From owner-proteins@hgmp.mrc.ac.uk Tue Feb 1 11:20:14 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id C4B8017A76; Tue, 1 Feb 2000 11:20:13 +0000 (GMT) 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 LAA03638 for ; Tue, 1 Feb 2000 11:20:10 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id LAA27734 for proteins-list@hgmp.mrc.ac.uk; Tue, 1 Feb 2000 11:20:09 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: BHKim X-Newsgroups: bionet.molbio.proteins Subject: Re: PMSF dire straits Date: Tue, 01 Feb 2000 12:21:34 +0100 Organization: Zentrum fuer Datenverarbeitung Lines: 19 Message-ID: <3896C1BE.64F273F2@uni-tuebingen.de> References: <86tjvs$81$1@bgtnsc03.worldnet.att.net> <3895BDFC.BBBA94A7@uni-konstanz.de> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: newsserv.zdv.uni-tuebingen.de 949403913 22092 134.2.89.90 (1 Feb 2000 11:18:33 GMT) X-Complaints-To: usenet@newsserv.uni-tuebingen.de X-Mailer: Mozilla 4.6 [de] (WinNT; I) X-Accept-Language: de To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk "Frank O. Fackelmayer" schrieb: > > HOWEVER, stability of PMSF is hardly ever a concern because its mode of action. > It covalently modifies the active center of many (serine) proteases, and makes > them proteolytically inactive. This modification is fast, and once it happened it > is irreversible, so you really donut have to care about breakdown of the excess > PMSF afterwards. > Frank I'm not sure, but I've heared that it can be reversible when there is reducing power (like DTT) in the solution. Byung-Hoon From owner-proteins@hgmp.mrc.ac.uk Tue Feb 1 16:13:37 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id EE00E17B60; Tue, 1 Feb 2000 16:13:34 +0000 (GMT) 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 QAA06915 for ; Tue, 1 Feb 2000 16:13:33 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id QAA01581 for proteins-list@hgmp.mrc.ac.uk; Tue, 1 Feb 2000 16:13:31 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: gregery1@indiatimes.com X-Newsgroups: bionet.molbio.proteins Subject: Only 14 Good Reps Needed ... Date: 1 Feb 2000 16:13:30 -0000 Organization: MRC Human Genome Mapping Project Resource Centre Lines: 13 Message-ID: X-Trace: niobium.hgmp.mrc.ac.uk 949421610 1578 193.62.192.80 (1 Feb 2000 16:13:30 GMT) X-Complaints-To: news@net.bio.net X-Received: from mail.confluent.fr (mail.confluent.fr [194.98.102.133]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with SMTP id QAA06904 for ; Tue, 1 Feb 2000 16:13:26 GMT X-Received: from comp6 (unverified [152.170.188.71]) by mail.confluent.fr (EMWAC SMTPRS 0.83) with SMTP id ; Tue, 01 Feb 2000 17:02:54 +0100 X-To: reps@hotmail.com To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk

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--- From owner-proteins@hgmp.mrc.ac.uk Tue Feb 1 20:13:14 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id C0EB817B37; Tue, 1 Feb 2000 20:13:13 +0000 (GMT) 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 UAA04795 for ; Tue, 1 Feb 2000 20:13:12 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id UAA04605 for proteins-list@hgmp.mrc.ac.uk; Tue, 1 Feb 2000 20:13:11 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: rwn Subject: Protease vs. proteinase X-Newsgroups: bionet.molbio.proteins Message-ID: <070dbf92.0e06949f@usw-ex0110-075.remarq.com> Lines: 8 Bytes: 295 X-Originating-Host: 192.38.35.212 Organization: http://www.remarq.com: The World's Usenet/Discussions Start Here X-Wren-Trace: eL+asrOq7afs7by5mvK8v6Lirruv9fui+uLp7vj4/rHj7LHs4K0= Date: Tue, 01 Feb 2000 13:05:41 -0800 X-Complaints-To: wrenabuse@remarq.com X-Trace: WReNphoon2 949435726 10.0.2.75 (Tue, 01 Feb 2000 12:08:46 PST) To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Can anyone give me an explanation when to use the term PROTEASE and when to use