pasteur.fr Xref: biosci bionet.molbio.proteins:3223 bionet.molbio.genome-program:1049 *********************************************** * CONGRES INTERNATIONAL SUR : * * LE CONTROLE MOLECULAIRE DE LA PROLIFERATION * * CELLULAIRE : CANCER ET THERAPIE * * MARRAKECH DU 26 AU 31 MARS 1995 * *********************************************** Ce congres est organise par l'association des biologistes marocains: BIOMATEC, ca se passera a Marrakech (MAROC) du 26 au 31 Mars 1995. Il comportera des conferences, des sessions plenieres, ainsi que des posters sur les sujets suivants: -controle moleculaire de la proliferation, differenciation et developpement. Apoptose, cycle cellulaire, reparation de l'ADN. -facteurs de croissance, cytokines, hormones et recepteurs. transduction des signaux, tyrosines kinases, phosphatases, evenements nucleaires, facteurs de transciptions et de replication. controle de l'expression genetique. -molecules d'adhesion cellulaires, matrice extra-cellulaire, migration, invasion et metastase. -oncogenes, genes suppresseurs et cancer. -therapies : topoisomerases, recepteurs de facteurs de croissance, tyrosines kinases et phosphatases, oncogenes et genes suppresseurs, oligonucleotides anti-sens, therapie genique, immunotherapie. Les conferenciers sont : -Dr Jacques POUSSEGUR -Dr Jean-Jacques LAWRENCE -Dr Jean-Paul THIERY -Dr Yves COURTOIS -Pr Claude HELENE -Dr Alain JACQUEMIN-SABLON -Dr Philippe JEANTEUR -Dr Dominique STEHELIN -Dr Jean-Francois DORE Le comite scientifique est forme par : -Dr Jacques POUSSEGUR -Dr Jean-Jacques LAWRENCE -Dr Jean-Paul THIERY -Pr Claude HELENE -Dr Philippe JEANTEUR -Dr Dominique STEHELIN La langue officielle du congres est le francais. Pour tout renseignement, contacter: Dr Hassan AIT HADDOU ( president de l'association BIOMATEC ) laboratoire de chimie de coordination 205, route de Narbonne 31077 TOULOUSE CEDEX telephone : 61 33 31 84 telecopie : 61 55 30 03 e-mail : aithad@lcctoul.lcc-toulouse.fr ou bien me contacter moi meme (Hassan BADRANE) par e-mail: hbadrane@pasteur.fr MERCI From owner-proteins@net.bio.net Thu Dec 08 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!bloom-beacon.mit.edu!gatech!howland.reston.ans.net!pipex!lyra.csx.cam.ac.uk!sunsite.doc.ic.ac.uk!daresbury!bioftp.unibas.ch!rc1.vub.ac.be!idefix.CS.kuleuven.ac.be!reks.uia.ac.be!news From: przemko@reks.uia.ac.be (Przemko) Subject: Re: Gel database Message-ID: <1994Dec8.122422.19431@reks.uia.ac.be> Sender: news@reks.uia.ac.be (USENET News System) Organization: University of Antwerp X-Newsreader: Date: Thu, 8 Dec 1994 12:24:22 GMT Lines: 32 In article <3biuuo$82@vixen.cso.uiuc.edu>, brazelto@ux1.cso.uiuc.edu (brazelton anthony d) writes: >brazelto@ux1.cso.uiuc.edu (brazelton anthony d) writes: > >s > >>I heard through the grape vine that there was a fancy graphics-oriented >>database program for protein gels. Has anyone heard anything about this? > >>Apparently with this program, one is able to search the database for >>protein candidates given MW or pI for given gel conditions. > >>Any help on elucidating this rumor would be welcome. > >>Thanks, > >>tony > >>Anthony D. Brazelton >>Neuronal Pattern Analysis >>Beckman Institute >>University of Illinois at Urbana-Champaign Hi! Well, there is "nothing" fancy about it. Just check the Expasy server. It is all (and much more) there (AND IT IS FREE!!!): http://expasy.hcuge.ch/ Hope it helps Przemk From owner-proteins@net.bio.net Thu Dec 08 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!bloom-beacon.mit.edu!spool.mu.edu!uwm.edu!lll-winken.llnl.gov!enews.sgi.com!decwrl!netcomsv!netcom.com!cjhixon From: cjhixon@netcom.com (Carl Hixon) Subject: HPLC Capability Question Message-ID: Organization: NETCOM On-line Communication Services (408 261-4700 guest) X-Newsreader: TIN [version 1.2 PL1] Date: Fri, 9 Dec 1994 18:20:14 GMT Lines: 8 My company currently performs ELISAs to determine protein concentration of a particulate suspension of 2 proteins. The question has been raised, "Would it be possible to measure the concentration of these two proteins using an HPLC technique?" Are there any HPLC experts out there who would care to chat about this? Thanks, Carl. From owner-proteins@net.bio.net Thu Dec 08 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!vixen.cso.uiuc.edu!ux1.cso.uiuc.edu!brazelto From: brazelto@ux1.cso.uiuc.edu (brazelton anthony d) Newsgroups: bionet.molbio.proteins Subject: Re: Gel database Date: 9 Dec 1994 19:38:17 GMT Organization: University of Illinois at Urbana Lines: 43 Message-ID: <3cabn9$a2@vixen.cso.uiuc.edu> References: <1994Dec8.122422.19431@reks.uia.ac.be> NNTP-Posting-Host: ux1.cso.uiuc.edu przemko@reks.uia.ac.be (Przemko) writes: >In article <3biuuo$82@vixen.cso.uiuc.edu>, brazelto@ux1.cso.uiuc.edu (brazelton >anthony d) writes: >>brazelto@ux1.cso.uiuc.edu (brazelton anthony d) writes: >> >>s >> >>>I heard through the grape vine that there was a fancy graphics-oriented >>>database program for protein gels. Has anyone heard anything about this? >> >>>Apparently with this program, one is able to search the database for >>>protein candidates given MW or pI for given gel conditions. >> >>>Any help on elucidating this rumor would be welcome. >> >>>Thanks, >> >>>tony >> >>>Anthony D. Brazelton >>>Neuronal Pattern Analysis >>>Beckman Institute >>>University of Illinois at Urbana-Champaign >Hi! >Well, there is "nothing" fancy about it. Just check the Expasy server. It is >all (and much more) there (AND IT IS FREE!!!): >http://expasy.hcuge.ch/ >Hope it helps >Przemk Actually, for netters info, the Expasy sever has many free analytical database programs, but the one I was looking for (Melanie II: a 2D-gel graphic database) is not free but must be purchased from BioRad, who distributes it. tony . From owner-proteins@net.bio.net Fri Dec 09 22:00:00 1994 Path: biosci!KB.USM.MY!asma From: asma@KB.USM.MY (Asma Ismail) Newsgroups: bionet.molbio.proteins Subject: glod colloidal system Date: 10 Dec 1994 21:39:00 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 9 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Hello dear netters, Could anyone explain to how gold colloid conjugated system wokrs!! Currently I am using HRP-conjugated antibody system for ELISA as well as for Dot EIA. I have been told that glod colloid system is much sensitive yet I do not know how this system works. It also stated that, the sensitivity of glod colloidal system could be enhanced via silver staining. Helps needed..... From owner-proteins@net.bio.net Fri Dec 09 22:00:00 1994 Path: biosci!FOX.NSTN.NS.CA!ewright From: ewright@FOX.NSTN.NS.CA ("Edwin Wright") Newsgroups: bionet.molbio.proteins Subject: Sequence Homology Date: 10 Dec 1994 16:15:59 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 19 Sender: daemon@net.bio.net Distribution: world Message-ID: <76807.ewright@fox.nstn.ns.ca> NNTP-Posting-Host: net.bio.net Does anyone know of an example of two proteins, one of which has a sequence length at least twice that of the other, where a significant portion of one sequence is homologous to a significant portion of the other? For example, consider Sequence A = 100 residues, Sequence B = 300 residues, where residues 10 - 40 and 60 - 90 of Sequence A are homologous to residues 120 - 150 and 200 - 230 of Sequence B. Regards, ------- Edwin Wright 85 Spinnaker Drive, A-602 Halifax, Nova Scotia CANADA B3N 3E3 Telephone: (902) 477-5037 Email: ewright@fox.nstn.ns.ca =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= From owner-proteins@net.bio.net Fri Dec 09 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!spool.mu.edu!caen!usenet.cis.ufl.edu!usenet.eel.ufl.edu!news.uoregon.edu!netnews.nwnet.net!news.u.washington.edu!carson.u.washington.edu!rfhoward From: Randall Howard Newsgroups: bionet.molbio.proteins Subject: Peptide acceptors for N-linked glycosylation reactions Date: Fri, 9 Dec 1994 12:03:09 -0800 Organization: University of Washington Lines: 9 Message-ID: NNTP-Posting-Host: carson.u.washington.