From owner-proteins@hgmp.mrc.ac.uk Mon May 1 03:50:20 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id B21B217AAD; Mon, 1 May 2000 03:50:19 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id DAA26188 for ; Mon, 1 May 2000 03:50:18 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id DAA10401 for proteins-list@hgmp.mrc.ac.uk; Mon, 1 May 2000 03:50:16 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: finance102@020.co.uk X-Newsgroups: bionet.molbio.proteins Subject: Work Smarter Not Harder Date: 1 May 2000 03:50:15 +0100 Organization: BIOSCI/MRC Human Genome Mapping Project Resource Centre Lines: 46 Message-ID: <200005010142.DAA12707@stones.bfh.fh-hof.de> Mime-Version: 1.0 Content-Type: multipart/mixed; boundary="----=_NextPart_000_1560_00003DCC.00007ED5" X-Trace: niobium.hgmp.mrc.ac.uk 957149416 10399 193.62.192.80 (1 May 2000 02:50:16 GMT) X-Complaints-To: news@net.bio.net X-Received: from stones.bfh.fh-hof.de (root@stones.bfh.fh-hof.de [194.95.60.53]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id DAA26164; Mon, 1 May 2000 03:50:13 +0100 (BST) X-Received: from tony (13-041.018.popsite.net [216.126.151.41]) by stones.bfh.fh-hof.de (8.8.8/8.8.8) with SMTP id DAA12707; Mon, 1 May 2000 03:42:31 +0200 X-Priority: 3 X-MSMail-Priority: Normal To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk ------=_NextPart_000_1560_00003DCC.00007ED5 Content-Type: text/html; LOOKING FOR GEMS!

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--- From owner-proteins@hgmp.mrc.ac.uk Mon May 1 23:11:57 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 6A1E617A43; Mon, 1 May 2000 23:11:56 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id XAA01032 for ; Mon, 1 May 2000 23:11:54 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id XAA03116 for proteins-list@hgmp.mrc.ac.uk; Mon, 1 May 2000 23:11:52 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f Message-ID: <390E0155.1ADB371F@freeuk.com> From: ChenHA X-Mailer: Mozilla 4.7 [en] (Win95; I) X-Accept-Language: en MIME-Version: 1.0 X-Newsgroups: bionet.molbio.proteins Subject: Re: Methods for protein-protein interface mapping References: <3908F0C7.5DCF34C7@nwu.edu> Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit Date: Mon, 01 May 2000 23:12:37 +0100 X-Complaints-To: abuse@freeuk.net X-Trace: nnrp3.clara.net 957219106 212.126.154.81 (Mon, 01 May 2000 23:11:46 BST) Lines: 18 To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Stoyan Smoukov wrote: > > I've been working on my thesis for a couple of years. One of the goals > is to map the interface bewteen two weakly-bound proteins. I know there > are some methods out there like NMR mapping, FRET, but is there any > other approaches? > Exhaustive mutagenesis (e.g. alanine scanning) is a common approach to defining the binding interface and characterising the binding interaction. See for example Cunningham BC, Wells JA J Mol Biol 1993 Dec 5;234(3):554-63 S. J. Davis, et al PNAS 1998 95: 5490-5494. From owner-proteins@hgmp.mrc.ac.uk Tue May 2 11:16:28 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id BFB0317A87; Tue, 2 May 2000 11:16:27 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id LAA22346 for ; Tue, 2 May 2000 11:16:25 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id LAA11528 for proteins-list@hgmp.mrc.ac.uk; Tue, 2 May 2000 11:16:23 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f X-Originating-Host: 130.37.84.107 Organization: http://www.remarq.com: The World's Usenet/Discussions Start Here Subject: Re: Fluorescent protein stain Lines: 34 From: rogier X-Newsgroups: bionet.molbio.proteins Message-ID: <049504f8.15a59940@usw-ex0105-036.remarq.com> References: <13ee84e6.7032373e@usw-ex0105-036.remarq.com> Bytes: 1202 X-Wren-Trace: eLOWvr+m4avg4LOusfagmqKlr7q0vvypubil/q+po766p/K8tvWzr6v8s7P9ovP0/Ozo8vy4nYKDhA== Date: Tue, 02 May 2000 03:15:29 -0700 X-Complaints-To: wrenabuse@remarq.com X-Trace: WReNphoon3 957263168 10.0.2.36 (Tue, 02 May 2000 03:26:08 PDT) To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk >Unfortunately that's a Coomasie blue reference but I'll have a >hunt for an EtBr reference OOPS!!! that's what happens if you keep your lablog on thousands of little yellow post-it notes... The Wu and Welsh paper was cited in the ethidium bromide story, which I pulled from Technical Tips Online: Igor V. Shevelev, Alexander N. Tretyakov, Oleg K. Kaboev; Staining of proteins after SDS-polyacrylamide gel electrophoresis by ethidium bromide; 8/1/1997. go to http://tto.trends.com/tto/index_of_TTO.shtml and key 'shevelev' in the search box (no, Elsevier is not paying me 5 bucks for this one :) Hope this also works during SDS-PAGE electrocution. If it does (or doesn't), let us know. Make 'em glow!!! Rogier Stuger Dept MicFizz, Free U of A E: rogier AT biogate.com _________________________ register at biowire. you get a free laserpointer for signing up and CASH for writing reviews, and I get 5 bucks from them if you sign up thru this URL: http://www.biowire.com/bw_jsp/gate_top.jsp?add=1&referrer=rogier * Sent from RemarQ http://www.remarq.com The Internet's Discussion Network * The fastest and easiest way to search and participate in Usenet - Free! From owner-proteins@hgmp.mrc.ac.uk Tue May 2 11:46:27 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 9E41F17B28; Tue, 2 May 2000 11:45:12 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id LAA27103 for ; Tue, 2 May 2000 11:44:29 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id LAA11765 for proteins-list@hgmp.mrc.ac.uk; Tue, 2 May 2000 11:44:11 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Simon Andrews X-Newsgroups: bionet.molbio.proteins Subject: Re: Methods for protein-protein interface mapping Date: Tue, 02 May 2000 11:35:29 +0100 Organization: BBSRC Biotechnology and Biological Sciences Research Council Lines: 13 Message-ID: <390EAF71.E7D2CD3F@bbsrc.ac.uk> References: <3908F0C7.5DCF34C7@nwu.edu> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.5 [en] (WinNT; I) X-Accept-Language: en To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Stoyan Smoukov wrote: > Also, i'm interested in finding out how many modeling programs are > available for protein-protein docking. There are plenty of commercial packages which will do this, but you may want to have a look at a freeware program called Hex, which will calculate protein-protein docking interactions. You can download it from; http://www.biochem.abdn.ac.uk/hex/ From owner-proteins@hgmp.mrc.ac.uk Tue May 2 21:19:36 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 5AAF817AC1; Tue, 2 May 2000 21:19:35 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id VAA14082 for ; Tue, 2 May 2000 21:19:32 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id VAA27671 for proteins-list@hgmp.mrc.ac.uk; Tue, 2 May 2000 21:19:31 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Chris LaRosa X-Newsgroups: bionet.molbio.proteins Subject: Re: Protein expression in Yeast Date: Tue, 02 May 2000 15:22:37 -0500 Organization: University of Nebraska-Lincoln Lines: 8 Message-ID: <390F390D.3B354D1E@biocomp.unl.edu> References: <0e54ac28.4018289a@usw-ex0109-068.remarq.com> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.72 [en] (Win95; I) X-Accept-Language: en To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk > > Is there a tip and/or hint to obtain transformants using a > 11 Kb plasmid? > Your kidding, right? From owner-proteins@hgmp.mrc.ac.uk Wed May 3 12:50:14 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id E420C17A7B; Wed, 3 May 2000 12:50:11 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id MAA26234 for ; Wed, 3 May 2000 12:50:07 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id MAA08863 for proteins-list@hgmp.mrc.ac.uk; Wed, 3 May 2000 12:50:06 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Rick Thorne X-Newsgroups: bionet.molbio.proteins Subject: Re: Problems with GST fusion protein Date: Wed, 03 May 2000 21:45:02 +1000 Organization: CRU Lines: 40 Message-ID: <3910113F.AEDD3288@mail.newcastle.edu.au> References: <39007DD9.C349D516@mcmail.vanderbilt.edu> Reply-To: rthorne@mail.newcastle.edu.au Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii; x-mac-type="54455854"; x-mac-creator="4D4F5353" Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.7C-CCK-MCD {C-UDP; EBM-APPLE} (Macintosh; I; PPC) X-Accept-Language: en To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Dear Eva All GST-fusions are different but I can offer you some guidelines which might help you. First step might be to alter the strain of bacteria you are using for a more specialized strain eg: BL21-DE3 from Stratagene (look at the web site to learn more about these). Alternatives are available from other companies (but I never used them). If this does not help, if at all practicable, make new construct/s with smaller bits of the protein you want to use. This sometimes removes parts of the molecule that encourage its wholesale destruction. regards, Rick Eva Chen wrote: > Is there anybody having experience on the purification of GST fusion > proteins? > Our lab doesn't have much experience and now there has been a serious > problem of degradation of the GST fusion protein. We have tried the > protease inhibitor from Sigma and Roche, and the whole purification > process was accomplished in a 4 C cold room. But the proteolysed > products are still there. > > Any suggestions are highly appreciated. Thank you all for your > attention! > > Eva Y. Chen > yi-hui.chen@mcmail.vanderbilt.edu > Jim Sutcliffe Lab > 615-936-3627 > Department of Molecular Physiology and Biophysics > Vanderbilt University School of Medicine > Nashville, TN 37232 > USA From owner-proteins@hgmp.mrc.ac.uk Thu May 4 13:00:18 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id C8D8E17A62; Thu, 4 May 2000 13:00:16 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id NAA17453 for ; Thu, 4 May 2000 13:00:09 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id NAA05151 for proteins-list@hgmp.mrc.ac.uk; Thu, 4 May 2000 13:00:07 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Petri Kursula X-Newsgroups: bionet.molbio.proteins Subject: Re: Problems with GST fusion protein Date: Thu, 4 May 2000 14:47:47 +0300 Organization: University of Oulu Lines: 53 Message-ID: References: <39007DD9.C349D516@mcmail.vanderbilt.edu> <3910113F.AEDD3288@mail.newcastle.edu.au> Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII X-Trace: ousrvr3.oulu.fi 957440869 21380 130.231.240.13 (4 May 2000 11:47:49 GMT) X-Complaints-To: news@news.oulu.fi X-Sender: pkursula@sun3.oulu.fi In-Reply-To: <3910113F.AEDD3288@mail.newcastle.edu.au> To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk The old tricks could also be useful: lower the induction temperature and IPTG concentration, and shorten the induction time.... Does the degradation occur during induction or purification? Pete On Wed, 3 May 2000, Rick Thorne wrote: > Dear Eva > > All GST-fusions are different but I can offer you some guidelines which > might help you. > > First step might be to alter the strain of bacteria you are using for a > more specialized strain eg: BL21-DE3 from Stratagene (look at the web > site to learn more about these). Alternatives are available from other > companies (but I never used them). > > If this does not help, if at all practicable, make new construct/s with > smaller bits of the protein you want to use. This sometimes removes > parts of the molecule that encourage its wholesale destruction. > > regards, > > Rick > > Eva Chen wrote: > > > Is there anybody having experience on the purification of GST fusion > > proteins? > > Our lab doesn't have much experience and now there has been a serious > > problem of degradation of the GST fusion protein. We have tried the > > protease inhibitor from Sigma and Roche, and the whole purification > > process was accomplished in a 4 C cold room. But the proteolysed > > products are still there. > > > > Any suggestions are highly appreciated. Thank you all for your > > attention! > > > > Eva Y. Chen > > yi-hui.chen@mcmail.vanderbilt.edu > > Jim Sutcliffe Lab > > 615-936-3627 > > Department of Molecular Physiology and Biophysics > > Vanderbilt University School of Medicine > > Nashville, TN 37232 > > USA > > From owner-proteins@hgmp.mrc.ac.uk Fri May 5 02:47:42 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id B66EE17A43; Fri, 5 May 2000 02:47:41 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id CAA04969 for ; Fri, 5 May 2000 02:47:39 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id CAA23968 for proteins-list@hgmp.mrc.ac.uk; Fri, 5 May 2000 02:47:38 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: "Toby Skinner" X-Newsgroups: bionet.molbio.proteins Subject: Pleas Help Quick Date: Fri, 5 May 2000 02:38:22 +0100 Organization: Customer of Planet Online Lines: 20 Message-ID: <8et97o$77a$1@news5.svr.pol.co.uk> X-Trace: news5.svr.pol.co.uk 957491256 7402 62.136.134.105 (5 May 2000 01:47:36 GMT) X-Complaints-To: abuse@theplanet.net X-Priority: 3 X-MSMail-Priority: Normal X-Newsreader: Microsoft Outlook Express 5.00.2615.200 X-MimeOLE: Produced By Microsoft MimeOLE V5.00.2615.200 To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Hello, I have a simlpe question and I really nead an answer quick. Any protein is made up of amino-acids, it is these that define the overall structure and therefore function of the protein. I know that amino-acids can be replaced by other amino-acids