PROTEINASE. Is there a formal distinction? Thanks RWN * Sent from AltaVista http://www.altavista.com Where you can also find related Web Pages, Images, Audios, Videos, News, and Shopping. Smart is Beautiful From owner-proteins@hgmp.mrc.ac.uk Tue Feb 1 22:59:45 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id B399117BAF; Tue, 1 Feb 2000 21:58:08 +0000 (GMT) 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 VAA16438 for ; Tue, 1 Feb 2000 21:58:03 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id VAA14345 for proteins-list@hgmp.mrc.ac.uk; Tue, 1 Feb 2000 21:58:01 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Colin Rasmussen X-Newsgroups: bionet.molbio.proteins Subject: Re: PMSF dire straits Date: Tue, 01 Feb 2000 15:58:01 -0600 Organization: University of Saskatchewan Lines: 20 Message-ID: <389756E8.C98692B2@pombe.usask.ca> References: <86tjvs$81$1@bgtnsc03.worldnet.att.net> 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: tribune.usask.ca 949442279 25956 128.233.77.115 (1 Feb 2000 21:57:59 GMT) X-Complaints-To: usenet@tribune.usask.ca X-Mailer: Mozilla 4.7 (Macintosh; U; PPC) X-Accept-Language: en To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk simm wrote: > Dear Protein experts, > > I am currently running experiments requiring the use of PMSF. I have to > prepare fresh > solutions each time which as you may know takes up time and wastes a lot of > the > reagent. Is there a way to prepare a solution which is stable in storage? > are there other similar protease inhibitors? I am specifically interested in > stability of PMSF in methanol, acetone and isopropanol over time. I have > heard some colleagues store such solutions for up to a month, but I need > confirmation from the scientific body. We make concentrated stock in isopropanol. They store just fine. Colin From owner-proteins@hgmp.mrc.ac.uk Tue Feb 1 23:16:13 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id E3C0317B9F; Tue, 1 Feb 2000 22:20:27 +0000 (GMT) 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 WAA21959 for ; Tue, 1 Feb 2000 22:20:25 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id WAA14589 for proteins-list@hgmp.mrc.ac.uk; Tue, 1 Feb 2000 22:20:24 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) X-Newsgroups: bionet.molbio.proteins Subject: Re: Protease vs. proteinase Date: Tue, 01 Feb 2000 22:21:01 GMT Organization: UW-Madison Lines: 7 Message-ID: <877m4o$96v8$1@news.doit.wisc.edu> References: <070dbf92.0e06949f@usw-ex0110-075.remarq.com> Mime-Version: 1.0 Content-Type: text/plain; charset=KOI8-R Content-Transfer-Encoding: 8bit X-Trace: news.doit.wisc.edu 949443544 302056 144.92.18.232 (1 Feb 2000 22:19:04 GMT) X-Complaints-To: abuse@doit.wisc.edu X-Newsreader: News Xpress 2.01 X-No-Archive: yes To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk :Can anyone give me an explanation when to use the term :PROTEASE and when to use PROTEINASE. Is there a formal :distinction? These are synonyms. - Dima From owner-proteins@hgmp.mrc.ac.uk Wed Feb 2 02:42:53 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id E11E317A6F; Wed, 2 Feb 2000 02:42:52 +0000 (GMT) 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 CAA21255 for ; Wed, 2 Feb 2000 02:42:51 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id CAA17890 for proteins-list@hgmp.mrc.ac.uk; Wed, 2 Feb 2000 02:42:50 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: turner@sprcore.caregroup.harvard.edu (bradley turner) X-Newsgroups: bionet.molbio.proteins Subject: Re: PMSF dire straits Date: 2 Feb 2000 02:42:49 -0000 Organization: MRC Human Genome Mapping Project Resource Centre Lines: 49 Message-ID: References: <86tjvs$81$1@bgtnsc03.worldnet.att.net> Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII X-Trace: niobium.hgmp.mrc.ac.uk 949459370 17888 193.62.192.80 (2 Feb 2000 02:42:50 