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII I am searching for a source(s) of small peptides that can function as an oligosaccharide acceptor of the N-linked glycosylation reaction. These peptides would therefore contain the sequence Asn-Xxx-Thr/Ser. They will be used in rabbit reticulocyte lysate-microsome coupled in vitro translation reactions. I would appreciate information about both possible academic and commercial sources. Thank you for any suggestions you have. Randy Howard From owner-proteins@net.bio.net Fri Dec 09 22:00:00 1994 Path: biosci!agate!library.ucla.edu!news.mic.ucla.edu!news.bc.net!vanbc.wimsey.com!unixg.ubc.ca!port47.annex4.net.ubc.ca!user From: seow@unixg.ubc.ca (Kah-Tong, Seow) Newsgroups: bionet.molbio.proteins Subject: Re: Getting Proteins out of the Periplasm Date: Sat, 10 Dec 1994 18:27:38 -0800 Organization: West East Centre, UBC Lines: 40 Distribution: world Message-ID: References: <3c7s30$kjl@mule.fhcrc.org> <3c9o54$neh@nrcnet0.nrc.ca> NNTP-Posting-Host: port47.annex4.net.ubc.ca In article <3c9o54$neh@nrcnet0.nrc.ca>, phi@nrcbsa.bio.nrc.ca (Dr. Barry Phipps) wrote: > Tim, > > Have you tried osmotic shock? It uses EDTA to compromise outer membrane > integrity and a sudden change in osmotic pressure (addition of 20% sucrose) to > release periplasmic contents. Quite effective. If interested, I can FAX you > a protocol and some refs. > > Barry > > > *************************************************************************** > Barry M. Phipps > Institute for Biological Sciences > National Research Council > Building M-54, Montreal Road > Ottawa, Ontario > CANADA K1A 0R6 > E-mail: phi@nrcbsa.bio.nrc.ca (132.246.240.13) > Phone: (613) 990-0889 Fax: (613) 941-4475 > **************************************************************************** Dear Barry, I am interested at the protocol, could I request it. Thank you. Kah-Tong, Seow West East Centre for Microbial Diversity Univ of British Columbia Vancouver. Fax (604) 224-0540 -- Kah-Tong, Seow West East Centre Univ of British Columbia Vancouver seow@unixg.ubc.ca From owner-proteins@net.bio.net Sat Dec 10 22:00:00 1994 Path: biosci!agate!sunsite.doc.ic.ac.uk!daresbury!not-for-mail From: BSS169@BANGOR.AC.UK Newsgroups: bionet.molbio.proteins Subject: Re: HPLC capability question. Date: 11 Dec 1994 15:22:01 -0000 Lines: 23 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <3cf5ep$9m7@mserv1.dl.ac.uk> Original-To: PROTEINS@dl.AC.UK In a message (sorry, I cannot remember its ID), someone asked IF IT'S POSSIBLE TO USE HPLC INSTEAD OF ELISA FOR QUANTITATION OF TWO PROTEINS IN A SUSPENSION. Theoretically saying, Yes, it's possible. If (1) yon can separate the two proteins from other proteins (if any) in suspension, (2) separation results are constant, and (3) you have already got the two proteins in high purity as standards. But I haven't seen any reason why you want to use HPLC instead of ELISA. You have been using ELISA which has advantages over HPLC in specificity, sensi- tivity, efficiency and cost for this task. If somebody asks me to do this job by ELISA for $1, or by HPLC for $50, I will difinitely choose the first one. Dan Wu School of Biol. Sci., Univ. of Wales, Bangor, UK email:bss169@bangor.ac.uk From owner-proteins@net.bio.net Sun Dec 11 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!swrinde!pipex!uunet!news.uiowa.edu!panda From: S. Fredricksen Newsgroups: bionet.molbio.proteins Subject: Re: Sequence Homology Date: 12 Dec 1994 23:31:46 GMT Organization: U of Iowa Panda System Lines: 26 Distribution: world Message-ID: <787275426-0-143314@blue.weeg.uiowa.edu> References: <76807.ewright@fox.nstn.ns.ca> Reply-To: sfredric@blue.weeg.uiowa.edu NNTP-Posting-Host: blue.weeg.uiowa.edu In note <76807.ewright@fox.nstn.ns.ca>, ewright@FOX.NSTN.NS.CA ("Edwin Wright") writes: >Does anyone know of an example of two proteins, one of which has a sequence >length at least twice that of the other, where a significant portion of one >sequence is homologous to a significant portion of the other? > >For example, consider Sequence A = 100 residues, Sequence B = 300 residues, >where residues 10 - 40 and 60 - 90 of Sequence A are homologous to residues >120 - 150 and 200 - 230 of Sequence B. > >Regards, >------- >Edwin Wright >85 Spinnaker Drive, A-602 >Halifax, Nova Scotia >CANADA B3N 3E3 > >Telephone: (902) 477-5037 > >Email: ewright@fox.nstn.ns.ca >=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= You might consider troponin C or calmodulin along with their corresponding globular domains. In addition to internal homology between, say, the 2 globular domains of troponin C, the two holo-proteins are very similar. Good Luck, Scott From owner-proteins@net.bio.net Sun Dec 11 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!gatech!swrinde!pipex!uunet!zib-berlin.de!informatik.tu-muenchen.de!lrz-muenchen.de!ipp-garching.mpg.de!alf.biochem.mpg.de!krasel From: krasel@alf.biochem.mpg.de (Cornelius Krasel) Newsgroups: bionet.molbio.proteins Subject: Re: Getting Proteins out of the Periplasm Date: 12 Dec 1994 21:15:14 GMT Organization: Rechenzentrum der Max-Planck-Gesellschaft in Garching Lines: 22 Distribution: world Message-ID: <3cieh2$2pbd@sat.ipp-garching.mpg.de> References: <3c7s30$kjl@mule.fhcrc.org> NNTP-Posting-Host: alf.biochem.mpg.de X-Newsreader: TIN [version 1.2 PL2] Tim Buss (tbuss@fred.fhcrc.org) wrote: > I'm expressing humanized antibody fragments in E.coli. The proteins are > concentrated into the periplasm and I'm looking for a good technique to > get them out, with minimal cell disruption. Many protocols make use of > lysozyme, but I cannot use this (it's an anti-lysozyme Fv). I am also > trying to avoid low pH as I think this may well disrupt the fragment. The following method is originally from the Plueckthun lab and was used for purification of a Fv fragment of McPC603 expressed in JM83: Add 2 ml / g wet cells of a solution of 1 mM EDTA, 160 mM NaCl, 200 mM boric acid (or was it borate? Can't recall ...), pH 8 with NaOH and stir for at least 30 min at 4 C. Centrifuge at 20000 rpm in a SS34 at 4 C. Filter supernatant through 0.4 um filter. You can leave the filtrate overnight at 4 C with no loss of antibody fragment. --Cornelius. -- /* Cornelius Krasel, Abt. Lohse, Genzentrum, D-82152 Martinsried, Germany */ /* email: krasel@alf.biochem.mpg.de fax: +49 89 8578 3795 */ /* "Science is the game you play with God to find out what His rules are." */ From owner-proteins@net.bio.net Sun Dec 11 22:00:00 1994 Path: biosci!daresbury!sunsite.doc.ic.ac.uk!pipex!oleane!jussieu.fr!univ-lyon1.fr!zaphod.crihan.fr!news.univ-rennes1.fr!univ-tours.fr!BOISSET From: boisset@univ-tours.fr Newsgroups: bionet.molbio.proteins Subject: PHOSPHORYLASE KINASE Date: 12 Dec 1994 17:49:22 GMT Organization: University Francois Rabelais - TOURS FRANCE Lines: 27 Message-ID: <3ci2f2$7g8@news.univ-rennes1.fr> Reply-To: BOISSET@univ-tours.fr NNTP-Posting-Host: rablai.univ-tours.fr )------------------------------------------------------------------------------( COLLABORATION ON PHOSPHORYLASE KINASE: IF INTERESTED CONTACT ME AT : Dr. Nicolas Boisset Laboratoire des Proteines Complexes URA CNRS 1334, Universite F. Rabelais 2 bis Boulevard Tonnelle 37032 Tours Cedex FRANCE Tel: (33) 47-37-66-84 Fax: (33) 47-36-61-29 email: BOISSET@UNIV-TOURS.FR Dear Netters, Does anyone working on the Biochemistry of the PHOSPHORYLASE KINASE would be interested into a collaboration on its quaternary structure or overall architecture? I am doing 3D reconstructions by cryoelectron microscopy and image processing on isolated particles and can study large macromolecular complexes with a resolution ca 30 Angstroems. Best regards, Nicolas Boisset From owner-proteins@net.bio.net