without effecting the function of the protein. So if I was able to generate a new say, Haemoglobin protein which has a significantly different amino-acid content, does anyone know if (in theory) it could be created in the lab and experimented apon? This is a serious question and has implications for some software I have written (dissertation) for finding amino-acid sequences which fold into the same user specified protein. The idea is that a useful but toxic protein could be enginered in the lab to be non-toxic. Any thanks is very appreciated. Toby From owner-proteins@hgmp.mrc.ac.uk Fri May 5 09:00:35 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 51CBA17A96; Fri, 5 May 2000 09:00:34 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id JAA27146 for ; Fri, 5 May 2000 09:00:32 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id JAA28299 for proteins-list@hgmp.mrc.ac.uk; Fri, 5 May 2000 09:00:31 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Frank Fuerst X-Newsgroups: bionet.molbio.proteins Subject: Re: Pleas Help Quick Date: Fri, 05 May 2000 10:00:23 +0200 Lines: 58 Message-ID: <8etv2s$9ode6$1@fu-berlin.de> References: <8et97o$77a$1@news5.svr.pol.co.uk> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: fu-berlin.de 957513628 10237382 141.89.201.37 (16 955) X-Mailer: Mozilla 4.51 [de]C-CCK-MCD QXW03200 (WinNT; I) X-Accept-Language: de,en To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Toby Skinner wrote: > > Hello, I have a simlpe question and I really nead an answer quick. Ahm, you no that this isn't your private helpdesk? > Any protein is made up of amino-acids, it is these that define the overall > structure and therefore function of the protein. I know that amino-acids > can be replaced by other amino-acids without effecting the function of the > protein. right. > So if I was able to generate a new say, Haemoglobin protein which has a > significantly different amino-acid content, does anyone know if (in theory) > it could be created in the lab and experimented apon? In principle, yes. You can alter a considerable percentage of the sequence (especially if your exchanges are conservative, i.e. the type of amino acid [aliphatic, acidic etc.] is not changed) without mayor effects on function and stability. But of course the really important residues in the active site cannot be altered as long as you still want your protein to function; in the hemoglobin case it would be at least the ligands of heme, and additionally some residues necessary for the cooperativity - otherwise you'll get a "tetrameric myoglobin". Created in the lab? genetic engineering, usually connected with heterologous expression (in E.coli, S. cervisiae etc.), would be a reallistic approach. Chemical synthesis of such a long polypeptide chain is not feasible. > This is a serious > question and has implications for some software I have written > (dissertation) for finding amino-acid sequences which fold into the same > user specified protein. My opinion is that everybody planning to write some bioinformatics software should first carefully read and understand a textbook specific for the relevant field (in this case, protein structure) and know some biochemist to talk to... > The idea is that a useful but toxic protein could > be enginered in the lab to be non-toxic. Toxicity of proteins often does not depend on the primary sequence, but on the specific function of the protein. So your non-toxic protein will probably no longer be usefull. There are some exceptions, e.g. (I think) the toxicity of gluten in patients with coeliac disease, but be careful. > Any thanks is very appreciated. Thanks. Yours, Frank -- Die Verwendung von mehreren Ausrufezeichen macht die Aussage nicht ausrufender sondern ausufernder. [Michael Bauer in dnq] From owner-proteins@hgmp.mrc.ac.uk Fri May 5 09:44:15 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 3F53C17A9A; Fri, 5 May 2000 09:44:14 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id JAA00536 for ; Fri, 5 May 2000 09:44:12 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id JAA28816 for proteins-list@hgmp.mrc.ac.uk; Fri, 5 May 2000 09:44:11 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Simon Andrews X-Newsgroups: bionet.molbio.proteins Subject: Re: Pleas Help Quick Date: Fri, 05 May 2000 09:42:12 +0100 Organization: BBSRC Biotechnology and Biological Sciences Research Council Lines: 84 Message-ID: <39128964.30CD1E11@bbsrc.ac.uk> References: <8et97o$77a$1@news5.svr.pol.co.uk> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.5 [en] (WinNT; I) X-Accept-Language: en To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Toby Skinner wrote: > > Hello, I have a simlpe question and I really nead an answer quick. Asking nicely is a better way to get a quick answer than demanding one (people on usenet will often pass over messages which start with a line like that....) > > Any protein is made up of amino-acids, it is these that define the overall > structure and therefore function of the protein. I know that amino-acids > can be replaced by other amino-acids without effecting the function of the > protein. This is true, to a certain extent - in a protein, many single amino acid substitutions will produce no apparent change in overall function, but it would be unlikely that a large number of even conservative changes would have no effect. In addition, certain amino acids would probably be absolutely required to maintain functionality. In haemoglobin, for instance, I would imagine that the amino acids which interact with the iron atoms in the molecule would be difficult to mutate without impairing the function, as would the groups which goverened the association of the subunits. > > So if I was able to generate a new say, Haemoglobin protein which has a > significantly different amino-acid content, does anyone know if (in theory) > it could be created in the lab and experimented apon? It depends what you mean by "generate". If you mean that you are going to computationally predict a completely new sequence which will adopt the same structure, and perform the same function as an existing enzyme then good luck!! The forces which govern the folding of a linear amino acid into a 3D structure are not well enough understood to be able to make definitive predictions about the final strucutre of a given sequence - let alone start with a structure and predict a sequence which would form it. It would be worth your while reading up the literature on prediction of protein folding before delving into this subject much further. Try looking at the best structure prediction methods, and their levels of inaccuracy, and you will appreciate the scale of the problem you are approaching. A overview of the forces which govern protein structure can be found at; http://www.med.unibs.it/~marchesi/pps97/course/section7/os_molf.html and a sensible review of techniques for structure prediction (and their inaccuracies) is at; http://www.embl-heidelberg.de/~rost/Papers/sisyphus.html What can be done, is to generate protein derivatives with varying numbers of mutations. This must usually be done in a stepwise mannar, evaluating the effect of single and double mutations biochemically, and then combining them. In our experience (trying to incorporate as many methionines as possible into a protein) you don't have to make a huge number of substitutions before things start to go wrong. > This is a serious > question and has implications for some software I have written > (dissertation) for finding amino-acid sequences which fold into the same > user specified protein. The idea is that a useful but toxic protein could > be enginered in the lab to be non-toxic. This would largely depend on why the protein was toxic in the first place. If the activity of the protein proved toxic then there's not much you can do about that. If you change the activity then you haven't got the same protein as you started with. On the other hand, the toxicity could come from the antigenic properties of the protein - and these could conceivably be altered without affecting the functionality - but this is not a trivial task. I think you should investigate the literature in this area some more, and prefereably find a molecular biologist / biochemist, with whom you can sit down and discuss your proposal I hope this helps TTFN Simon. From owner-proteins@hgmp.mrc.ac.uk Fri May 5 16:15:07 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 445B417A9E; Fri, 5 May 2000 16:15:05 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id QAA10121 for ; Fri, 5 May 2000 16:15:03 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id QAA03710 for proteins-list@hgmp.mrc.ac.uk; Fri, 5 May 2000 16:15:02 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: hroychow@nmsu.edu ("Hiranya S. Roychowdhury") X-Newsgroups: bionet.molbio.proteins Subject: Re: Pleas Help Quick Date: 5 May 2000 16:15:00 +0100 Organization: BIOSCI/MRC Human Genome Mapping Project Resource Centre Lines: 49 Message-ID: <200005051514.JAA185556@nestor.NMSU.Edu> Mime-Version: 1.0 Content-Type: text/plain; charset="us-ascii" X-Trace: niobium.hgmp.mrc.ac.uk 957539701 3708 193.62.192.80 (5 May 2000 15:15:01 GMT) X-Complaints-To: news@net.bio.net X-Received: from bubba.NMSU.Edu (bubba.NMSU.Edu [128.123.3.39]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id QAA10108 for ; Fri, 5 May 2000 16:14:56 +0100 (BST) X-Received: from dns1.NMSU.Edu (dns1.NMSU.Edu [128.123.3.5]) by bubba.NMSU.Edu (8.9.3/8.9.0) with ESMTP id JAA23148; Fri, 5 May 2000 09:14:44 -0600 (MDT) X-Received: from nestor.NMSU.Edu (nestor.NMSU.Edu [128.123.34.146]) by dns1.NMSU.Edu (8.9.3/8.9.0) with ESMTP id JAA23200; Fri, 5 May 2000 09:14:46 -0600 (MDT) X-Received: from peaberry.nmsu.edu (peaberry.NMSU.Edu [128.123.96.3]) by nestor.NMSU.Edu (8.9.3/8.9.0) with SMTP id JAA185556; Fri, 5 May 2000 09:14:46 -0600 X-Sender: hroychow@cnmailsvr.nmsu.edu X-Mailer: Windows Eudora Light Version 1.5.2 X-To: "Toby Skinner" X-Cc: proteins@hgmp.mrc.ac.uk To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Proteins are more complex than just their sum of amino acids. Whether one retains its functionality by just having one or a few aa's altered depends on a lot of different factors: 1. physicochemical property of the target aa; 2. location of the aa in the polypeptide (IOW, the secondary structure of the region, eg H,C or E); 3. neighboring aa's; 4. its functional significance in the tert. structure of the prot., etc. When you change the aa's of a protein, say "A", "significantly", chances are that the resultant protein will no longer be "A". The functionality of a protein depends generally on some select group of aa within the core. It is possible to have "similar" catalytic or prosthetic properties in a grossly altered protein, and that is not uncommon, evolutionarily speaking. For example, there are numerous kinases that have about 25% aa identities among themselves. This reflects the variability in substrates. Similarly, check the structures of hemoglobin and leghemoglobin and you may have some answers to your question. I think the LgHgb may be in PDB. At 02:38 AM 5/5/00 +0100, Toby Skinner wrote: >Hello, I have a simlpe question and I really nead an answer quick. > >Any protein is made up of amino-acids, it is these that define the overall >structure and therefore function of the protein. I know that amino-acids >can be replaced by other amino-acids without effecting the function of the >protein. > >So if I was able to generate a new say, Haemoglobin protein which has a >significantly different amino-acid content, does anyone know if (in theory) >it could be created in the lab and experimented apon? This is a serious >question and has implications for some software I have written >(dissertation) for finding amino-acid sequences which fold into the same >user specified protein. The idea is that a useful but toxic protein could >be enginered in the lab to be non-toxic. > >Any thanks is very appreciated. > >Toby > > > Dr. Hiranya Sankar Roychowdhury GENE LAB/ EPPWS New Mexico State University Las Cruces, NM 88003 Ph. (505) 646-5785 hroychow@nmsu.edu --- From owner-proteins@hgmp.mrc.ac.uk Fri May 5 16:18:46 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id A297C17A9E; Fri, 5 May 2000 16:18:45 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id QAA10557 for ; Fri, 5 May 2000 16:18:42 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id QAA03717 for proteins-list@hgmp.mrc.ac.uk; Fri, 5 May 2000 16:18:41 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Oliver X-Newsgroups: bionet.molbio.proteins Subject: detecting pH in protein containing samples Date: Fri, 05 May 2000 17:19:50 +0200 Organization: Johannes Gutenberg-Universitaet Mainz, Germany Lines: 13 Message-ID: <3912E696.EFC8A2D6@biophysik.biologie.uni-mainz.de> Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Trace: bambi.zdv.Uni-Mainz.DE 957539917 11419 134.93.248.74 (5 May 2000 15:18:37 GMT) X-Complaints-To: usenet@mail.uni-mainz.de X-Mailer: Mozilla 4.7 [de] (Win95; I) X-Accept-Language: de To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Hello, I try to use a common pH-electrode (Ingold) to measure pH changes upon oxygenation of hemocyanin of spiders. The problem is, that I have to use concentrations between 22 to 32 g/l. The protein is denaturated inside the diaphragm of the electrode after a short time. Does anybody use the Friscolyt-electrolyt (Mettler-Toledo) to solve that problem? I also donīt know how to change the electrolyte. Itīs a mico-electrode and capillary forces prevent simple change by pipette. Thank you for any help, Oliver From owner-proteins@hgmp.mrc.ac.uk Fri May 5 21:33:10 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 7AD7617A72; Fri, 5 May 2000 21:33:09 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id VAA04292 for ; Fri, 5 May 2000 21:33:07 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id VAA16274 for proteins-list@hgmp.mrc.ac.uk; Fri, 5 May 2000 21:33:06 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: jlb@ncgr.org ("Jeff L. Blanchard") X-Newsgroups: bionet.molbio.proteins Subject: Deadline for Madison Bioinformatics Courses Date: 5 May 2000 21:33:05 +0100 Organization: BIOSCI/MRC Human Genome Mapping Project Resource Centre Lines: 118 Message-ID: <200005052032.OAA10361@gallina.ncgr.org> Mime-Version: 1.0 Content-Type: TEXT/plain; charset=us-ascii X-Trace: niobium.hgmp.mrc.ac.uk 957558786 16272 193.62.192.80 (5 May 2000 20:33:06 GMT) X-Complaints-To: news@net.bio.net Content-MD5: ucQ1QW74sEbyVYyjfuWMkQ== X-Received: from madison.ncgr.org (ncgr.ncgr.org [216.161.34.2]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id VAA04272 for ; Fri, 5 May 2000 21:32:59 +0100 (BST) X-Received: from gallina.ncgr.org (gallina [172.19.68.25]) by madison.ncgr.org (8.9.0/8.9.0) with ESMTP id OAA04597 for ; Fri, 5 May 2000 14:32:28 -0600 (MDT) X-Received: from gallina (gallina [172.19.68.25]) by gallina.ncgr.org (8.9.1b+Sun/8.9.0) with SMTP id OAA10361 for ; Fri, 5 May 2000 14:32:25 -0600 (MDT) X-Reply-To: "Jeff L. Blanchard" X-To: proteins@hgmp.mrc.ac.uk X-Mailer: dtmail 1.3.0 @(#)CDE Version 1.3.2 SunOS 5.7 sun4u sparc To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk This summer the BioPharmaceutical Technology Center Institute in Madison, WI will be offering the following two bioinformatics-related courses. The application deadline for the courses is this Monday May 7th. After this date applicants will be accepted on a first come/ space available basis. However since the classes are limited to 16 participants additional space will be scarce, especially for "Techniques in Bioinformatics and Comparative Genomics". For more information see http://www.btci.org/courses/CoursesList/courses.htm or contact Jeff Blanchard at the address below. -------------------------------------------------------------------------------- Techniques in Bioinformatics and Comparative Genomics June 18 - June 23, 2000 Objectives and Goals: This five-day intensive computer laboratory course is designed to help participants construct a working library of bioinformatic tools and resources. The format will combine lectures and interactive problem-solving sessions with each participant working individually at a computer. An emphasis will be placed on analysis of practical problems posed by the participants. The flow of the course will move from traditional sequence analysis techniques to the opportunities afforded by the imminent flood of genomic information. Afternoon research seminars by the instructors will sample academic and private sector visions of bioinformatics and highlight creative approaches to utilizing genomic data. (Please note: this course is designed to provide an overview of the field; detailed training in specific packages will not be provided.) Instructors include: Jeff Blanchard, Ph.D., Research Scientist, National Center for Genome Resources Tim Burland, Ph.D., Vice President and General Manager, DNASTAR Barbara Butler, Ph.D., Bioinformatics Training and Education Group Leader, Genetics Computer Group Ross Overbeek, Ph.D., Senior Computer Scientist, Integrated Genomics Ann Palmenberg, Ph.D., Professor, Institute for Molecular Virology Department of Biochemistry,University of Wisconsin Michael Slater, Ph.D., Senior Scientist, Promega Corporation Jeff Thorne, Ph.D., Assistant Professor, Department of Statistics, North Carolina State University ---------------------------------------------------------------------- Database Design and Development for Genomics Research June 29 - July 1, 2000 Course Overview: Databases have been the quiet intermediary of molecular biology research and in their current state offer a wonderful example of using the web to share experimental results and knowledge. In recent years there has been a proliferation of individual and community oriented databases that contain DNA sequence, expression, structural, enzymatic, phenotypic, organismal and other types of data. Many scientists and private companies now face the challenge of integrating their data into these heterogeneous databases and/or synthesizing databases before they can finally get to the heart of a research question. This three-day "hands on" workshop will start with a session on "What are databases?" and move onto to database issues in molecular biology related to storing, integrating, visualizing, analyzing, synthesizing and presenting data. The workshop is geared for people in molecular biology lab groups and computer scientists getting into bioinformatics. The format will combine discussions with problem-solving sessions and each participant will work individually at a computer. Some specific topics include: An introduction to database systems and biological databases Using object-oriented principles to construct a relational database Developing and using gene expression databases Genomic Data Warehouse - Integrating data for Complex Analysis Integrating undisclosed/private and public data Development of Mouse Databases at the Jackson Laboratory A Distributed Sequence Annotation System Ontologies for Molecular Biology Databases Instructors include: Jeff Blanchard, Ph.D., Research Scientist, National Center for Genome Resources Carol Bult, Ph.D., Research Scientist, Mouse Genome Informatics Group, The Jackson Laboratory Allan Dickerman, Program Manager, National Center for Genome Resources Elizabeth Shoop, Ph.D., Research Associate, Computational Biology Center, University of Minnesota Lincoln Stein, Ph.D., Assistant Professor, Cold Spring Harbor Laboratory Jennifer Weller, Ph.D. Program Manager, National Center for Genome Resources There will probably be an additional instructor from the private sector. Matteo di Tommaso from Genetics Computer Group was originally scheduled to participate. However, his is in the process of moving to Celera and because of the time of his move will not be able to make it. ---------------------------------------------------------------------- The BioPharmaceutical Technology Center Institute (BTCI) is a not-for-profit organization operated exclusively for educational, scientific and cultural purposes. One goal of BTCI is to promote the exchange of scientific, educational and cultural information between industry, educators and the general public by providing facilities and resources to support conferences, seminars, classes, and electronic distribution of programs. _____________________________________ Jeffrey L. Blanchard Research Scientist National Center for Genome Resources 2935 Rodeo Park Drive East Santa Fe, NM 87505 tel: 505-995-4405 fax: 505-995-4432 http://www.ncgr.org/about/staff/jlb --- From owner-proteins@hgmp.mrc.ac.uk Sat May 6 02:30:10 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 343B417A78; Sat, 6 May 2000 02:30:09 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id CAA28835 for ; Sat, 6 May 2000 02:30:08 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id CAA19596 for proteins-list@hgmp.mrc.ac.uk; Sat, 6 May 2000 02:30:06 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: chetirk@my-deja.com X-Newsgroups: bionet.molbio.proteins Subject: membrane topology control? Date: Sat, 06 May 2000 01:17:32 GMT Organization: Deja.com - Before you buy. Lines: 13 Message-ID: <8evrr3$9jk$1@nnrp1.deja.com> X-Article-Creation-Date: Sat May 06 01:17:32 2000 GMT X-Http-User-Agent: Mozilla/4.0 (compatible; MSIE 5.01; Windows 98) X-Http-Proxy: 1.1 x33.deja.com:80 (Squid/1.1.22) for client 134.193.11.215 X-MyDeja-Info: XMYDJUIDchetirk To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Hi, I'm looking for a GOOD control of membrane (microsomal)topology. Does anybody knows well known microsomal protein(s) to which antibodies are commercially available? Any suggestions are highly appreciated. Serg Sent via Deja.com http://www.deja.com/ Before you buy. From owner-proteins@hgmp.mrc.ac.uk Sat May 6 16:30:38 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 1122517A9B; Sat, 6 May 2000 16:30:37 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id QAA03050 for ; Sat, 6 May 2000 16:30:36 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id QAA29446 for proteins-list@hgmp.mrc.ac.uk; Sat, 6 May 2000 16:30:35 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: "Toby Skinner" X-Newsgroups: bionet.molbio.proteins Subject: Thanks to everyone who replied Date: Sat, 6 May 2000 16:21:22 +0100 Organization: Customer of Planet Online Lines: 21 Message-ID: <8f1dqo$3k9$1@newsg1.svr.pol.co.uk> X-Trace: newsg1.svr.pol.co.uk 957627032 3721 62.136.97.23 (6 May 2000 15:30:32 GMT) X-Complaints-To: abuse@theplanet.net X-Priority: 3 X-MSMail-Priority: Normal X-Newsreader: Microsoft Outlook Express 5.00.2615.200 X-MimeOLE: Produced By Microsoft MimeOLE V5.00.2615.200 To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk This is just a quick thanks to everyone who replied to my mail a few days ago, I realise it was a bit rash at places and I appologise if anyone found it a bit pushy. Anyway, I have read the replies and the information is very useful, as it happens I have read a lot of approachs to protein folding but each one seems to be fairly different, I am using the Chou & Fasman rules, not because their particulary accurate but it was the easiest to understand (I am a Compuer Science & AI student after all). If you happen to have a bit of time then I would really appreciate it if you would be able to comment on the results of my program trying to generate a new (possibly) haemoglobin protein. I realise it will be inacurate but because I'm not a biochemist I wont realise exactly why it wont be perfect. If you can help me then drop me a mail to my home address (tobyski@frogpad.freeserve.co.uk). Thanks again. Toby From owner-proteins@hgmp.mrc.ac.uk Sat May 6 18:42:50 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 481A917A7C; Sat, 6 May 2000 18:42:50 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id SAA08871 for ; Sat, 6 May 2000 18:42:48 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id SAA01014 for proteins-list@hgmp.mrc.ac.uk; Sat, 6 May 2000 18:42:45 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Eva Chen X-Newsgroups: bionet.molbio.proteins Subject: Re: Problems with GST fusion protein Date: Sat, 06 May 2000 12:40:55 -0500 Organization: Vanderbilt University Medical Center Lines: 16 Message-ID: <39145923.D2F831F7@mcmail.vanderbilt.edu> References: <39007DD9.C349D516@mcmail.vanderbilt.edu> <3910113F.AEDD3288@mail.newcastle.edu.au> Reply-To: yi-hui.chen@mcmail.vanderbilt.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii; x-mac-type="54455854"; x-mac-creator="4D4F5353" Content-Transfer-Encoding: 7bit X-Trace: news.vanderbilt.edu 957634627 3692 160.129.249.196 (6 May 2000 17:37:07 GMT) X-Complaints-To: usenet@news.vanderbilt.edu X-Mailer: Mozilla 4.72 (Macintosh; I; PPC) X-Accept-Language: en,pdf To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk > Thank you for all of your kind replies and suggestions. Meanwhile our lab is still doing several trials and see what will happen. Our PI would prefer to set any change of the E. coli strain and/or vectors as the last thing we would do. And I will try other reagents first. Much thanks again! > Eva Y. Chen > > yi-hui.chen@mcmail.vanderbilt.edu > > Jim Sutcliffe Lab > > 615-936-3627 > > Department of Molecular Physiology and Biophysics > > Vanderbilt University School of Medicine > > Nashville, TN 37232 > > USA From owner-proteins@hgmp.mrc.ac.uk Mon May 8 06:06:01 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id B76C017A6C; Mon, 8 May 2000 06:06:00 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id GAA10628 for ; Mon, 8 May 2000 06:05:59 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id GAA12684 for proteins-list@hgmp.mrc.ac.uk; Mon, 8 May 2000 06:05:57 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: income@china.com X-Newsgroups: bionet.molbio.proteins Subject: $$$Cash in on the Intenet Explosion$$$ Date: 8 May 2000 06:05:56 +0100 Organization: BIOSCI/MRC Human Genome Mapping Project Resource Centre Lines: 40 Message-ID: <200005080448.NAA03085@cyber.sktelecom.com> Mime-Version: 1.0 Content-Type: multipart/mixed; boundary="----=_NextPart_000_6402_00000180.00007AA6" X-Trace: niobium.hgmp.mrc.ac.uk 957762356 12681 193.62.192.80 (8 May 2000 05:05:56 GMT) X-Complaints-To: news@net.bio.net X-Received: from cyber.sktelecom.com (cyber.sktelecom.com [203.236.1.18]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id GAA10620 for ; Mon, 8 May 2000 06:05:49 +0100 (BST) X-Received: from erik (max1-96.atlanta.corecomm.net [216.214.97.224]) by cyber.sktelecom.com (8.8.8/8.8.8) with SMTP id NAA03085; Mon, 8 May 2000 13:48:37 +0900 (KST) X-Priority: 3 X-MSMail-Priority: Normal X-Reply-To: internet@china.com To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk ------=_NextPart_000_6402_00000180.00007AA6 Content-Type: text/html; The Internet and E-Commerce are here to stay!