Sun Dec 11 22:00:00 1994 Path: biosci!MITVMA.MIT.EDU!W_SHAO%UVMVAX.BITNET From: W_SHAO%UVMVAX.BITNET@MITVMA.MIT.EDU ("Wei Shao\", ADDRESS: IN%\"w_shao@uvmvax.uvm.edu") Newsgroups: bionet.molbio.proteins Subject: signoff Date: 12 Dec 1994 13:36:50 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 1 Sender: daemon@net.bio.net Distribution: world Message-ID: <01HKK9P7MDGG000JTX@uvmvax.uvm.edu> NNTP-Posting-Host: net.bio.net Signoff From owner-proteins@net.bio.net Sun Dec 11 22:00:00 1994 Path: biosci!sb.com!Seth_M_Fisher%notes From: Seth_M_Fisher%notes@sb.com (Seth M Fisher) Newsgroups: bionet.molbio.proteins Subject: re: Amino Acid Question Date: 12 Dec 1994 06:56:51 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 13 Sender: daemon@net.bio.net Distribution: world Message-ID: <9412121756.AA1275@pho018.sb.com> NNTP-Posting-Host: net.bio.net dyan writes: >Now I'm told that the notion of amino acid combinations was bogus, made up >by Adele Davis who wrote 'Diet for a Small Planet' and that one does not >need the complement of amino acids. I find this hard to believe - since >they'd worked out the structure of protein decades before my texts were >written, I find it hard to believe they got it so wrong in 1980. Francis Moore Lappe wrote 'Diet for a Small Planet' not Adele Davis. Who told you it was bogus? You can experiment by eating only rice as a source of protein and seeing what happens. From owner-proteins@net.bio.net Sun Dec 11 22:00:00 1994 Path: biosci!agate!howland.reston.ans.net!pipex!oleane!jussieu.fr!univ-lyon1.fr!cri.ens-lyon.fr!mac-adp.ibcp.fr!user From: adp@ibcp.fr (adp@ibcp.fr) Newsgroups: bionet.molbio.proteins Subject: XXII FORUM OF YOUNG INVESTIGATORS - French SFBBM Followup-To: bionet.molbio.proteins Date: Mon, 12 Dec 1994 15:20:58 +0000 Organization: Institut de Chimie et Biologie des Proteines - France Lines: 65 Message-ID: NNTP-Posting-Host: mac-adp.ibcp.fr Mime-Version: 1.0 Content-Type: text/plain; charset=ISO-8859-1 Content-Transfer-Encoding: 8bit Newsgroup: From: forumsfbbm@ibcp.fr Subject: 22nd Forum of young investigators - French society of biochemistry and molecular biology ******************************************************** XXIInd FORUM OF YOUNG INVESTIGATORS- FRENCH SOCIETY OF BIOCHEMISTRY AND MOLECULAR BIOLOGY ____________________ GRENOBLE (France) 4-7 July 1995 ******************************************************** The 2nd Forum of young investigators will be held in Grenoble the 4-7 july 1995. Every year, young researchers (less than 35 years old) in the fields of biochemistry, molecular biology and cellular biology, meet to exchange their technical and scientific views at this multidisciplinary congress. Participants belong to diverse horizons. They must be less than 35 years old, pre- or post-doctoral students or have recently taken a fixed position, industrials, medical or pharmaceutical researchers. Most of them are French-speaking researchers, but other European or non-European researchers are welcome. One of the most prominent feature of the Forum consists in giving the younger researchers priority over oral communications, which are highly recommended. This year, participants with oral communications should be below 35 years old and will speek for 15 minutes (plus 5 minutes discussion). For infrastructure reasons, the number of accepted participants will be reduced this year. Registration fees should be about 500 FF for a young researcher below 35 and member of the French SFBBM. ********************************************************* PRE-REGISTRATION FORM This form should be sent together with the summary of your communication (in triplicate) PRIOR TO MARCH 1st, 1995. (See address below). Registrations will not be taken into account after this deadline. FAMILY NAME_______________________FIRST NAME_______________ ADDRESS__________________________________________________ _________________________________________________________ _________________________________________________________ ZIP CODE___________CITY________________COUNTRY_____________ TELEPHONE______________________FAX_______________________ DATE OF BIRTH_________________ PROFESSIONNAL STATUTE_____________________________________ COMMUNICATION : ( ) ORAL ( ) POSTER WORSHOPS AND ROUND TABLES (mark the themes that best fit to your work) ( ) Rational conception of therapeutic drugs (O. Diedberg) ( ) Micro-analysis of proteic sample (J. Gagnon & J. Garin) ( ) Chemical models of enzymes (M. Fontecave) ( ) Signal transduction in bacteria (P.M. Vignais) ( ) Radiopharmaceutical markers ( M. Comet) ( ) Endocytosis ( M. Satre) ( ) HIV (J-M. Seigneurin) ( ) Molecular cytogenetics (P. Jalbert) ( ) Monomer G-proteins (P.V. Vignais) ( ) Hormones and plant development ( M. Herzog) ( ) Metabolic NMR ( R. Douce) ( ) Dynamics of cellular adhesion (M. Block) ********************************************************* Co-ordinator: Dr Jacques COVES - XXII Forum des Jeunes Chercheurs - Universite Joseph Fourier - LEDSS 5 - Chimie Recherches - BP 53 - F-38041 GRENOBLE CEDEX 9 - Tel (33) 76 63 57 56 - Fax (33) 76 51 43 82 - E-mail coves@ledss.grenet.fr From owner-proteins@net.bio.net Sun Dec 11 22:00:00 1994 Path: biosci!agate!howland.reston.ans.net!pipex!uunet!comp.vuw.ac.nz!waikato!status.gen.nz!iconz.co.nz!dyan From: dyan@iconz.co.nz (Dyan Campbell) Newsgroups: bionet.molbio.proteins Subject: Amino Acid Question Date: 12 Dec 1994 11:49:28 GMT Organization: Public Access Internet, Auckland New Zealand Lines: 27 Message-ID: <3chdc8$d37@status> NNTP-Posting-Host: iconz.co.nz X-Newsreader: TIN [version 1.2 PL2] Hello, this is a really basic question from a layperson - there is a running argument in the cooking newsgroup about amino acid combinations, and available protein. I'm hoping someone here can help settle it. The information I have may be out of date - I took a couple years' sciences in college, and at that time I think we were taught that humans require 8 of the 20 amino acids (since the other 12 can be synthesised) and infants require 9. (This information would have been about '79-'80) In the text books I still have, (i.e. Keeton - Biological Science) it says that neither beans nor rice on their own do not supply the full range of amino acids, but in combination do make up the full complement of amino acids for complete protein. Now I'm told that the notion of amino acid combinations was bogus, made up by Adele Davis who wrote 'Diet for a Small Planet' and that one does not need the complement of amino acids. I find this hard to believe - since they'd worked out the structure of protein decades before my texts were written, I find it hard to believe they got it so wrong in 1980. Can anyone she any light on the controversy regarding beans and rice?!? Thanks in advance cheers dyan From owner-proteins@net.bio.net Sun Dec 11 22:00:00 1994 Path: biosci!agate!howland.reston.ans.net!pipex!sunsite.doc.ic.ac.uk!daresbury!imbb1.imbb.forth.gr!nefeli.imbb.forth.gr!SKOUFOS From: skoufos@nefeli.imbb.forth.gr Newsgroups: bionet.molbio.proteins Subject: Re: Sequence Homology Date: Mon, 12 Dec 1994 18:51:23 GMT Organization: Imbb-forth,CREtE, creece Lines: 26 Distribution: world Message-ID: <00988D4F.EA27F659@nefeli.imbb.forth.gr> References: <76807.ewright@fox.nstn.ns.ca> Reply-To: skoufos@nefeli.imbb.forth.gr NNTP-Posting-Host: nefeli.imbb.forth.gr In article <76807.ewright@fox.nstn.ns.ca>, ewright@FOX.NSTN.NS.CA ("Edwin Wright") writes: >Does anyone know of an example of two proteins, one of which has a sequence >length at least twice that of the other, where a significant portion of one >sequence is homologous to a significant portion of the other? > >For example, consider Sequence A = 100 residues, Sequence B = 300 residues, >where residues 10 - 40 and 60 - 90 of Sequence A are homologous to residues >120 - 150 and 200 - 230 of Sequence B. One