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 --- From owner-proteins@hgmp.mrc.ac.uk Mon May 8 13:24:12 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id ED97C17A89; Mon, 8 May 2000 13:24:11 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id NAA16541 for ; Mon, 8 May 2000 13:24:09 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id NAA17941 for proteins-list@hgmp.mrc.ac.uk; Mon, 8 May 2000 13:24:08 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Simon Andrews X-Newsgroups: bionet.molbio.proteins Subject: Re: Thanks to everyone who replied Date: Mon, 08 May 2000 13:09:36 +0100 Organization: BBSRC Biotechnology and Biological Sciences Research Council Lines: 34 Message-ID: <3916AE80.F6963861@bbsrc.ac.uk> References: <8f1dqo$3k9$1@newsg1.svr.pol.co.uk> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.5 [en] (WinNT; I) X-Accept-Language: en To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Toby Skinner wrote: > > This is just a quick thanks to everyone who replied to my mail a few days > ago, I realise it was a bit rash at places and I appologise if anyone found > it a bit pushy. Anyway, I have read the replies and the information is very > useful, as it happens I have read a lot of approachs to protein folding but > each one seems to be fairly different, I am using the Chou & Fasman rules, > not because their particulary accurate but it was the easiest to understand > (I am a Compuer Science & AI student after all). The Chou and Fasman rules are a *very* simplified approach to the problem of protein folding. They have been superseded several times since their creation. They are also only predictors of secondary strucutre, and will not relate this to tertiary structure. If you are using them merely to make suggestions for conservative mutations, then you could look at one of the standard PAM or BLOSUM scoring matrices. These are simply tabulated values which relate to the frequency with which a given amino acid is observed to be substited with all other amino acids in groups of homologous proteins. This should also be very easy to incorporate into a program. > > If you happen to have a bit of time then I would really appreciate it if you > would be able to comment on the results of my program trying to generate a > new (possibly) haemoglobin protein. I realise it will be inacurate but > because I'm not a biochemist I wont realise exactly why it wont be perfect. If you have something reasonally concise which describes exactly what you have done then why not post it here? I'm sure people would be happy to comment on it. Simon. From owner-proteins@hgmp.mrc.ac.uk Mon May 8 15:34:00 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 42C4E17A5A; Mon, 8 May 2000 15:33:58 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id PAA28988 for ; Mon, 8 May 2000 15:33:56 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id PAA19535 for proteins-list@hgmp.mrc.ac.uk; Mon, 8 May 2000 15:33:55 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: rjones@post.cis.smu.edu ("Rick Jones") X-Newsgroups: bionet.molbio.proteins Subject: Postdoc position: Polycomb-group-dependent transcriptional silencing Date: 8 May 2000 15:33:54 +0100 Organization: BIOSCI/MRC Human Genome Mapping Project Resource Centre Lines: 69 Message-ID: Mime-Version: 1.0 Content-Type: multipart/alternative; boundary="MS_Mac_OE_3040619735_88923_MIME_Part" X-Trace: niobium.hgmp.mrc.ac.uk 957796434 19533 193.62.192.80 (8 May 2000 14:33:54 GMT) X-Complaints-To: news@net.bio.net X-Received: from post.cis.smu.edu (root@post.cis.smu.edu [129.119.64.23]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id PAA28980 for ; Mon, 8 May 2000 15:33:47 +0100 (BST) X-Received: by post.cis.smu.edu (Smail-3.2.0.96 1997-Jun-2 #26) id ; Mon, 8 May 2000 09:31:00 -0500 (CDT) X-Mailer: Microsoft Outlook Express Macintosh Edition - 4.5 (0410) X-To: proteins@hgmp.mrc.ac.uk X-Priority: 3 To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk > THIS MESSAGE IS IN MIME FORMAT. Since your mail reader does not understand this format, some or all of this message may not be legible. --MS_Mac_OE_3040619735_88923_MIME_Part Content-type: text/plain; charset="US-ASCII" Content-transfer-encoding: 7bit Postdoctoral position to study Polycomb-group-dependent transcriptional silencing An NIH-funded postdoctoral position is available immediately to study the molecular/biochemical mechanisms by which Drosophila Polycomb-group (PcG) proteins maintain the transcriptional silence of target genes. To date, at least three multimeric PcG protein complexes have been identified. However, little is known about the biochemical activities of these complexes and the mechanisms by which they alter chromatin structure and maintain transcriptional silence of target genes. Current and future projects in our lab include purification and characterization of protein complexes that contain the PcG protein Enhancer of zeste [E(z)], and to initiate functional analysis of the components of these complexes. More information and a list of recent publications from our lab is available at the following web page: http://www.smu.edu/~biology/jones.html. Individuals with expertise in protein biochemistry, chromatin protein analysis, and/or molecular biology are encouraged to send an application, including a C.V. and the names and addresses of three referees, to: Dr. Richard Jones, Dept. of Biological Sciences, Southern Methodist University, Dallas, TX 75275-0376. email: rjones@mail.smu.edu --MS_Mac_OE_3040619735_88923_MIME_Part Content-type: text/html; charset="US-ASCII" Content-transfer-encoding: quoted-printable Postdoc position: Polycomb-group-dependent transcriptional silencing= Postdoctoral position to study Polycomb-group-dependent = transcriptional silencing

An NIH-funded postdoctoral position is available immediately to study the m= olecular/biochemical mechanisms by which Drosophila Polycomb-group (P= cG) proteins maintain the transcriptional silence of target genes.  To = date, at least three multimeric PcG protein complexes have been identified. =  However, little is known about the biochemical activities of these com= plexes and the mechanisms by which they alter chromatin structure and mainta= in transcriptional silence of target genes.  Current and future project= s in our lab include purification and characterization of protein complexes = that contain the PcG protein Enhancer of zeste [E(z)], and to initiate funct= ional analysis of the components of these complexes.  More information = and a list of recent publications from our lab is available at the following= web page: http://www.smu.edu/~biology/jones.html.  

Individuals with expertise in protein biochemistry, chromatin protein analy= sis, and/or molecular biology are encouraged to send an application, includi= ng a C.V. and the names and addresses of three referees, to:

Dr. Richard Jones, Dept. of Biological Sciences, Southern Methodist Univers= ity, Dallas, TX 75275-0376.  email: rjones@mai= l.smu.edu --MS_Mac_OE_3040619735_88923_MIME_Part-- --- From owner-proteins@hgmp.mrc.ac.uk Tue May 9 15:15:51 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 981CF17B2E; Tue, 9 May 2000 15:15:50 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id PAA03799 for ; Tue, 9 May 2000 15:15:47 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id PAA15777 for proteins-list@hgmp.mrc.ac.uk; Tue, 9 May 2000 15:15:46 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: radha@imtech.ernet.in (Radha Chauhan) X-Newsgroups: bionet.molbio.proteins Subject: Guanidium unfolding Date: 9 May 2000 15:15:44 +0100 Organization: BIOSCI/MRC Human Genome Mapping Project Resource Centre Lines: 33 Message-ID: <39181CCF.5E9980F6@lion.imtech.ernet.in> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: niobium.hgmp.mrc.ac.uk 957881745 15771 193.62.192.80 (9 May 2000 14:15:45 GMT) X-Complaints-To: news@net.bio.net X-Received: from imtech.chd.nic.in (imtech.chd.nic.in [164.100.244.206]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with SMTP id PAA03694 for ; Tue, 9 May 2000 15:14:58 +0100 (BST) X-Received: from imtech.ernet.in by imtech.chd.nic.in (5.65v4.0/1.1.19.2/30Apr00-1133PM) id AA31853; Tue, 9 May 2000 19:47:47 +0500 X-Received: from [202.141.121.119] by imtech.ernet.in (5.65v4.0/1.1.19.2/30Apr00-1130PM) id AA09400; Tue, 9 May 2000 19:41:38 -0400 X-Mailer: Mozilla 4.04 [en] (Win95; I) X-To: proteins@hgmp.mrc.ac.uk, bhupesh@imtech.ernet.in To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Hi all! I am a novice with protein unfolding problems and am facing a strange problem: I wanted to unfold my protein with guanidium chloride. As a test, I added some protein (25 ug/ml) to 4 M guanidium in 20 mM tris ph 7.5. When I meaured fluorescence, the native protein gave a good signal, so did a small signal for the blank (buffer + 4 M guanidium). However, on with the test (protein+ 4 M guanidium), I got a perfect straight line at 0 intensity units. I wonder what's wrong? Any suggestions? Thanks in advance for all help, Bhupesh. 9-5-00. ****************************************** * * * Bhupesh Taneja, SRF (Graduate student) * * Institute of Microbial Technology, * * Sector 39-A, * * Chandigarh - 160 036. (INDIA). * * Ph: +91 172 695226 extn 494 * * * * Email : bhupesh@lion.imtech.ernet.in * * bhupesht@yahoo.com * http://www.bragg.imtech.ernet.in/bhupesh * * ****************************************** --- From owner-proteins@hgmp.mrc.ac.uk Tue May 9 15:25:33 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id D0BF517B33; Tue, 9 May 2000 15:25:32 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id PAA05020 for ; Tue, 9 May 2000 15:25:30 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id PAA15927 for proteins-list@hgmp.mrc.ac.uk; Tue, 9 May 2000 15:25:29 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: bhupesh@imtech.ernet.in (Bhupesh Taneja) X-Newsgroups: bionet.molbio.proteins Subject: Guanidium unfolding (fwd) Date: 9 May 2000 15:25:28 +0100 Organization: BIOSCI/MRC Human Genome Mapping Project Resource Centre Lines: 26 Message-ID: Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII X-Trace: niobium.hgmp.mrc.ac.uk 957882329 15925 193.62.192.80 (9 May 2000 14:25:29 GMT) X-Complaints-To: news@net.bio.net X-Received: from imtech.chd.nic.in (imtech.chd.nic.in [164.100.244.206]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with SMTP id PAA04805 for ; Tue, 9 May 2000 15:23:49 +0100 (BST) X-Received: from imtech.ernet.in by imtech.chd.nic.in (5.65v4.0/1.1.19.2/30Apr00-1133PM) id AA32173; Tue, 9 May 2000 19:52:20 +0500 X-Received: by imtech.ernet.in (5.65v4.0/1.1.19.2/30Apr00-1130PM) id AA09495; Tue, 9 May 2000 19:46:18 -0400 X-To: proteins@hgmp.mrc.ac.uk To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Hi all! This is with reference to my earlier mail. Please forward all replies to any of the addresses below and not to the "radha@imtech..... " address. Thanks again! Bhupesh. ****************************************** * * * Bhupesh Taneja, SRF (Graduate student) * * Institute of Microbial Technology, * * Sector 39-A, * * Chandigarh - 160 036. (INDIA). * * Ph: +91 172 695226 extn 494 * * * * Email : bhupesh@lion.imtech.ernet.in * * bhupesht@yahoo.com * http://www.bragg.imtech.ernet.in/bhupesh * * ****************************************** --- From owner-proteins@hgmp.mrc.ac.uk Tue May 9 17:21:35 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id A2DBD17B37; Tue, 9 May 2000 17:21:34 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id RAA20488 for ; Tue, 9 May 2000 17:21:32 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id RAA17479 for proteins-list@hgmp.mrc.ac.uk; Tue, 9 May 2000 17:21:32 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Luc CAMOIN X-Newsgroups: bionet.molbio.proteins Subject: reduction of cystines in proteins with tri-n-butylphosphine Date: Tue, 09 May 2000 18:23:02 +0200 Organization: CITI2 - Universite Rene Descartes, Paris Message-ID: Mime-Version: 1.0 Content-Type: text/plain; charset="US-ASCII" Content-Transfer-Encoding: 7bit User-Agent: Microsoft Outlook Express Macintosh Edition - 5.0 (1513) Lines: 14 To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Dear Netters, I'am looking advice about protein reduction of cystines in proteins with tri-n-butylphosphine. Does anyone can send me a detailed protocol to reduction/alkylation including any safety measure when using this reagent? Thanks in advance Luc From owner-proteins@hgmp.mrc.ac.uk Tue May 9 23:30:14 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 2070C17A78; Tue, 9 May 2000 23:30:12 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id XAA26489 for ; Tue, 9 May 2000 23:30:11 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id XAA00555 for proteins-list@hgmp.mrc.ac.uk; Tue, 9 May 2000 23:30:09 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: lca1@cornell.edu (Larry) X-Newsgroups: bionet.molbio.proteins Subject: Engineering a disulfide bond Date: 9 May 2000 22:21:13 GMT Organization: University of Wisconsin, Madison Lines: 10 Message-ID: <8fa30p$nb6$1@news.doit.wisc.edu> Mime-Version: 1.0 Content-Type: Text/Plain; charset=US-ASCII X-Trace: news.doit.wisc.edu 957910873 23910 128.104.48.185 (9 May 2000 22:21:13 GMT) X-Complaints-To: abuse@doit.wisc.edu X-Newsreader: WinVN 0.99.9 (Beta 3) (x86 32bit) To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk My thesis work involves studying the protein-protein interactions between two enzymes. One of the interaction domains is a short 69 amino acid coiled-coil motif. We've cloned and expressed this region in E. coli but want to engineer a disulfide bond to "close" off the loop (and make the motif more rigid). I know that disulfide bond formation is subject to many variables (correct phi, psi angles, etc.). Is there a computer or web-based program that will help me predict where I can insert two cysteines to create this disulfide bond? Thanks. From owner-proteins@hgmp.mrc.ac.uk Wed May 10 10:30:10 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 3AC4117A56; Wed, 10 May 2000 10:30:08 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id KAA12538 for ; Wed, 10 May 2000 10:30:07 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id KAA08296 for proteins-list@hgmp.mrc.ac.uk; Wed, 10 May 2000 10:30:05 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: James Fethiere X-Newsgroups: bionet.molbio.proteins Subject: Deglycosylation Date: Wed, 10 May 2000 11:28:01 +0200 Organization: MPI-Med Forschung Lines: 56 Message-ID: <39192BA1.CFE80F99@mpimf-heidelberg.mpg.de> Mime-Version: 1.0 Content-Type: multipart/alternative; boundary="------------93CC3DE7439F5380DEBEFEE4" X-Trace: news.urz.uni-heidelberg.de 957950881 26633 149.217.48.25 (10 May 2000 09:28:01 GMT) X-Complaints-To: usenet@news.urz.uni-heidelberg.de X-Mailer: Mozilla 4.07C-SGI [en] (X11; I; IRIX64 6.5 IP30) To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk --------------93CC3DE7439F5380DEBEFEE4 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit Somebody on this list once suggested to me to increase NaCl up to 0.7 M when deglycosylating with EndoF in order to avoid precipitation of my glycoprotein. I would like to know if Sialidases are also resistant to the high salt concentration. P.S. I am using a combination of EndoF2 and Sialidase C in 250 mM Na Acetate buffer pH 5 Thank you James -- ************************************************************************ * Dr. James Fethiere * * MPI-Medical Research Int'l Tel: +49-6221-486154 * * Ion Channel Structure Group Int'l Fax: +49-6221-486437 * * Jahnstrasse 29 email: james@mpimf-heidelberg.mpg.de * * 69120, Heidelberg * * Germany * ************************************************************************ --------------93CC3DE7439F5380DEBEFEE4 Content-Type: text/html; charset=us-ascii Content-Transfer-Encoding: 7bit Somebody on this list once suggested to me to increase NaCl up to 0.7 M when deglycosylating with EndoF in order to avoid precipitation of my glycoprotein.  I would like to know if Sialidases are also resistant to the high salt concentration.

P.S.  I am using a combination of EndoF2 and  Sialidase C in 250 mM Na Acetate buffer pH 5

Thank you

James 

-- 
************************************************************************
* Dr. James Fethiere                                                   *
* MPI-Medical Research            Int'l Tel: +49-6221-486154           *
* Ion Channel Structure Group     Int'l Fax: +49-6221-486437           *
* Jahnstrasse 29                  email: james@mpimf-heidelberg.mpg.de *
* 69120, Heidelberg                                                    *
* Germany                                                              *
************************************************************************
  --------------93CC3DE7439F5380DEBEFEE4-- From owner-proteins@hgmp.mrc.ac.uk Wed May 10 11:24:13 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id B36E317A56; Wed, 10 May 2000 11:24:12 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id LAA17837 for ; Wed, 10 May 2000 11:24:08 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id LAA08934 for proteins-list@hgmp.mrc.ac.uk; Wed, 10 May 2000 11:24:08 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: llewelyn wyn hynes X-Newsgroups: bionet.molbio.proteins Subject: TKX1 cells Date: Wed, 10 May 2000 11:22:37 +0100 Organization: BBSRC Biotechnology and Biological Sciences Research Council Lines: 8 Message-ID: <3919386D.5105BDD7@bbsrc.ac.uk> Mime-Version: 1.0 Content-Type: text/plain; charset=gb2312 Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.07 [en] (WinNT; I) To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk I am interested to contact people that have worked with the Stratagene cells TKX1, that allow tyrosine phosphorylation of a protein of interest in bacteria. We are having diffculties with the slow growth of the cells, and little expression of our protein in these cells. Many thanks. Donald Richards e-mail: donald.richards@bbsrc.ac.uk From owner-proteins@hgmp.mrc.ac.uk Wed May 10 16:36:11 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id E285117AC7; Wed, 10 May 2000 16:36:10 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id QAA21149 for ; Wed, 10 May 2000 16:36:08 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id QAA13047 for proteins-list@hgmp.mrc.ac.uk; Wed, 10 May 2000 16:36:07 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: DOCTORHIM@aol.com X-Newsgroups: bionet.molbio.proteins Subject: Re: Deglycosylation Date: 10 May 2000 16:36:06 +0100 Organization: BIOSCI/MRC Human Genome Mapping Project Resource Centre Lines: 6 Message-ID: Mime-Version: 1.0 Content-Type: text/plain; charset="US-ASCII" Content-Transfer-Encoding: 7bit X-Trace: niobium.hgmp.mrc.ac.uk 957972967 13045 193.62.192.80 (10 May 2000 15:36:07 GMT) X-Complaints-To: news@net.bio.net X-Received: from imo21.mx.aol.com (imo21.mx.aol.com [152.163.225.65]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id QAA21132 for ; Wed, 10 May 2000 16:36:03 +0100 (BST) X-Received: from DOCTORHIM@aol.com by imo21.mx.aol.com (mail_out_v26.7.) id i.a7.3b5f898 (3962); Wed, 10 May 2000 11:34:46 -0400 (EDT) X-To: james@mpimf-heidelberg.mpg.de, proteins@hgmp.mrc.ac.uk X-Mailer: AOL 4.0 for Mac - Post-GM sub 53 To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk James, I assume you are using the recombinant Clostridium perfringens sialidase from Prozyme(Sialidase C). This enzyme will work perfectly well in 0.7 M NaCl. How well does the Endo F2 work at this concentration? --- From owner-proteins@hgmp.mrc.ac.uk Wed May 10 16:54:34 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id D678717A9C; Wed, 10 May 2000 16:54:33 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id QAA23083 for ; Wed, 10 May 2000 16:54:31 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id QAA13216 for proteins-list@hgmp.mrc.ac.uk; Wed, 10 May 2000 16:54:30 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: hroychow@NMSU.Edu ("Hiranya S. Roychowdhury") X-Newsgroups: bionet.molbio.proteins Subject: Re: Engineering a disulfide bond Date: 10 May 2000 16:54:29 +0100 Organization: BIOSCI/MRC Human Genome Mapping Project Resource Centre Lines: 25 Message-ID: <200005101437.IAA76102@nestor.NMSU.Edu> Mime-Version: 1.0 Content-Type: text/plain; charset="us-ascii" X-Trace: niobium.hgmp.mrc.ac.uk 957974069 13214 193.62.192.80 (10 May 2000 15:54:29 GMT) X-Complaints-To: news@net.bio.net X-Received: from bubba.NMSU.Edu (bubba.NMSU.Edu [128.123.3.39]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id QAA23067 for ; Wed, 10 May 2000 16:54:26 +0100 (BST) X-Received: from dns1.NMSU.Edu (dns1.NMSU.Edu [128.123.3.5]) by bubba.NMSU.Edu (8.9.3/8.9.0) with ESMTP id JAA19385; Wed, 10 May 2000 09:54:10 -0600 (MDT) X-Received: from nestor.NMSU.Edu (nestor.NMSU.Edu [128.123.34.146]) by dns1.NMSU.Edu (8.9.3/8.9.0) with ESMTP id IAA28134; Wed, 10 May 2000 08:37:54 -0600 (MDT) X-Received: from peaberry.nmsu.edu (peaberry.NMSU.Edu [128.123.96.3]) by nestor.NMSU.Edu (8.9.3/8.9.0) with SMTP id IAA76102; Wed, 10 May 2000 08:37:53 -0600 X-Sender: hroychow@cnmailsvr.nmsu.edu X-Mailer: Windows Eudora Light Version 1.5.2 X-To: lca1@cornell.edu (Larry) X-Cc: proteins@hgmp.mrc.ac.uk To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk I have not used it yet, but the PredictProtein server (http://cubic.bioc.columbia.edu/predictprotein) may be able to to help you. At 10:21 PM 5/9/00 GMT, Larry wrote: >My thesis work involves studying the protein-protein interactions >between two enzymes. One of the interaction domains is a short 69 >amino acid coiled-coil motif. We've cloned and expressed this region >in E. coli but want to engineer a disulfide bond to "close" off the >loop (and make the motif more rigid). I know that disulfide bond formation >is subject to many variables (correct phi, psi angles, etc.). Is >there a computer or web-based program that will help me predict where >I can insert two cysteines to create this disulfide bond? Thanks. > > > Dr. Hiranya Sankar Roychowdhury GENE LAB/ EPPWS New Mexico State University Las Cruces, NM 88003 Ph. (505) 646-5785 hroychow@nmsu.edu --- From owner-proteins@hgmp.mrc.ac.uk Wed May 10 18:11:22 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 6007317A53; Wed, 10 May 2000 18:11:20 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id SAA00560 for ; Wed, 10 May 2000 18:11:18 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id SAA14267 for proteins-list@hgmp.mrc.ac.uk; Wed, 10 May 2000 18:11:17 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: "Gary" X-Newsgroups: bionet.molbio.proteins Subject: Stripping nitrocellulose membranes containing phosphoprotein Date: Wed, 10 May 2000 13:06:01 -0400 Organization: Wayne State University Lines: 11 Message-ID: <8fc4th$hhu$1@cwis-1.wayne.edu> Mime-Version: 1.0 Content-Type: text/plain; charset=US-ASCII. Content-Transfer-Encoding: 7bit X-Trace: cwis-1.wayne.edu 957978353 17982 146.9.12.243 (10 May 2000 17:05:53 GMT) X-Complaints-To: usenet@REMOVE_THIS_TO_E-MAIL.wayne.edu X-Priority: 3 X-MSMail-Priority: Normal X-Newsreader: Microsoft Outlook Express 5.00.2919.6600 X-MimeOLE: Produced By Microsoft MimeOLE V5.00.2919.6600 To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk I'm trying to strip a nitrocellulose membrane [10 mM b-mercaptoethanol, 0.1% SDS, 20 mM Tris, pH 6.5] to restain it with another antibody . The signals I 'm looking for are phosphoproteins. I appear to be losing more signal than I would expect. Is there a chance that I'm hydrolyzing the phosphate group during stripping? How do I avoid this? Thanks. Gary From owner-proteins@hgmp.mrc.ac.uk Thu May 11 00:20:30 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 6ECB417A68; Thu, 11 May 2000 00:20:29 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id AAA02775 for ; Thu, 11 May 2000 00:20:27 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id AAA27332 for proteins-list@hgmp.mrc.ac.uk; Thu, 11 May 2000 00:20:26 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: hines@pharm.med.upenn.edu (John Hines) X-Newsgroups: bionet.molbio.proteins Subject: light activation of Calphostin C Date: Wed, 10 May 2000 19:18:08 -0400 Organization: University of Pennsylvania Lines: 21 Message-ID: To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk I ordered some Calphostin C (the PKC inhibitor) to use in signal transduction experiments in transfected COS cells. I found out that this compound must be "activated by light" before it will achieve maximum inhibition. What does this mean? Does it mean I have to: - expose the stock solution to light before adding it to the cells, and then incubate the cells in the dark? - incubate the cells in the presence of light while they are exposed to the inhibitor? I guess I was a bit naive -- if anyone works with Calphostin C in experiments like this, I'd love to hear how you "light-activate" the compound. John hines@pharm.med.upenn.edu From owner-proteins@hgmp.mrc.ac.uk Thu May 11 11:43:05 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 03B1917AAB; Thu, 11 May 2000 11:43:04 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id LAA22830 for ; Thu, 11 May 2000 11:43:03 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id LAA06013 for proteins-list@hgmp.mrc.ac.uk; Thu, 11 May 2000 11:43:00 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: "Gabor E. Tusnady" X-Newsgroups: bionet.molbio.proteins Subject: Re: Engineering a disulfide bond Date: Thu, 11 May 2000 12:42:59 +0200 Organization: IIF Lines: 36 Message-ID: References: <200005101437.IAA76102@nestor.NMSU.Edu> Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII In-Reply-To: <200005101437.IAA76102@nestor.NMSU.Edu> To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Hi, There was an article about the effect of the sequential environment of cysteins to the disulfide forming potential of cysteins: Fiser, A., Cserzo, M., Tudos, E., and Simon, I. (1992) Different sequence environment of cysteins and half cystines in proteins: Application to predict disulfide forming residues, FEBS Letters 302, 117-120. There is no html page or program, but using the table given in the article you can easily calculate the potential of the disulfide forming. Regards, Gabor Gabor E. Tusnady, PhD | e-mail: tusi@enzim.hu Institute of Enzymology, BRC | www: http://www.enzim.hu/~tusi Hungarian Academy of Sciences | tel: (36-1) 466-6533/158 H-1113 Budapest Karolina ut 29, HUNGARY | fax: (36-1) 466-5465 ************************************************************************** At 10:21 PM 5/9/00 GMT, Larry wrote: >My thesis work involves studying the protein-protein interactions >between two enzymes. One of the interaction domains is a short 69 >amino acid coiled-coil motif. We've cloned and expressed this region >in E. coli but want to engineer a disulfide bond to "close" off the >loop (and make the motif more rigid). I know that disulfide bond formation >is subject to many variables (correct phi, psi angles, etc.). Is >there a computer or web-based program that will help me predict where >I can insert two cysteines to create this disulfide bond? Thanks. From owner-proteins@hgmp.mrc.ac.uk Thu May 11 13:36:36 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 4063D17A65; Thu, 11 May 2000 13:36:34 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id NAA04418 for ; Thu, 11 May 2000 13:36:32 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id NAA07383 for proteins-list@hgmp.mrc.ac.uk; Thu, 11 May 2000 13:36:31 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: vale@ssmain.uniss.it (Valeria Maida) X-Newsgroups: bionet.molbio.proteins Subject: blue native PAGE and molecular mass analisys Date: 11 May 2000 13:36:30 +0100 Organization: BIOSCI/MRC Human Genome Mapping Project Resource Centre Lines: 15 Message-ID: <4.3.0.20000511110838.00a97660@ssmain.uniss.it> Mime-Version: 1.0 Content-Type: text/plain; charset="us-ascii"; format=flowed X-Trace: niobium.hgmp.mrc.ac.uk 958048591 7381 193.62.192.80 (11 May 2000 12:36:31 GMT) X-Complaints-To: news@net.bio.net X-Received: from herakles.uci.kun.nl (herakles.uci.kun.nl [131.174.93.48]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id NAA04400 for ; Thu, 11 May 2000 13:36:28 +0100 (BST) X-Received: from g. kappe.fmw.kun.nl (m38-106.fmw.kun.nl [131.174.144.235]) by mailserver.uci.kun.nl (PMDF V5.2-31 #42470) with ESMTP id <0FUE0010GB0GUS@mailserver.uci.kun.nl> for proteins@net.bio.net; Thu, 11 May 2000 14:36:16 +0200 (MET DST) X-Sender: vale@ssmain.uniss.it X-To: proteins@hgmp.mrc.ac.uk X-Mailer: QUALCOMM Windows Eudora Version 4.3 To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Hallo everybody! I'm working on a protein that likely forms tetramers and dimers in equilibrium. The monomer molecular mass is