example I can think of is that of _Drosophila_ Delta and of _C. elegans_ Lag-2. Eventhough Delta is more that twice the size of Lag-2, they share at least two large domains of high degree of similarity. The functions of the two proteins are homologues (i.e. they are probably both ligands to similar receptors; Notch and GLP-1 respectively). The highly similar domains are probably homologous but the two proteins probably are not. Emmanuel Emmanuel Skoufos, Ph.D. Insect Molecular Biology Group Institute of Molecular Biology and Biotechnology P. O. Box 1527 Heraklion, Crete GR71110 Greece From owner-proteins@net.bio.net Sun Dec 11 22:00:00 1994 Path: biosci!agate!sunsite.doc.ic.ac.uk!lyra.csx.cam.ac.uk!dh124 From: dh124@cus.cam.ac.uk (D. Hodges) Newsgroups: bionet.molbio.proteins Subject: Re: Getting Proteins out of the Periplasm Date: 12 Dec 1994 08:20:47 GMT Organization: University of Cambridge, England Lines: 25 Distribution: world Message-ID: <3ch14v$b3l@lyra.csx.cam.ac.uk> References: <3c7s30$kjl@mule.fhcrc.org> NNTP-Posting-Host: grus.cus.cam.ac.uk X-Newsreader: TIN [version 1.2 PL2] Tim Buss (tbuss@fred.fhcrc.org) wrote: : I'm expressing humanized antibody fragments in E.coli. The proteins are : concentrated into the periplasm and I'm looking for a good technique to : get them out, with minimal cell disruption. Many protocols make use of : lysozyme, but I cannot use this (it's an anti-lysozyme Fv). I am also : trying to avoid low pH as I think this may well disrupt the fragment. : Any tried and tested methods you folks would be willing to share???? : Tim. The method I've used is to resuspend the cells in 0.5M sucrose, 0.1M tris 1mM EDTA, pH 8.2. Add an equal volume of ice cold H2O. Leave on ice for 5 min. Then add MgSO4 to 20 mM (optional). Centrifuge at 10,000 rpm for 20 minutes and the supernatant should be mostly periplasm. If you repeat it, you can get a bit more out, but the cells start to lyse. It works but I don't know how it compares with other methods. Good luck Debbie -- --------------------------------------------------------------------------- Debbie Hodges dh124@cus.cam.ac.uk Fax 0223 217838 Rheumatology Research Unit, Addenbrookes Hospital, Hills Road, Cambridge, UK. ---------------------------------------------------------------------------- I never could get the hang of Thursdays (A.Dent). From owner-proteins@net.bio.net Sun Dec 11 22:00:00 1994 Path: biosci!agate!howland.reston.ans.net!pipex!sunsite.doc.ic.ac.uk!daresbury!bioftp.unibas.ch!rc1.vub.ac.be!imol2.vub.ac.be!thomas From: thomas@ultr5.vub.ac.be (Thomas hamelryck) Newsgroups: bionet.molbio.proteins Subject: Where is jellyroll in Con A ? Date: 12 Dec 1994 13:03:53 GMT Organization: Instituut voor Moleculaire Biologie en Biotechnologie - VUB Lines: 15 Message-ID: <3chhnq$b6k@imol2.vub.ac.be> NNTP-Posting-Host: ultr5.vub.ac.be X-Newsreader: TIN [version 1.2 PL2] The lectin Concanavalin A is often reported having a jellyroll supersecondary structure. But I can't see how this jellyroll motif could be present when I look at the topology of Concanavalin A. According to me, Concanavalin A simply does not posses a jellyroll fold, but a beta-sandwich fold. I think the mistake emerged because a *different* protein from Canavalia ensiformis, canavalin, *does* have a jellyroll fold. The similarity between the two names could have caused the mistake. Am I right, or am I wrong ? Has anyone got a different opinion, or has anyone noticed the mistake before ? -Thomas. From owner-proteins@net.bio.net Sun Dec 11 22:00:00 1994 Path: biosci!newshost.lanl.gov!news.ttu.edu!aurora.LaTech.edu!darwin.sura.net!ukma!occ!ndlc.occ.uky.edu!daviseg From: daviseg@ndlc.occ.uky.edu (Eric Davis) Newsgroups: bionet.molbio.proteins Subject: BIOLOGY IMAGES Date: 12 Dec 1994 22:43:39 GMT Organization: The NDLC's Internet Gateway Lines: 5 Message-ID: <3cijmr$ac6@sparc.occ.uky.edu> NNTP-Posting-Host: ndlc.occ.uky.edu X-Newsreader: TIN [version 1.2 PL2] Does anyone know where I could find GIF's or JPEG's of biology-related images? Thank you. Eric Davis From owner-proteins@net.bio.net Sun Dec 11 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!gatech!howland.reston.ans.net!vixen.cso.uiuc.edu!news.uoregon.edu!gaia.ucs.orst.edu!owl.csrv.uidaho.edu!raven.csrv.uidaho.edu!mahad881 From: mahad881@raven.csrv.uidaho.edu (Mahadevan Brinda) Newsgroups: bionet.molbio.proteins Subject: rotofer problems and questions Date: 6 Dec 1994 18:16:52 GMT Organization: University of Idaho, Moscow, Idaho Lines: 5 Distribution: world Message-ID: <3c29qk$cuo@owl.csrv.uidaho.edu> NNTP-Posting-Host: raven.csrv.uidaho.edu X-Newsreader: TIN [version 1.2 PL2] I have been having problems with the 18ml chamber of the Biorad rotofer unit. Anybody has some suggestions or comments on the same? The difficulties I have been facing are voltage fluctuations, bubbles and low recovery of purified protein. From owner-proteins@net.bio.net Sun Dec 11 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!gatech!howland.reston.ans.net!news.sprintlink.net!bga.com!news From: cmwalden@bga.com Reply-To: cmwalden@bga.com NNTP-Posting-Host: edwin-b4.aip.realtime.net X-Newsreader: WinVN 0.92.6+ I know that this doesn't fit in with any discussions-- but I'm not sure were else to put it. I'm trying to locate John Priestle. He works in Switzerland with protien synthesis. I know that he has been recognized for some software that graphically represents protiens. If anyone knows his e-mail address, or could suggest a better place to search, I'd appreciate it. Chris or cmwalden@bga.com From owner-proteins@net.bio.net Sun Dec 11 22:00:00 1994 Path: biosci!agate!garnet.berkeley.edu!owens From: owens@garnet.berkeley.edu () Newsgroups: bionet.molbio.proteins Subject: protein volumes? Date: 12 Dec 1994 23:03:40 GMT Organization: University of California, Berkeley Lines: 12 Message-ID: <3ciksc$cid@agate.berkeley.edu> NNTP-Posting-Host: garnet.berkeley.edu hi- does anyone know of a source for protein volumes in the crystalline form? (i.e. an online database maybe?) densities would work also, except that any error would be multiplied massively when I multiply by MW. thanks for any help you can give me! christine owens owens@garnet.berkeley.edu From owner-proteins@net.bio.net Mon Dec 12 22:00:00 1994 Path: biosci!MANI.CBS.UMN.EDU!npv From: npv@MANI.CBS.UMN.EDU (Nora Plesofsky-Vig) Newsgroups: bionet.molbio.proteins Subject: electrophoretic resolution of 25 kDa protein Date: 13 Dec 1994 14:24:15 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 15 Sender: daemon@net.bio.net Distribution: world Message-ID: <9412132222.AA00899@mani.cbs.umn.edu> Reply-To: nora@molbio.cbs.umn.edu NNTP-Posting-Host: net.bio.net Has anyone had experience with resolving proteins in the 25 kDa range by SDS gel electrophoresis? Specifically, would adding urea (6 or 8 M) to the gel and/or sample increase the sharpness of the protein bands? Would the tricene gel system that is used for smaller proteins help to separate proteins in the 25 kDa range? I am trying to isolate a minor protein that binds to a glutathione-S-transferase fusion protein, and I need to get bands that are as sharp as possible. If you have any relevant experience with electrophoretic systems that you have found to be useful, I would appreciate hearing about your results. Thanks. Nora Plesofsky-Vig nora@molbio.cbs.umn.edu From owner-proteins@net.bio.net Mon Dec 12 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!vixen.cso.uiuc.edu!newsrelay.iastate.edu!gw1.phibred.com!dalmiabk.phibred.com!dalmiabk From: bipin dalmia Subject: Re: Storage of purified proteins Message-ID: X-Xxmessage-Id: X-Xxdate: Tue, 13 Dec 94 16:06:10 GMT Sender: news@phibred.com (USENET News System) Organization: pioneer hi-bred X-Useragent: Version 1.1.3 References: <9412132042.AB12297@usa.net> Date: Tue, 13 Dec 1994 22:12:23 GMT Lines: 26 In article <9412132042.AB12297@usa.net> Gene Holowachuk, geneh@USA.NET writes: >Can anyone suggest some guidelines for the storage of highly purified >recombinantly produced proteins. We have purified a couple of cytokines >produced in E. coli, about 1 mg/l. What are the best ways for short term >and long term storage - 50% glycerol, lyophilized, -70 oC, etc. We have >found loss of activity when stored at 4 oC for longer than 1 week. Any >suggestions will be appreciated. Cheers :-)) i use the following concoction for my purified proteins. should work with most proteins. 