around 12. I would like to move the equilibrium towards the dimers or tetramer but I have many difficult by gel filtration to obtain the separation of the two species: tipically on a Superose12 the sample elute in the trimer position instead of having two defined peaks. I've also tried to use a S-100 resin but results are not always clear. Now I'm trying a longer and sharper coloumn, but I heard about blue native PAGE as a system to resolve different species in solution. Anyone has experience in that and could give some suggestion? I don't know anything about this tecnique. Thank you very much Valeria --- From owner-proteins@hgmp.mrc.ac.uk Thu May 11 15:39:44 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 9971217A43; Thu, 11 May 2000 15:39:43 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id PAA18593 for ; Thu, 11 May 2000 15:39:41 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id PAA08952 for proteins-list@hgmp.mrc.ac.uk; Thu, 11 May 2000 15:39:40 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: " J. Martinez-Irujo" X-Newsgroups: bionet.molbio.proteins Subject: Re: blue native PAGE and molecular mass analisys Date: Thu, 11 May 2000 16:43:48 +0200 Organization: Universidad de Navarra Lines: 30 Message-ID: <391AC723.5C28263E@unav.es> References: <4.3.0.20000511110838.00a97660@ssmain.uniss.it> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: cti2.cti.unav.es 958055975 25797 159.237.64.64 (11 May 2000 14:39:35 GMT) X-Complaints-To: usenet@cti2.cti.unav.es X-Mailer: Mozilla 4.5 [en] (Win95; I) X-Accept-Language: es-ES To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Valeria Maida wrote: > Hallo everybody! > I'm working on a protein that likely forms tetramers and dimers in > equilibrium. The monomer molecular mass is around 12. I would like to move > the equilibrium towards the dimers or tetramer but I have many difficult by > gel filtration to obtain the separation of the two species: tipically on a > Superose12 the sample elute in the trimer position instead of having two > defined peaks. For this protein I would try a Superdex 75 filtration column; it gives much better resolution than Superose 12 (try several sal concentrations). Bad news are that this column is very expensive, and sometimes you may need to use two of them in tandem (for example Superdex 75 + Superdex 200), specially when separating dimer/monomer species. If native electrophoresis works well in your hands, please send a mail to this newsgroup, it could help many people... -- Juan J. Martinez Irujo Departamento de Bioquimica Universidad de Navarra Pamplona, Spain. From owner-proteins@hgmp.mrc.ac.uk Fri May 12 09:42:09 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id CF71517A93; Fri, 12 May 2000 09:42:07 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id JAA17829 for ; Fri, 12 May 2000 09:42:04 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id JAA00604 for proteins-list@hgmp.mrc.ac.uk; Fri, 12 May 2000 09:42:03 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Frank Fuerst X-Newsgroups: bionet.molbio.proteins Subject: Re: blue native PAGE and molecular mass analisys Date: Fri, 12 May 2000 10:42:19 +0200 Lines: 22 Message-ID: <8fgg4p$aoada$1@fu-berlin.de> References: <4.3.0.20000511110838.00a97660@ssmain.uniss.it> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: fu-berlin.de 958120921 11282858 141.89.201.37 (16 955) X-Mailer: Mozilla 4.51 [de]C-CCK-MCD QXW03200 (WinNT; I) X-Accept-Language: de,en To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Valeria Maida wrote: > > Hallo everybody! > I'm working on a protein that likely forms tetramers and dimers in > equilibrium. The monomer molecular mass is around 12. I would like to move > the equilibrium towards the dimers or tetramer but I have many difficult by > gel filtration to obtain the separation of the two species: tipically on a > Superose12 the sample elute in the trimer position instead of having two > defined peaks. It is also possible that the kinetics of association and dissociation is faster than the retention time - then you'll never see dimeric or tetrameric peaks. This is because one molecule is and runs as a dimer for a fraction (depending on the equilibrium constant) of the time, and a tetramer for the rest. If dimers and tetramers each account for about half of the protein, you'll see a broad peak with the retention time of a trimer... Yours, Frank -- Die Verwendung von mehreren Ausrufezeichen macht die Aussage nicht ausrufender sondern ausufernder. [Michael Bauer in dnq] From owner-proteins@hgmp.mrc.ac.uk Fri May 12 11:57:29 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 8DC7E17AC2; Fri, 12 May 2000 11:57:28 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id LAA02186 for ; Fri, 12 May 2000 11:57:26 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id LAA02344 for proteins-list@hgmp.mrc.ac.uk; Fri, 12 May 2000 11:57:25 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f Message-ID: <391BE3E4.CBC2290A@freeuk.com> From: ChenHA X-Mailer: Mozilla 4.7 [en] (Win95; I) X-Accept-Language: en MIME-Version: 1.0 X-Newsgroups: bionet.molbio.proteins Subject: Re: blue native PAGE and molecular mass analisys References: <4.3.0.20000511110838.00a97660@ssmain.uniss.it> <8fgg4p$aoada$1@fu-berlin.de> Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit Lines: 24 Date: Fri, 12 May 2000 11:58:44 +0100 X-Complaints-To: abuse@freeuk.net X-Trace: nnrp3.clara.net 958129044 212.126.153.98 (Fri, 12 May 2000 11:57:24 BST) To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Frank Fuerst wrote: > > It is also possible that the kinetics of association and dissociation > is faster than the retention time - then you'll never see dimeric or > tetrameric peaks. This is because one molecule is and runs as a dimer > for a fraction (depending on the equilibrium constant) of the time, > and a tetramer for the rest. If dimers and tetramers each account for > about half of the protein, you'll see a broad peak with the retention > time of a trimer... > That was my first reaction when I read that it came out a single peak. It may be possible to change the equilibrium by changing the pH and/or buffer composition. Very often this kind of rapid equilibrium is mediated by charged species, therefore changing the pH can shift the equilibrium. There are a number of papers on that, so do a paper search. > Yours, Frank > -- > Die Verwendung von mehreren Ausrufezeichen macht die Aussage nicht > ausrufender sondern ausufernder. [Michael Bauer in dnq] From owner-proteins@hgmp.mrc.ac.uk Fri May 12 12:30:11 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 163FF17AC2; Fri, 12 May 2000 12:30:09 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id MAA05840; Fri, 12 May 2000 12:30:07 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id MAA02755; Fri, 12 May 2000 12:30:06 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Tim Barnett X-Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: NAD coupling Date: Fri, 12 May 2000 07:14:45 -0400 Organization: Emory University Lines: 14 Message-ID: <391BE7A5.E2656FC8@bimcore.emory.edu> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: lendl.cc.emory.edu 958130215 28910 170.140.250.3 (12 May 2000 11:16:55 GMT) X-Complaints-To: abuse@emory.edu X-Mailer: Mozilla 4.5 [en]C-CCK-MCD (Win98; I) X-Accept-Language: en To: methods@hgmp.mrc.ac.uk, proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk I am after a method for coupling NAD to some sort of carrier molecule in order to prevent it from passively diffusing into cells. Any help would be greatly appreciated. -- Tim Barnett, PhD Postdoctoral Research Fellow Department of Microbiology and Immunology Rollins Research Center Emory University Atlanta, GA 30322 Ph: 404 727 0522 From owner-proteins@hgmp.mrc.ac.uk Fri May 12 16:30:22 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 57E8617ABC; Fri, 12 May 2000 16:30:21 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id QAA01862 for ; Fri, 12 May 2000 16:30:19 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id QAA05873 for proteins-list@hgmp.mrc.ac.uk; Fri, 12 May 2000 16:30:17 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Tim Barnett X-Newsgroups: bionet.molbio.proteins Subject: linking fusion proteins Date: Fri, 12 May 2000 11:17:08 -0700 Organization: Emory University Lines: 17 Message-ID: <391C4AA4.73E4F8C9@bimcore.emory.edu> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: lendl.cc.emory.edu 958145071 840 170.140.48.33 (12 May 2000 15:24:31 GMT) X-Complaints-To: abuse@emory.edu X-Mailer: Mozilla 4.61 [en] (Win98; I) X-Accept-Language: en To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk I am interested in designing a fusion of two proteins connected via a short amino acid linker region. Are there any rules of thumb for doing this, and if so, are there any good review articles discussing them. Thanks.... -- Tim Barnett, PhD Postdoctoral Research Fellow Department of Microbiology and Immunology Rollins Research Center Emory University 1510 Clifton Road Atlanta, GA 30322 Ph: (404) 727 0522 From owner-proteins@hgmp.mrc.ac.uk Sat May 13 02:50:22 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 3F6AE17A80; Sat, 13 May 2000 02:50:22 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id CAA18937 for ; Sat, 13 May 2000 02:50:20 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id CAA22039 for proteins-list@hgmp.mrc.ac.uk; Sat, 13 May 2000 02:50:19 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f Message-ID: <391CB52B.6813E47B@freeuk.com> From: ChenHA X-Mailer: Mozilla 4.7 [en] (Win95; I) X-Accept-Language: en MIME-Version: 1.0 X-Newsgroups: bionet.molbio.proteins Subject: Re: linking fusion proteins References: <391C4AA4.73E4F8C9@bimcore.emory.edu> Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit Lines: 30 Date: Sat, 13 May 2000 02:51:39 +0100 X-Complaints-To: abuse@freeuk.net X-Trace: nnrp4.clara.net 958182617 212.126.154.47 (Sat, 13 May 2000 02:50:17 BST) To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Tim Barnett wrote: > > I am interested in designing a fusion of two proteins connected via a > short amino acid linker region. Are there any rules of thumb for doing > this, and if so, are there any good review articles discussing them. > Thanks.... Can't think of any review articles on such an issue but you can always try a literature search. However, in general, you should try to avoid having a linker that can form some kind of structure, i.e. it should be a flexible loop. As such, you should think of residues that are frequently found in loops such as, IIRC, glycines, serines etc. Alternatively you can use the kind of linker sequences such as those found in GST fusion protein (since they obviously worked). > -- > Tim Barnett, PhD > Postdoctoral Research Fellow > Department of Microbiology and Immunology > Rollins Research Center > Emory University > 1510 Clifton Road > Atlanta, GA 30322 > > Ph: (404) 727 0522 From owner-proteins@hgmp.mrc.ac.uk Sun May 14 02:30:09 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 5B05C17A63; Sun, 14 May 2000 02:30:08 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id CAA19730 for ; Sun, 14 May 2000 02:30:06 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id CAA17012 for proteins-list@hgmp.mrc.ac.uk; Sun, 14 May 2000 02:30:04 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: M Malten X-Newsgroups: bionet.molbio.proteins Subject: synthetic substrate for protease assay Date: Sat, 13 May 2000 21:05:52 -0400 Organization: University of Waterloo Lines: 8 Message-ID: <391DFBF0.275B00A0@engmail.uwaterloo.ca> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.7 [en] (Win95; U) X-Accept-Language: en To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Hi, I am searching for a special synthetic substrate for a proteolytic assay (oligopeptide linked to a chromophore). I already checked Bachem, Calbiochem and Sigma, but they do not supply this substrate. Do you know other suppliers of synthetical substrates? Thanks, Marco From owner-proteins@hgmp.mrc.ac.uk Sun May 14 20:26:08 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 5982C17A43; Sun, 14 May 2000 20:26:07 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id UAA11237 for ; Sun, 14 May 2000 20:26:05 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id UAA29743 for proteins-list@hgmp.mrc.ac.uk; Sun, 14 May 2000 20:26:03 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: "Meißner Markus" X-Newsgroups: bionet.molbio.proteins Subject: Re: Stripping nitrocellulose membranes containing phosphoprotein Date: Sun, 14 May 2000 21:27:14 +0200 Organization: T-Online Lines: 22 Message-ID: <391EFE12.ED69FAB9@t-online.de> References: <8fc4th$hhu$1@cwis-1.wayne.edu> Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Trace: news.t-online.com 958332081 18 25421 320050865995-0001 000514 19:21:21 X-Complaints-To: abuse@t-online.de X-Sender: 320050865995-0001@t-dialin.net X-Mailer: Mozilla 4.05 [de]C-DT (Win95; I) To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Gary schrieb: > > I'm trying to strip a nitrocellulose membrane [10 mM b-mercaptoethanol, 0.1% > SDS, 20 mM Tris, pH 6.5] to restain it with another antibody . The signals > I 'm looking for are phosphoproteins. I appear to be losing more signal than > I would expect. Is there a chance that I'm hydrolyzing the phosphate group > during stripping? How do I avoid this? Thanks. > > Gary Hi Gary ! I do also a lot stripping, and Iīm using the same protocol as You describe here. Although Iīm not looking for Phosphoproteins, I encountered the same problem that the signal strength is decreasing due to stripping the Blot. Therefore I guess that itīs rather a general problem of destroying the relevant epitopes the antibodoies recognizes than a problem of hydrolyzing the Phosphates........ Hope this information helps You something..... Markus From owner-proteins@hgmp.mrc.ac.uk Mon May 15 06:41:34 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id A373617A5D; Mon, 15 May 2000 06:41:33 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id GAA20234 for ; Mon, 15 May 2000 06:41:31 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id GAA15438 for proteins-list@hgmp.mrc.ac.uk; Mon, 15 May 2000 06:41:30 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Tatjana Schwabe X-Newsgroups: bionet.molbio.proteins Subject: MALDI-software Date: Wed, 10 May 2000 22:21:25 +0200 Organization: Ruhr-Universitaet Bochum, Rechenzentrum Lines: 11 Message-ID: <3919C4C5.6591A722@ruhr-uni-bochum.de> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: sunu789.rz.ruhr-uni-bochum.de 958369285 8039 134.147.7.67 (15 May 2000 05:41:25 GMT) X-Mailer: Mozilla 4.7 [en] (WinNT; I) X-Accept-Language: en To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Hello! I am looking for an additional newsgroup concerned with MALDI-TOF MS and software used. Does anyone have a recommendation? Thank you very much, Tatjana From owner-proteins@hgmp.mrc.ac.uk Mon May 15 07:55:12 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id CD50917A5D; Mon, 15 May 2000 07:55:11 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id HAA23154 for ; Mon, 15 May 2000 07:55:09 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id HAA16275 for proteins-list@hgmp.mrc.ac.uk; Mon, 15 May 2000 07:55:08 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Peeter Toomik X-Newsgroups: bionet.molbio.proteins Subject: Re: synthetic substrate for protease assay Date: Mon, 15 May 2000 08:52:32 +0300 Lines: 16 Message-ID: <391F90A0.F15556DC@ut.ee> References: <391DFBF0.275B00A0@engmail.uwaterloo.ca> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: kadri.ut.ee 958373448 22883 193.40.8.208 (15 May 2000 06:50:48 GMT) X-Complaints-To: usenet@news.ut.ee X-Mailer: Mozilla 4.7 [en] (Win98; I) X-Accept-Language: en,pdf To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk http://www.chromogenix.se , and if you cannot find what you need in their catalog, then probably their distributor can help you. Peeter Toomik M Malten wrote: > Hi, > > I am searching for a special synthetic substrate for a proteolytic assay > (oligopeptide linked to a chromophore). I already checked Bachem, > Calbiochem and Sigma, but they do not supply this substrate. Do you know > other suppliers of synthetical substrates? > > Thanks, Marco From owner-proteins@hgmp.mrc.ac.uk Mon May 15 13:09:20 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 0C76017A5B; Mon, 15 May 2000 13:09:19 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id NAA23140 for ; Mon, 15 May 2000 13:09:18 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id NAA20243 for proteins-list@hgmp.mrc.ac.uk; Mon, 15 May 2000 13:09:16 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: vale@ssmain.uniss.it (Valeria Maida) X-Newsgroups: bionet.molbio.proteins Subject: &replyto=391BE3E4.CBC2290A@freeuk.com&subject=Re: blue native PAGE and molecular mass analisys Date: 15 May 2000 13:09:15 +0100 Organization: BIOSCI/MRC Human Genome Mapping Project Resource Centre Lines: 15 Message-ID: <4.3.0.20000515140623.00a74210@ssmain.uniss.it> Mime-Version: 1.0 Content-Type: text/plain; charset="us-ascii"; format=flowed X-Trace: niobium.hgmp.mrc.ac.uk 958392555 20240 193.62.192.80 (15 May 2000 12:09:15 GMT) X-Complaints-To: news@net.bio.net X-Received: from herakles.uci.kun.nl (herakles.uci.kun.nl [131.174.93.48]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id NAA23127 for ; Mon, 15 May 2000 13:09:11 +0100 (BST) X-Received: from g. kappe.fmw.kun.nl (m38-106.fmw.kun.nl [131.174.144.235]) by mailserver.uci.kun.nl (PMDF V5.2-31 #42470) with ESMTP id <0FUL00DA1OF5NN@mailserver.uci.kun.nl> for proteins@net.bio.net; Mon, 15 May 2000 14:09:06 +0200 (MET DST) X-Sender: vale@ssmain.uniss.it X-To: proteins@hgmp.mrc.ac.uk X-Mailer: QUALCOMM Windows Eudora Version 4.3 To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk I agree (I mean there is a fast equilibrium between the two species) but as I want to prepare a sample for NMR, I can NOT play too much with pH and also with salt concentration. By the way, what I observed is that increasing the amount of NaCl the gel filtration peak move towards the dimeric form but NMR told us that the dimeric specie seems a molten globule one, even if the tetramer is well folded. So my problem is to have one specie (dimer or tetramer) AND well folded AND, at the same time, omogeneous. Gel filtration seems not good to test omogeneity, maybe either BN-page is not, I don't know. Any idea how to move the equilibrium towards a well folded tetramer or dimer (not using chaotrops)? thank you for the suggestions Vale --- From owner-proteins@hgmp.mrc.ac.uk Tue May 16 00:40:12 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 99C9917A5F; Tue, 16 May 2000 00:40:10 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id AAA27436 for ; Tue, 16 May 2000 00:40:08 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id AAA07153 for proteins-list@hgmp.mrc.ac.uk; Tue, 16 May 2000 00:40:06 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: phains@NOSPAMproteome.org.au (Peter Hains) X-Newsgroups: bionet.molbio.proteins Subject: Re: MALDI-software Date: Mon, 15 May 2000 22:35:39 GMT Organization: APAF Lines: 34 Message-ID: <8fq1kb$c1b$1@sunb.ocs.mq.edu.au> References: <3919C4C5.6591A722@ruhr-uni-bochum.de> X-Newsreader: News Xpress 2.01 To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Dear Tatjana, try sci.techniques.mass-spec Peter. In article <3919C4C5.6591A722@ruhr-uni-bochum.de>, Tatjana Schwabe wrote: >Hello! > >I am looking for an additional newsgroup concerned with MALDI-TOF MS >and software used. Does anyone have a recommendation? > >Thank you very much, > >Tatjana > > > I'm afraid I don't have a clever saying to put here. Remove NOSPAM to e-mail me. Peter Hains (PhD) Ph. +61 2 9850 6216 Australian Proteome Analysis Facility Fax. +61 2 9850 6200 Level 4 Building F7B Macquarie University, Sydney 2109 From owner-proteins@hgmp.mrc.ac.uk Tue May 16 01:44:53 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 04E7117A5F; Tue, 16 May 2000 01:44:51 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id BAA00147 for ; Tue, 16 May 2000 01:44:49 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id BAA07942 for proteins-list@hgmp.mrc.ac.uk; Tue, 16 May 2000 01:44:48 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f Message-ID: <39209A58.D42B369C@freeuk.com> From: ChenHA X-Mailer: Mozilla 4.7 [en] (Win95; I) X-Accept-Language: en MIME-Version: 1.0 X-Newsgroups: bionet.molbio.proteins Subject: Re: blue native PAGE andmolecular mass analisys References: <4.3.0.20000515140623.00a74210@ssmain.uniss.it> Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit Lines: 69 Date: Tue, 16 May 2000 01:46:16 +0100 X-Complaints-To: abuse@freeuk.net X-Trace: nnrp3.clara.net 958437886 212.126.151.211 (Tue, 16 May 2000 01:44:46 BST) To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Valeria Maida wrote: > > I agree (I mean there is a fast equilibrium between the two species) but as > I want to prepare a sample for NMR, I can NOT play too much with pH and > also with salt concentration. You can play about with the pH and salt concentration for NMR, as long as your protein doesn't precipitate at different pH. High pH and salt concentration is not good for NMR, but still doable. Everything depends on your protein, although for a tetramer, I suppose you must optimise your condition, but I think there could be a reasonable pH range within which you could try (say 2-7?) > By the way, what I observed is that > increasing the amount of NaCl the gel filtration peak move towards the > dimeric form but NMR told us that the dimeric specie seems a molten globule > one, even if the tetramer is well folded. This tells me that the protein-protein interaction is mediated by charged species. I would therefore suspect histidines, glutamates or aspartates. At a simplistic level, if it is glutamates or aspartates, at low pH when these residues are protonated, you should get only dimer. If it is histidines, you can try higher pH. I don't know how you can tell if the dimeric specie is a molten globule (this is just my ignorance, but is there such a thing as dimeric molten globule?). Part of the structure (probably the protein-protein interaction site) may simply be more flexible as a dimer, but fold into a stable structure when forming tetramer. Are you just looking at chemical shift changes, or by other kinds of analysis? > So my problem is to have one > specie (dimer or tetramer) AND well folded AND, at the same time, > omogeneous. Gel filtration seems not good to test omogeneity, maybe either > BN-page is not, I don't know. > Any idea how to move the equilibrium towards a well folded tetramer or > dimer (not using chaotrops)? If I'm correct in my thinking, there is no point in separating the dimer and tetramer by gel filtration, simply because if they are in equilibrium, given enough time, you will get both dimer and tetramers reappearing. Therefore I think playing with pH and salt concentration will be useful. If it is charge-charge interaction, low salt and buffer concentration will enhance such interaction. You can therefore use minimal buffer concentration (say 0 - 5 mM ?) and see if you get more tetramer. Also, as I said before, changing the pH is likely to shift the equilibrium if it is mediated by charged specie, therefore the easiest thing to do (do a pH titration, increasing/decreasing by ~0.5 pH unit for each titration point). You can also try mutagenesis of specific residue which can enhance or destroy the protein-protein interaction. However, that may be a lot of work if you are not familiar with it. Crosslinking of the subunits is possible but might be difficult or the result useless. Other people may have better suggestions. > thank you for the suggestions > Vale > > --- From owner-proteins@hgmp.mrc.ac.uk Tue May 16 05:41:32 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 0CC2717A5F; Tue, 16 May 2000 05:41:31 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id FAA17659; Tue, 16 May 2000 05:41:30 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id FAA10599; Tue, 16 May 2000 05:41:25 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Isidore Rigoutsos X-Newsgroups: bionet.software,bionet.molbio.proteins Subject: pattern discovery servers, software, and downloadable content Date: Tue, 16 May 2000 00:50:25 -0400 Organization: IBM TJ Watson Research Center Lines: 36 Message-ID: <3920D391.928F8F45@us.ibm.com> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: news.btv.ibm.com 958452070 29542 9.2.101.30 (16 May 2000 04:41:10 GMT) X-Complaints-To: news@btv.ibm.com X-Mailer: Mozilla 4.7 [en] (Win95; U) X-Accept-Language: en To: bio-software@hgmp.mrc.ac.uk, proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Dear Colleagues, On behalf of my group, I would like to inform you that we recently released a set of web engines that provide access to several algorithms that we have developed and published. The web engines are accompanied by a collection of training data sets as well as a detailed on-line tutorial, and allow you to carry out a number of tasks that include pattern discovery in proteins and dna, multiple sequence alignment, association discovery (e.g. gene expression analysis, etc.), text mining in unformatted natural text, etc. In addition to the web