50 mM Tris-HCl, pH 7.5 50 % Glycerol 250 mM NaCl 1 mM EDTA 1 mM DTT 1 mM PMSF 0.1 % tween-20 (or triton x-100) 0.05 % NaN3 divide in small aliquots and store at -80 C. flash freezing may help but is not critical with the 50 % glycerol. for shorter-term storage, use the same mixture as above but store at -20 C. bip From owner-proteins@net.bio.net Mon Dec 12 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!news.moneng.mei.com!uwm.edu!lll-winken.llnl.gov!enews.sgi.com!decwrl!netcomsv!netcom.com!brom From: brom@netcom.com (Brad Romney) Subject: FAQ Message-ID: Organization: NETCOM On-line Communication Services (408 261-4700 guest) X-Newsreader: TIN [version 1.2 PL1] Date: Tue, 13 Dec 1994 21:17:49 GMT Lines: 9 Is there a FAQ with the bionet USENET groups? Maybe a FAQ of common favorite protocols that readers find useful. Brad -- From owner-proteins@net.bio.net Mon Dec 12 22:00:00 1994 Path: biosci!USA.NET!geneh From: geneh@USA.NET (Gene Holowachuk) Newsgroups: bionet.molbio.proteins Subject: Storage of purified proteins Date: 13 Dec 1994 12:42:51 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 16 Sender: daemon@net.bio.net Distribution: world Message-ID: <9412132042.AB12297@usa.net> NNTP-Posting-Host: net.bio.net Can anyone suggest some guidelines for the storage of highly purified recombinantly produced proteins. We have purified a couple of cytokines produced in E. coli, about 1 mg/l. What are the best ways for short term and long term storage - 50% glycerol, lyophilized, -70 oC, etc. We have found loss of activity when stored at 4 oC for longer than 1 week. Any suggestions will be appreciated. Cheers :-)) =========================================================================== ** Gene Holowachuk | Voice: 607/547-3937 ** ** Research Institute | Fax : 607/547-3061 (attn Gene H) ** ** Mary Imogene Bassett Hospital | email: geneh@research.mibh.org ** ** One Atwell Road | : geneh@usa.net ** ** Cooperstown, NY 13326 | : geneh@cscns.com ** =========================================================================== From owner-proteins@net.bio.net Mon Dec 12 22:00:00 1994 Path: biosci!adam.cc.sunysb.edu!citlab.bio.sunysb.edu!user From: ghoshroy@mcbsgi.bio.sunysb.edu (Soumitra Ghoshroy) Newsgroups: bionet.cellbiol,bionet.molbio.proteins,bionet.plants Subject: Plant Phosphatases Date: Tue, 13 Dec 1994 15:21:29 -0500 Organization: SUNY Stony Brook Lines: 9 Message-ID: NNTP-Posting-Host: citlab.bio.sunysb.edu Xref: biosci bionet.cellbiol:1265 bionet.molbio.proteins:3255 bionet.plants:4682 I need some general information about plant phosphatases (especially ATPases). Does anyone know of a good review on phosphatases specifically from plants? Are there any groups who work on them primarily? Any genes cloned? Information pertaining to cellular location (i.e. membrane-bound, cytosolic, etc.) would be especially helpful. Respond by posting or via e-mail, as you choose. Thanks in advance. James Gergel jgergel@mcbsgi.bio.sunysb.edu From owner-proteins@net.bio.net Mon Dec 12 22:00:00 1994 Path: biosci!agate!boulder!bloom-beacon.mit.edu!uhog.mit.edu!europa.eng.gtefsd.com!howland.reston.ans.net!news.cac.psu.edu!news.pop.psu.edu!psuvax1!news.cc.swarthmore.edu!mac04.lang.swarthmore.edu!user From: ehom1@cc.swarthmore.edu (Erik Forbes Y. Hom) Newsgroups: bionet.molbio.proteins Subject: Q: about "Look" structural modeling package Date: 13 Dec 1994 18:04:07 GMT Organization: Swarthmore College, Swarthmore PA, USA Lines: 21 Message-ID: NNTP-Posting-Host: mac04.lang.swarthmore.edu Mime-Version: 1.0 Content-Type: text/plain; charset=US-ASCII Content-Transfer-Encoding: 7bit Hi netters, has/does anyone here use the 3D "segment-matching" based structural prediction package for proteins called "Look"? (based on Stanford's Michael Levitt's work, among others). If so, what do you think about it? What do you use it for and how does the package deliver? Any comments on this would be much appreciated. Thanks in advance for your help. Yours, erik -- Erik Forbes Y. Hom '95 Laboratory of Neurobiology Swarthmore College 500 College Avenue Swarthmore, PA 19081-1397, USA Lab: 610-328-7788 Dorm: 610-690-5689 ehom1@cc.swarthmore.edu (or erikhom@sccs.swarthmore.edu) From owner-proteins@net.bio.net Mon Dec 12 22:00:00 1994 Path: biosci!agate!howland.reston.ans.net!pipex!uunet!dziuxsolim.rutgers.edu!er7.rutgers.edu!not-for-mail From: alkaiser@er7.rutgers.edu (Alan Kaiser) Newsgroups: bionet.molbio.proteins Subject: Re: rotofer problems and questions Date: 13 Dec 1994 12:09:39 -0500 Organization: Rutgers University Lines: 45 Distribution: world Message-ID: <3ckkgj$34u@er7.rutgers.edu> References: <3c29qk$cuo@owl.csrv.uidaho.edu> NNTP-Posting-Host: er7.rutgers.edu In <3c29qk$cuo@owl.csrv.uidaho.edu> mahad881@raven.csrv.uidaho.edu (Mahadevan Brinda) writes: >I have been having problems with the 18ml chamber of the Biorad rotofer >unit. Anybody has some suggestions or comments on the same? The >difficulties I have been facing are voltage fluctuations, bubbles and low >recovery of purified protein. I have also experienced these same problems when using the 18ml Rotofor cell. I found it almost impossible to remove all of the air from the cell alfter loading the sample. This is probably the cause of the voltage fluctuations that you have seen. The low recovery of your protein may also be related to this problem since the voltage fluctuations will tend to cause the pH gradient to not form properly. To help you answer the question about recovery ask yourself these two questions 1. What was the final voltage when you collected the sample from the cell and was the voltage stable for at least a half hour before you collected it? and 2. Is there any way that you can check each resulting fraction for activity (despite the pH differences). I would try dialyzing the fractions to the optimal pH of your protein and then check for activity to see where your protein is. My guess is that you are getting low recovery due to the fact that the pH gradient has not formed properly. Therefore, your protein has not focused at its pI and when you collect the fractions you are only seeing a fraction of your protein at the expected pI. To get around the bubble problem I ran two 60ml runs and pooled the active fractions around the pI of interest (I'm lucky, I can see activity at almost any pH). Dilute the pooled active fractions to a volume sufficient to load the sample back into the 60ml cell and run that. I had no problems when I tried this approach but my starting sample is easy to obtain. What is your starting sample? The reason I ask is that the voltage fluctuations may in part be due to a high salt concentration. I had to dialyze my sample against water before I could get it to work well. Are you losing protein due to precipitation? This would account for the low recovery. I had this problem (it's common around the pI) and I got around it by using 4M urea and 15% glycerol. This solved my precipitation problems. Anyway, I hope this helps. Let me know how things turn out. From owner-proteins@net.bio.net Mon Dec 12 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!agate!dog.ee.lbl.gov!news.cs.utah.edu!emba-news.uvm.edu!brianf From: brianf@med.uvm.edu (Brian Foley) Subject: Re: Amino Acid Question Message-ID: <1994Dec13.160513.18694@emba.uvm.edu> Sender: news@emba.uvm.edu Organization: EMBA Computer Facility, University of Vermont X-Newsreader: TIN [version 1.2 PL1] References: <3chdc8$d37@status> Date: Tue, 13 Dec 1994 16:05:13 GMT Lines: 128 Dyan Campbell (dyan@iconz.co.nz) wrote: : Hello, this is a really basic question from a layperson - there is a : running argument in the cooking newsgroup about amino acid combinations, : and available protein. I'm hoping someone here can help settle it. : ... some deleted : In the text books I still have, (i.e. Keeton - Biological Science) it : says that neither beans nor rice on their own do not supply the full range : of amino acids, but in combination do make up the full complement of : amino acids for complete protein. : Can anyone she any light on the controversy regarding beans and rice?!? : dyan All plants animals and bacteria have at least some of all 20 amino acids in their proteins. However, beans as rice have "major seed storage proteins" which are stored in these seeds for the purpose of nourishing the seedling. The storage proteins thus make up the bulk of the protein found in these seeds (there is also a lot of starch) and these proteins do not contain "significant amounts" of all 20 amino acids. The amino acids that are under-represented in rice are present in beans, and the amino acids which are under-represented in beans are present in rice. I have not checked this out for myself, this is a layman's explanation. But you could look up the protein sequences of "major seed storage proteins" for beans and rice and check it out if you really need first hand proof. Oh, what the heck... here are a couple of rice and bean major seed storage proteins, see for yourself: Definition 19 kDa globulin precursor Protein Name: 19 kDa globulin precursor gi|20159: [ Whole ] NCBI Seq ID: 20159 Citation REF [1] Shorrosh,B.S., Wen,L., Zen,K.C., Huang,J.K., Pan,J.S., Hermodson,M.A., Tanaka,K., Muthukrishnan,S. & Reeck,G.R. (1992). A novel cereal storage protein: molecular genetics of the 19 kDa globulin of rice. Plant Mol. Biol. 18, 151-154. MEDLINE identifier: 92119226 Organism Oryza sativa (rice) Method conceptual translation Coding region gi|20158: 29..589 Sequence 186 aa 1 maskvvffaa almaamvais gahvsesemr frdrqcqrev qdspldacrq 51 vldrqltgre rfqpmfrrpg alglrmqccq qlqdvsrecr caairrmvrs 101 yeesmpmple qgwsssssey yggegssseq gyygegssee gyygeqqqqp 151 gmtrvrltra rqyaaqlpsm crvepqqcsi faagqy Definition glutelin NCBI Seq ID: 20208 Citation REF [1] Data Submission: Fumio,T. (1990). Citation REF [2] Takaiwa,F. & Oono,K. (1991). Genomic DNA sequences of two new genes for new storage protein glutelin in rice. Jpn. J. Genet. 66, 161-171. Organism Oryza sativa (rice) Method conceptual translation Coding region gene: GluA-3. gi|20207: 985..1312 gi|20207: 1393..1667 gi|20207: 1779..2258 gi|20207: 2336..2743 Sequence 496 aa 1 matikfpivf fvvclfllcn gslaqllsqs tsqwqssrrg sprecrfdrl 51 qafepirtvr sqagtteffd vsnelfqctg vfvvrrviep rglllphysn 101 gatlvyviqg rgitgptfpg cpetyqqqfq qseqdqqleg qsqshkfrde 151 hqkihrfqqg dvvalpagva hwcyndgdap ivaiyvtdiy nsanqldprh 201 rdfflagnnk igqqlyryea rdnsknvfgg fsvellseal gissgvarql 251 qcqndqrgei vrvehglsll qpyaslqeqq qeqvqsrdyg qtqyqqkqlq 301 gscsngldet fctmrvrqni dnpnladtyn pragrityln gqkfpilnlv 351 qmsavkvnly qnallspfwn inahsvvyit qgrarvqvvn nngktvfdge 401 lrrgqlliip qhhvvikkaq regcsyialk tnpdsmvshm agknsifral 451 pddvvanayr isreearrlk hnrgdelgvf tpshayksyq disvsa Definition phaseolin Citation REF [2] Data Submission: Kami,J.A. (1993). Organism Phaseolus lunatus (lima bean) Method conceptual translation Coding region partial: . gi|403583: 1..1273 function: seed storage protein. gene: Phs. * Partial Sequence 423 aa 1 rrvpllllgi lflaslsasf aislrehnes qdnpfyfssd nswqtlfknq 51 yghirvlqrf dqrserlqnl edyrlvefms kpetlllpqq adaefllvvr 101 sgsallalvk pggtiiyslk qqdtlkipag tifflinpen nedlriikla 151 mtvnnpqiqd fflssteaqq sylygfskhi ldasfnspie kinrllfaee 201 grqegvivni gsdliqelsr haksssrksl dhnsldisne wgnltdivyn 251 sldvlltyve ikegglfvph ynskaivilv veegvakvel vgpkrekesl 301 eletyradvs egdvfvipaa fpfaikaisn vnftsfgina nnnyrifltg 351 kggptgkedn iisaginpdv lglmfpgsge dvqklfnnqn lshfvngsyh 401 knahpheqeq qkqqkgrkga fvy Definition phaseolin Protein Name: phaseolin gi|403596: [ Whole ] NCBI Seq ID: 403596 Citation REF [1] [11] Citation REF [2] Data Submission: Kami,J.A. (1993). Organism Phaseolus vulgaris (French bean) Method conceptual translation Coding region function: seed storage protein. gi|403595: 1..1293 gene: Phs. Sequence 430 aa 1 mmrarvplll lgilflasls asfatslree eesqdnpfyf nsdnswntlf 51 knqyghirvl qrfdqqskrl qnledyrlve frskpetlll pqqadaelll 101 vvrsgsailv lvkpddrrey ffltsdnpif sdhqkipagt ifylvnpdpk 151 edlriiqlam pvnnpqihdf flssteaqqs ylqefskhil easfnskfee 201 inrvlfaeeg qqegvivnid seqieelskh aksssrksls kqdntignef 251 gnltertdns lnvlissmem kegalfvphy yskaivilvv negeahvelv 301 gpkgnketle fesyraelsk ddvfvipaay pvaikatsnv nftgfginan 351 nnnrnllagk tdnvissigs aldgkdvlgl tfsgsgeevm klinkqsgsy 401 fvdghhhqqe qqkgshqqeq qkgrkgafvy a = Alanine c = Cysteine and so on... -- ******************************************************************** * Brian Foley * If we knew what we were doing * * Molecular Genetics Dept. * it wouldn't be called research * * University of Vermont * * ******************************************************************** From owner-proteins@net.bio.net Mon Dec 12 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!agate!dog.ee.lbl.gov!news.cs.utah.edu!emba-news.uvm.edu!brianf From: brianf@med.uvm.edu (Brian Foley) Subject: Re: Sequence Homology Message-ID: <1994Dec13.153538.18098@emba.uvm.edu> Sender: news@emba.uvm.edu Organization: EMBA Computer Facility, University of Vermont X-Newsreader: TIN [version 1.2 PL1] References: <76807.ewright@fox.nstn.ns.ca> Date: Tue, 13 Dec 1994 15:35:38 GMT Lines: 60 Edwin Wright (ewright@FOX.NSTN.NS.CA) wrote: : Does anyone know of an example of two proteins, one of which has a sequence : length at least twice that of the other, where a significant portion of one : sequence is homologous to a significant portion of the other? : For example, consider Sequence A = 100 residues, Sequence B = 300 residues, : where residues 10 - 40 and 60 - 90 of Sequence A are homologous to residues : 120 - 150 and 200 - 230 of Sequence B. It depends on what you mean by "homologous". If you really mean "has sequence similarity to" then there are many examples. The elongation factor 1-alpha has a high degree of sequence similarity to the amino terminal end of the elongation factor 2. Elongation factor 2 has a long carboxy terminus that is not found in elongation factor 1-alpha. The low molecular weight GTP-binding proteins have some similarity to both EF-1a and EF-2, but not as extensive as between EF-1a and EF-2. If by "homologous" you mean "identical function", then all of these proteins are known to bind GTP and hydrolyze it, but in doing so they cause vastly different effects on cells. EF-1a and EF-2 are both needed for protein synthesis, while most other GTP-binding proteins are needed for signal transduction, protein transport and other functions. The other tricky part of your question is the word "significantly". Deciding what is "significant" is not easy in protein similarity searches. The homeo box found in developmental proteins is a quite small region of each of the homeotic proteins, yet it is found again and again in developmental regulation. If such a region of similarity was found in proteins of diverse functions we would not consider it "significant", but when it occurs in several proteins with similar function it stands out as very significant. Likewise the GTP-binding domains found in GTP-binding proteins are very small GxxxxGK[S/T] -- DxxG -- NKxD just 8 identical amino acids and 1 conservative substitution spread over an 150 amino acid region. Yet using this pattern to search the non-redundant protein database will pull up almost all the GTP-binding proteins, and very few false-positives. The ProSite database is a collection of such "signature patterns" which can be used to get all the members of various protein "families". : Regards, : ------- : Edwin Wright : 85 Spinnaker Drive, A-602 : Halifax, Nova Scotia : CANADA B3N 3E3 : Telephone: (902) 477-5037 : Email: ewright@fox.nstn.ns.ca : =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= -- ******************************************************************** * Brian Foley * If we knew what we were doing * * Molecular Genetics Dept. * it wouldn't be called research * * University of Vermont * * ******************************************************************** From owner-proteins@net.bio.net Mon Dec 12 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!agate!dog.ee.lbl.gov!news.cs.utah.edu!emba-news.uvm.edu!brianf From: brianf@med.uvm.edu (Brian Foley) Subject: Re: Amino Acid Homology Message-ID: <1994Dec13.145411.17133@emba.uvm.edu> Sender: news@emba.uvm.edu Organization: EMBA Computer Facility, University of Vermont X-Newsreader: TIN [version 1.2 PL1] References: <37234.ewright@fox.nstn.ns.ca> <3c1ui7$176@network.ucsd.edu> Date: Tue, 13 Dec 1994 14:54:11 GMT Lines: 59 : In article <37234.ewright@fox.nstn.ns.ca> Edwin Wright, : ewright@FOX.NSTN.NS.CA writes: : >What is the latest word on amino acid homology with respect to : > conservative : >versus moderate or radical amino acid substitutions in protein sequences? There are a number of matrices available now for computing the SIMILARITY (not homology) of amino acid substitutions. The Dayhoff table is shown below, but there are also BLOSUM, PAM250 and many other such matrices in use. Each particular matirix is best suited to a particular application. For example one may be better for estimating phylogenetic distance, another may be better for attempting to calculate conservation of secondary structures. It is not wise to just score a perfect match as "1.0" and a mismatch as "0" or "-1.0" when computing peptide SIMILARITIES. A matrix is much more sensitive because it can give variable scores for variable degrees of disimilarity. Isoleucine and Leucine are much more similar to each other than are Valine and Aspartic Acid. Dayhoff table (Schwartz, R. M. and Dayhoff, M. O. [1979] in Atlas of Protein Sequence and Structure, Dayhoff, M. O. Ed, pp. 353-358, National Biomedical Research Foundation, Washington D.C.) rescaled by dividing each value by the sum of its row and column, and normalizing to a mean of 0 and standard deviation of 1.0. The value for FY (Phe-Tyr) = RW = 1.425. Perfect matches are set to 1.5 and no matches on any row are better than perfect matches. Table used by Gribskov and Burgess NAR 14(16) 6745-6763 December 29, 1986 12:46 This table is clipped off after Proline in order to fit on the NEWS page: A B C D E F G H I K L M N P 1.5 0.2 0.3 0.3 0.3 -0.5 0.7 -0.1 0.0 0.0 -0.1 0.0 0.2 0.5 A 1.1 -0.4 1.1 0.7 -0.7 0.6 0.4 -0.2 0.4 -0.5 -0.3 1.1 0.1 B 1.5 -0.5 -0.6 -0.1 0.2 -0.1 0.2 -0.6 -0.8 -0.6 -0.3 0.1 C 1.5 1.0 -1.0 0.7 0.4 -0.2 0.3 -0.5 -0.4 0.7 0.1 D 1.5 -0.7 0.5 0.4 -0.2 0.3 -0.3 -0.2 0.5 0.1 E 1.5 -0.6 -0.1 0.7 -0.7 1.2 0.5 -0.5 -0.7 F 1.5 -0.2 -0.3 -0.1 -0.5 -0.3 0.4 0.3 G 1.5 -0.3 0.1 -0.2 -0.3 0.5 0.2 H 1.5 -0.2 0.8 0.6 -0.3 -0.2 I 1.5 -0.3 0.2 0.4 0.1 K 1.5 1.3 -0.4 -0.3 L 1.5 -0.3 -0.2 M 1.5 0.0 N 1.5 P -- ******************************************************************** * Brian Foley * If we knew what we were doing * * Molecular Genetics Dept. * it wouldn't be called research * * University of Vermont * * ******************************************************************** From owner-proteins@net.bio.net Mon Dec 12 22:00:00 1994 Path: biosci!agate!howland.reston.ans.net!pipex!uunet!zib-berlin.de!news.th-darmstadt.de!terra.wiwi.uni-frankfurt.de!zeus.rbi.informatik.uni-frankfurt.de!news.dfn.de!scsing.switch.ch!sun.rediris.es!power.ci.uv.es!usenet From: Mireille Moulard Newsgroups: bionet.molbio.proteins Subject: Re: Method to quantify number of cysteine residues Date: 13 Dec 1994 09:19:07 GMT Organization: uv Lines: 13 Distribution: world Message-ID: <3cjoub$tk7@power.ci.uv.es> NNTP-Posting-Host: biogen.geneti.uv.es X-Newsreader: Hi! I have never try this by myself but I can give you references. People use the DTNB to detect the reduced thiol groups. - Ellman, G.L. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 1959, vol. 82, p. 70-77. - Cavallini et al. Determination of disulphide goups in proteins. Nature. 1966, vol. 212, p.294-295. I hope this help. Mireille From owner-proteins@net.bio.net Mon Dec 12 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!gatech!howland.reston.ans.net!news.moneng.mei.com!uwm.edu!msunews!harbinger.cc.monash.edu.au!bunyip.cc.uq.oz.au!munnari.oz.au!news.unimelb.EDU.AU!ucsvc.ucs.unimelb.edu.au!lugb!news Newsgroups: bionet.molbio.proteins Subject: insoluble proteins? Message-ID: <1994Dec13.043139.1550@lugb.latrobe.edu.au> From: bioab@lure.latrobe.edu.au (ann) Date: Tue, 13 Dec 1994 04:31:39 GMT Sender: news@lugb.latrobe.edu.au (News System) Organization: Latrobe University X-Newsreader: WinVN 0.92.6 Lines: 5 It seems that the majority of proteins from Plasmodium falciparum (malaria) that we have seen are Triton insoluble. Most of the proteins in other cells seem to be Triton soluble (in my limited experience). Are there any sugestions as to why this may be. Are there any other possibilities for solubilisation (that would still allow chromatography if possible?) From owner-proteins@net.bio.net Mon Dec 12 22:00:00 1994 Path: biosci!agate!news.ucdavis.edu!bullwinkle!szdong From: szdong@bullwinkle.ucdavis.edu (Jian Dong) Newsgroups: bionet.molbio.proteins Subject: Biotinylated Nucleotides for PCR Date: 13 Dec 1994 22:18:49 GMT Organization: University of California, Davis Lines: 6 Message-ID: <3cl6k9$358@mark.ucdavis.edu> NNTP-Posting-Host: bullwinkle.ucdavis.edu X-Newsreader: TIN [version 1.2 PL2] I am looking for information using PCR for incorporating biotinylated nucleotides into probe DNA for Northern blotting (i. e. ratios of biotinylated to non-biotinylated, and sensitivity). I am planning to use the biotinylated PCR fragment as a probe to detect mRNA via chemilumensence. I would appreciate any information or conditions regarding this type of method. From owner-proteins@net.bio.net Tue Dec 13 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!spool.mu.edu!uwm.edu!news.doit.wisc.edu!sweetprotein.ahabs.wisc.edu!user From: ming@ahabs.wisc.edu (Ding Ming) Newsgroups: bionet.molbio.proteins Subject: Re: Glycoprotein stain? Date: Wed, 14 Dec 1994 12:25:46 -0600 Organization: University of Wisconsin-Madison Lines: 14 Message-ID: References: NNTP-Posting-Host: sweetprotein.ahabs.wisc.edu In article , nwinegar@tuzo.erin.utoronto.ca wrote: > Is anyone aware of a stain which can be used to selectively stain > glycoproteins in an SDS/native PAGE gel? > There is a commercial kit sold be GLYKO, a Novato, CA based company called Glyko's FACE, which you can use to identify glycoproteins in the gels. Their phone # is 800-334-5956. +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ Dr. Ding Ming University of Wisconsin-Madison +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ From owner-proteins@net.bio.net Tue Dec 13 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!news2.near.net!cat.cis.Brown.EDU!cis-ts3-slip10.cis.brown.edu!user From: Stephen_Lasky@brown.edu (Stephen R. Lasky, Ph.D.) Newsgroups: bionet.molbio.proteins Subject: Re: Glycoprotein stain? Date: 14 Dec 1994 18:16:05 GMT Organization: Roger Williams Hospital/Brown U. Lines: 31 Message-ID: References: NNTP-Posting-Host: cis-ts3-slip10.cis.brown.edu In article , nwinegar@tuzo.erin.utoronto.ca wrote: > Is anyone aware of a stain which can be used to selectively