servers, you can download code (in executable form) for our algorithms and associated utilities for three platforms (AIX, Windows 9x/NT, Sun/Solaris). Finally, we have also made available Bio-Dictionaries(TM) for 17 complete genomes. The web servers, executable code, training data sets, tutorial, and Bio-Dictionaries(TM) can be accessed through the group's main web page at http://www.research.ibm.com/bioinformatics/ We hope that you will find these tools useful and welcome your feedback on how we can improve our site and web engines. Sincerely, Isidore Rigoutsos Manager, Bioinformatics & Pattern Discovery Group IBM Thomas J Watson Research Center P.O. Box 218 Yorktown Heights, NY 10598, USA. Phone: (914) 945-1384 FAX: (914) 945-4104 From owner-proteins@hgmp.mrc.ac.uk Wed May 17 15:35:23 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 0221917A59; Wed, 17 May 2000 15:35:21 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id PAA12817 for ; Wed, 17 May 2000 15:35:19 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id PAA14117 for proteins-list@hgmp.mrc.ac.uk; Wed, 17 May 2000 15:35:18 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Chan Hei X-Newsgroups: bionet.molbio.proteins Subject: 26S proteasome assay kit Date: Wed, 17 May 2000 22:29:26 +0800 Organization: The Chinese University of Hong Kong Lines: 8 Message-ID: <3922ACC6.F4627349@mailserv.cuhk.edu.hk> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: justice.csc.cuhk.edu.hk 958573817 22289 137.189.255.197 (17 May 2000 14:30:17 GMT) X-Complaints-To: abuse@cuhk.edu.hk X-Mailer: Mozilla 4.72 [en] (Win98; I) X-Accept-Language: en,pdf To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Hi, i'm searching for assay kits of 26S proteasome, I've checked Affinity Research company but it's quite expensive and no details provided. Is there other suppliers for this kit, esp. fluorometric ones. Thanks for your help! Anna From owner-proteins@hgmp.mrc.ac.uk Wed May 17 17:29:34 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 8F7E217A43; Wed, 17 May 2000 17:29:33 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id RAA27372 for ; Wed, 17 May 2000 17:29:31 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id RAA15631 for proteins-list@hgmp.mrc.ac.uk; Wed, 17 May 2000 17:29:29 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: "Kunal Mukhopadhyay" X-Newsgroups: bionet.molbio.proteins Subject: SDS Page Date: Wed, 17 May 2000 12:30:24 -0400 Organization: Penn State University, Center for Academic Computing Lines: 45 Message-ID: <8fuhan$17ie@r02n01.cac.psu.edu> X-Priority: 3 X-MSMail-Priority: Normal X-Newsreader: Microsoft Outlook Express 5.00.2919.6700 X-MimeOLE: Produced By Microsoft MimeOLE V5.00.2919.6700 To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Hello again fellow scientists A note of gratitude to all who replied to my earlier query on BamHI. I was pleased to see that it worked and now am awaiting the confirmation (sequence report). In the meanwhile I had another query : While running SDS Page gels for my proteins, I find occasionally a "squirm" towards the end of the gel totally ruining the gel. I thought it might be due to the heat generated during the electrophoresis. However when running two gels parallel on different units, one ends up with Squirms, the other doesn't. Does anyone face similar problems? Is there a way to minimize the effect? I'll try to put up a picture of the gel if I can on the web. Thanks Kunal -- **************************************************************************** ****** What does it profit a man if he gains the world... but loses his soul? **************************************************************************** ****** Kunal Mukhopadhyay 52, 152 Davey Lab, Department Of Chemistry Pennsylavania State University, State College, PA 16802 USA (814) 865-1273 email : kunalm@psu.edu >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> >> From owner-proteins@hgmp.mrc.ac.uk Wed May 17 19:23:47 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 4B9ED17AE3; Wed, 17 May 2000 19:23:47 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id TAA08658 for ; Wed, 17 May 2000 19:23:45 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id TAA17077 for proteins-list@hgmp.mrc.ac.uk; Wed, 17 May 2000 19:23:43 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: "Kunal Mukhopadhyay" X-Newsgroups: bionet.molbio.proteins Subject: SDS page - the images Date: Wed, 17 May 2000 14:24:43 -0400 Organization: Penn State University, Center for Academic Computing Lines: 34 Message-ID: <8fuo11$1ftm@r02n01.cac.psu.edu> X-Priority: 3 X-MSMail-Priority: Normal X-Newsreader: Microsoft Outlook Express 5.00.2919.6700 X-MimeOLE: Produced By Microsoft MimeOLE V5.00.2919.6700 To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Here are the images of the gels that I mentioned in my earlier post. www.personal.psu.edu/kxm66/gels/tiffa1.jpg www.personal.psu.edu/kxm66/gels/tiffa2.jpg They certainly are nothing to brag about! Any comments would be appreciated. P.S Yes...I have run better ones, just in case anyone may wonder. -- **************************************************************************** ****** What does it profit a man if he gains the world... but loses his soul? **************************************************************************** ****** Kunal Mukhopadhyay 52, 152 Davey Lab, Department Of Chemistry Pennsylavania State University, State College, PA 16802 USA (814) 865-1273 email : kunalm@psu.edu From owner-proteins@hgmp.mrc.ac.uk Wed May 17 20:10:17 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 9FF5417A43; Wed, 17 May 2000 20:10:16 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id UAA14241 for ; Wed, 17 May 2000 20:10:14 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id UAA17567 for proteins-list@hgmp.mrc.ac.uk; Wed, 17 May 2000 20:10:12 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Nick Theodorakis X-Newsgroups: bionet.molbio.proteins Subject: Re: SDS page - the images Date: Wed, 17 May 2000 18:58:08 GMT Organization: Deja.com - Before you buy. Lines: 33 Message-ID: <8fuq3n$pi0$1@nnrp1.deja.com> References: <8fuo11$1ftm@r02n01.cac.psu.edu> X-Article-Creation-Date: Wed May 17 18:58:08 2000 GMT X-Http-User-Agent: Mozilla/4.7 (Macintosh; I; PPC) X-Http-Proxy: 1.0 x33.deja.com:80 (Squid/1.1.22) for client 128.151.71.1 X-MyDeja-Info: XMYDJUIDntheo To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk In article <8fuo11$1ftm@r02n01.cac.psu.edu>, "Kunal Mukhopadhyay" wrote: > Here are the images of the gels that I mentioned in my earlier post. > > www.personal.psu.edu/kxm66/gels/tiffa1.jpg > > www.personal.psu.edu/kxm66/gels/tiffa2.jpg > > They certainly are nothing to brag about! Any comments would be appreciated. > > P.S Yes...I have run better ones, just in case anyone may wonder. > My first guess is that you have an air bubble trapped on the bottom plate, disrupting the current flow around that region of the gel. What kind of a gel setup do you use? The older ones that formed the bottom by putting a spacer inside the plates were prone to this. In the "olden days" we used to keep either a syringe with a bent needle or a "bent" pasteur pipet around so that we could squirt buffer up into that space after putting the gel in the tank in order to flush the bubbles away. Nick -- _______________________________________________ Nick Theodorakis nicholas_theodorakis@urmc.rochester.edu Sent via Deja.com http://www.deja.com/ Before you buy. From owner-proteins@hgmp.mrc.ac.uk Wed May 17 20:17:53 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 4758417AC7; Wed, 17 May 2000 20:17:52 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id UAA14863 for ; Wed, 17 May 2000 20:17:50 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id UAA17692 for proteins-list@hgmp.mrc.ac.uk; Wed, 17 May 2000 20:17:49 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Chris LaRosa X-Newsgroups: bionet.molbio.proteins Subject: Re: SDS Page Date: Wed, 17 May 2000 14:21:02 -0500 Organization: University of Nebraska-Lincoln Lines: 5 Message-ID: <3922F11E.151D04D7@biocomp.unl.edu> References: <8fuhan$17ie@r02n01.cac.psu.edu> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.72 [en] (Win95; I) X-Accept-Language: en To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk the squirms look like bubbles. One gel appears to have a huge bubble probably at the gel, buffer interface on the bottom, like another commenter mentioned, the other looks like a bubble formed in the middle of the gel. Even a tiny bubble can cause are large disruption in gel polymerization. From owner-proteins@hgmp.mrc.ac.uk Wed May 17 21:44:32 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id CF32117B00; Wed, 17 May 2000 21:44:30 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id VAA21740 for ; Wed, 17 May 2000 21:44:29 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id VAA27492 for proteins-list@hgmp.mrc.ac.uk; Wed, 17 May 2000 21:44:28 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: hroychow@nmsu.edu ("Hiranya S. Roychowdhury") X-Newsgroups: bionet.molbio.proteins Subject: Re: SDS page - the images Date: 17 May 2000 21:44:27 +0100 Organization: BIOSCI/MRC Human Genome Mapping Project Resource Centre Lines: 56 Message-ID: <200005172044.OAA295134@nestor.NMSU.Edu> Mime-Version: 1.0 Content-Type: text/plain; charset="us-ascii" X-Trace: niobium.hgmp.mrc.ac.uk 958596267 27490 193.62.192.80 (17 May 2000 20:44:27 GMT) X-Complaints-To: news@net.bio.net X-Received: from bubba.NMSU.Edu (bubba.NMSU.Edu [128.123.3.39]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id VAA21728 for ; Wed, 17 May 2000 21:44:23 +0100 (BST) X-Received: from dns1.NMSU.Edu (dns1.NMSU.Edu [128.123.3.5]) by bubba.NMSU.Edu (8.9.3/8.9.0) with ESMTP id OAA28008; Wed, 17 May 2000 14:44:16 -0600 (MDT) X-Received: from nestor.NMSU.Edu (nestor.NMSU.Edu [128.123.34.146]) by dns1.NMSU.Edu (8.9.3/8.9.0) with ESMTP id OAA02379; Wed, 17 May 2000 14:44:17 -0600 (MDT) X-Received: from peaberry.nmsu.edu (peaberry.NMSU.Edu [128.123.96.3]) by nestor.NMSU.Edu (8.9.3/8.9.0) with SMTP id OAA295134; Wed, 17 May 2000 14:44:16 -0600 X-Sender: hroychow@cnmailsvr.nmsu.edu X-Mailer: Windows Eudora Light Version 1.5.2 X-To: Nick Theodorakis X-Cc: proteins@hgmp.mrc.ac.uk To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk I wonder! A few air bubbles would certainly distort the run, but the picture is like nothing I have seen before (and I have more than a little experience in SDS-PAGE). Is it possible that somehow the gel is polymerizing at significantly diffrent rate in the middle? More appropriately, the the gel seems to be of a higher %-age (hence the retardation) where the stained band is "frowning" so much. At 06:58 PM 5/17/00 GMT, Nick Theodorakis wrote: >In article <8fuo11$1ftm@r02n01.cac.psu.edu>, > "Kunal Mukhopadhyay" wrote: >> Here are the images of the gels that I mentioned in my earlier post. >> >> www.personal.psu.edu/kxm66/gels/tiffa1.jpg >> >> www.personal.psu.edu/kxm66/gels/tiffa2.jpg >> >> They certainly are nothing to brag about! Any comments would be appreciated. >> >> P.S Yes...I have run better ones, just in case anyone may wonder. >> > >My first guess is that you have an air bubble trapped on the bottom >plate, disrupting the current flow around that region of the gel. > >What kind of a gel setup do you use? The older ones that formed the >bottom by putting a spacer inside the plates were prone to this. In the >"olden days" we used to keep either a syringe with a bent needle or a >"bent" pasteur pipet around so that we could squirt buffer up into that >space after putting the gel in the tank in order to flush the bubbles >away. > >Nick > >-- >_______________________________________________ >Nick Theodorakis >nicholas_theodorakis@urmc.rochester.edu > > >Sent via Deja.com http://www.deja.com/ >Before you buy. > Dr. Hiranya Sankar Roychowdhury GENE LAB/ EPPWS New Mexico State University Las Cruces, NM 88003 Ph. (505) 646-5785 hroychow@nmsu.edu --- From owner-proteins@hgmp.mrc.ac.uk Wed May 17 21:50:48 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 20E8217AB6; Wed, 17 May 2000 21:50:46 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id VAA22169 for ; Wed, 17 May 2000 21:50:44 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id VAA27499 for proteins-list@hgmp.mrc.ac.uk; Wed, 17 May 2000 21:50:43 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Chris LaRosa X-Newsgroups: bionet.molbio.proteins Subject: Re: SDS page - the images Date: Wed, 17 May 2000 15:53:56 -0500 Organization: University of Nebraska-Lincoln Lines: 14 Message-ID: <392306E4.B1AD6922@biocomp.unl.edu> References: <200005172044.OAA295134@nes