stain > glycoproteins in an SDS/native PAGE gel? > > Thanx for any help > > > -- > ________________________________ > Neil Winegarden > University Of Toronto > nwinegar@credit.erin.utoronto.ca Seems to me that I remember that Periodic Acid Schiff's base (PAS) stains glycoproteins specifically. Don't have a reference, but that's a lead for you. SRLasky -- ********************************************************************* Stephen R. Lasky, Ph.D. Roger Williams Medical Center/Brown University Phone: 401-456-6572 Fax: 401-456-6569 e-mail: Stephen_Lasky@brown.edu ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ "To me at least, 'Yuck' doesn't capture the full essence of death by neurotoxin." -Dick Dunn ********************************************************************* From owner-proteins@net.bio.net Tue Dec 13 22:00:00 1994 Path: biosci!MANI.CBS.UMN.EDU!npv From: npv@MANI.CBS.UMN.EDU (Nora Plesofsky-Vig) Newsgroups: bionet.molbio.proteins Subject: re:insoluble proteins Date: 14 Dec 1994 10:12:40 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 10 Sender: daemon@net.bio.net Distribution: world Message-ID: <9412141810.AA01233@mani.cbs.umn.edu> Reply-To: nora@molbio.cbs.umn.edu NNTP-Posting-Host: net.bio.net I do not know why the proteins in Plasmodium should be uniquely insoluble in Triton. Perhaps the concentration of protein in your solubilization reaction is too high and diluting the cell extract would help. There are other detergents that might prove useful as an alternative to Triton, such as CHAPS, which is zwitterionic, and the nonionic octyl glucoside or octyl-thioglucoside, its more stable derivative. Nora Plesofsky-Vig From owner-proteins@net.bio.net Tue Dec 13 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!uhog.mit.edu!europa.eng.gtefsd.com!howland.reston.ans.net!news.moneng.mei.com!uwm.edu!news.doit.wisc.edu!sweetprotein.ahabs.wisc.edu!user From: ming@ahabs.wisc.edu (Ding Ming) Newsgroups: bionet.molbio.proteins Subject: Re: electrophoretic resolution of 25 kDa protein Date: Wed, 14 Dec 1994 11:00:12 -0600 Organization: University of Wisconsin-Madison Lines: 17 Distribution: world Message-ID: References: <9412132222.AA00899@mani.cbs.umn.edu> NNTP-Posting-Host: sweetprotein.ahabs.wisc.edu In article <9412132222.AA00899@mani.cbs.umn.edu>, nora@molbio.cbs.umn.edu wrote: > help to separate proteins in the 25 kDa range? I am trying to isolate > a minor protein that binds to a glutathione-S-transferase fusion > protein, and I need to get bands that are as sharp as possible. > If you really want to have super-sharp bands, you better work with IEF rather than SDS-PAGE. Tricine gel has a better resolution in low molecular weight range ( less than 10,000), but still hard to beat the IEF in regard of resolution. But smaller proteins do not behave well on IEF generally, but I think yours will be ok. +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ Dr. Ding Ming University of Wisconsin-Madison +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ From owner-proteins@net.bio.net Tue Dec 13 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!bloom-beacon.mit.edu!spool.mu.edu!umn.edu!maroon.tc.umn.edu!rosto001 From: rosto001@maroon.tc.umn.edu (Alexander P Rostovtsev) Subject: Re: glod colloidal system Message-ID: Sender: news@news.cis.umn.edu (Usenet News Administration) Nntp-Posting-Host: maroon.tc.umn.edu Organization: University of Minnesota, Twin Cities References: Date: Wed, 14 Dec 1994 16:05:14 GMT Lines: 19 asma@KB.USM.MY (Asma Ismail) writes: >Hello dear netters, >Could anyone explain to how gold colloid conjugated system wokrs!! >Currently I am using HRP-conjugated antibody system for ELISA as well as >for Dot EIA. I have been told that glod colloid system is much sensitive >yet I do not know how this system works. It also stated that, the >sensitivity of glod colloidal system could be enhanced via silver staining. >Helps needed..... Anal.Biochem.(1984)142,79 Cytochem.(1983)31,433 Anal.Biochem.(1984)142,221 Detection limit ~2 ng rabbit IgG by dot-blot. Expensive. Dr. Alexander Rostovtsev U of Minnesota From owner-proteins@net.bio.net Tue Dec 13 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!agate!spool.mu.edu!torn!utnut!erin.utoronto.ca!tuzo.erin!nwinegar From: nwinegar@tuzo.erin (Neil Winegarden) Subject: Glycoprotein stain? Message-ID: Sender: news@credit.erin.utoronto.ca (Usenet News) Nntp-Posting-Host: tuzo.erin Reply-To: nwinegar@tuzo.erin.utoronto.ca Organization: Erindale College, University of Toronto, Canada Date: Wed, 14 Dec 1994 16:14:12 GMT Lines: 11 Is anyone aware of a stain which can be used to selectively stain glycoproteins in an SDS/native PAGE gel? Thanx for any help -- ________________________________ Neil Winegarden University Of Toronto nwinegar@credit.erin.utoronto.ca From owner-proteins@net.bio.net Tue Dec 13 22:00:00 1994 Path: biosci!agate!spool.mu.edu!uwm.edu!lll-winken.llnl.gov!enews.sgi.com!decwrl!tribune.usask.ca!canopus.cc.umanitoba.ca!loewen5.microbio.umanitoba.ca!ahillar From: ahillar@your-servername.Lan1.UManitoba.CA Newsgroups: bionet.molbio.proteins Subject: Re: insoluble proteins? Date: Wed, 14 Dec 1994 16:04:09 GMT Organization: University of Manitoba Lines: 15 Message-ID: References: <1994Dec13.043139.1550@lugb.latrobe.edu.au> NNTP-Posting-Host: loewen5.microbio.umanitoba.ca X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] In article <1994Dec13.043139.1550@lugb.latrobe.edu.au> bioab@lure.latrobe.edu.au (ann) writes: >Subject: insoluble proteins? >From: bioab@lure.latrobe.edu.au (ann) >Date: Tue, 13 Dec 1994 04:31:39 GMT >It seems that the majority of proteins from Plasmodium falciparum (malaria) >that we have seen are Triton insoluble. Most of the proteins in other cells >seem to be Triton soluble (in my limited experience). Are there any >sugestions as to why this may be. Are there any other possibilities >for solubilisation (that would still allow chromatography if possible?) You might try either CHAPS or Brij 58. Alex From owner-proteins@net.bio.net Tue Dec 13 22:00:00 1994 Path: biosci!agate!howland.reston.ans.net!vixen.cso.uiuc.edu!aries!lwalsh From: lwalsh@aries.scs.uiuc.edu (Laura Walsh) Newsgroups: bionet.molbio.proteins Subject: Re: Where is jellyroll in Con A ? Date: 14 Dec 94 15:28:27 GMT Organization: University of Illinois at Urbana Lines: 25 Message-ID: References: <3chhnq$b6k@imol2.vub.ac.be> NNTP-Posting-Host: aries.scs.uiuc.edu thomas@ultr5.vub.ac.be (Thomas hamelryck) writes: >The lectin Concanavalin A is often reported having a jellyroll >supersecondary structure. But I can't see how this jellyroll >motif could be present when I look at the topology of Concanavalin A. >According to me, Concanavalin A simply does not posses a jellyroll >fold, but a beta-sandwich fold. I think the mistake emerged because >a *different* protein from Canavalia ensiformis, canavalin, *does* have a >jellyroll fold. The similarity between the two names could have >caused the mistake. >Am I right, or am I wrong ? >Has anyone got a different opinion, or has anyone noticed the mistake >before ? >-Thomas. I would tend to agree with you. In my classification system, I would put Canavalin into the double stranded beta cylinder class, which is the jelly roll fold. Concanavalin A, on the other hand, I put in the regular Greek anti beta class, along with the lectins. Laura Walsh lwalsh@aries.scs.uiuc.edu -- Laura Walsh From owner-proteins@net.bio.net Tue Dec 13 22:00:00 1994 Path: biosci!BORCIM.WUSTL.EDU!saboteur From: saboteur@BORCIM.WUSTL.EDU Newsgroups: bionet.molbio.proteins Subject: re: 25kDa resolution Date: 14 Dec 1994 07:10:45 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 10 Sender: daemon@net.bio.net Distribution: world Message-ID: <199412141510.AA00872@wugate.wustl.edu> NNTP-Posting-Host: net.bio.net I am suprised you cannot resolve this wi