GMT) X-Complaints-To: news@net.bio.net X-Received: from sprcore.caregroup.harvard.edu (sprcore.bidmc.harvard.edu [134.174.112.128]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with SMTP id CAA21248 for ; Wed, 2 Feb 2000 02:42:46 GMT X-Received: by sprcore.caregroup.harvard.edu (4.1/SMI-4.1) id AA29221; Tue, 1 Feb 00 21:45:00 EST X-To: simm X-Cc: proteins@hgmp.mrc.ac.uk X-In-Reply-To: <86tjvs$81$1@bgtnsc03.worldnet.att.net> To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Hello Margaret, I've always prepared 1.0M stock solutions of PMSF in DMSO and stored aliquots at -20oC. I've used them for years. Hope this helps, Brad Turner **************************************************************** Bradley Turner Beth Israel Deaconess Medical Center Harvard Medical School 617-667-1215 phone Division of Gastroenterology 617-667-2767 fax Room Dana 536 bsturner@biosun.harvard.edu 330 Brookline Avenue bturner@caregroup.harvard.edu Boston, MA 02215 turner@sprcore.caregroup.harvard.edu **************************************************************** On Fri, 28 Jan 2000, simm wrote: > Dear Protein experts, > > I am currently running experiments requiring the use of PMSF. I have to > prepare fresh > solutions each time which as you may know takes up time and wastes a lot of > the > reagent. Is there a way to prepare a solution which is stable in storage? > are there other similar protease inhibitors? I am specifically interested in > stability of PMSF in methanol, acetone and isopropanol over time. I have > heard some colleagues store such solutions for up to a month, but I need > confirmation from the scientific body. > > Kindly, please respond to my email address: > simm@worldnet.att.net > > Best regards, > > Dr. Margaret Simm, PhD > > > --- From owner-proteins@hgmp.mrc.ac.uk Wed Feb 2 05:39:27 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id C006217B85; Wed, 2 Feb 2000 05:39:25 +0000 (GMT) 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 FAA09388 for ; Wed, 2 Feb 2000 05:39:20 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id FAA20068 for proteins-list@hgmp.mrc.ac.uk; Wed, 2 Feb 2000 05:39:18 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: timour@itte.kz (Timour Ivashchenko) X-Newsgroups: bionet.molbio.proteins Subject: Help to find software Date: 2 Feb 2000 05:39:18 -0000 Organization: MRC Human Genome Mapping Project Resource Centre Lines: 13 Message-ID: <008401bf6d40$70302080$4e8013d4@timour> X-Trace: niobium.hgmp.mrc.ac.uk 949469958 20066 193.62.192.80 (2 Feb 2000 05:39:18 GMT) X-Complaints-To: news@net.bio.net X-Received: from ss20.itte.kz (ss20.itte.kz [212.19.128.8]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id FAA09372 for ; Wed, 2 Feb 2000 05:39:16 GMT X-Received: from timour ([212.19.128.75]) by ss20.itte.kz (Netscape Mail Server v2.02) with SMTP id AAA27991 for ; Wed, 2 Feb 2000 11:36:16 +0600 X-To: X-Priority: 3 X-MSMail-Priority: Normal X-Mailer: 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 Dear All! Can anybody tell me where to get free software which can calculate the following parameters based on amino acid sequences: 1. Kyte&Doolittle Hydrophobicity priofile 2. Protein secondary structure Sincerely, Timour --- From owner-proteins@hgmp.mrc.ac.uk Wed Feb 2 13:51:10 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id F17C317A5A; Wed, 2 Feb 2000 13:51:09 +0000 (GMT) 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 NAA25786 for ; Wed, 2 Feb 2000 13:51:07 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id NAA26389 for proteins-list@hgmp.mrc.ac.uk; Wed, 2 Feb 2000 13:51:06 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Irina Marianovsky X-Newsgroups: bionet.molbio.proteins Subject: Finding an E.coli strain Date: Wed, 2 Feb 2000 15:43:16 +0200 Organization: The Hebrew University of Jerusalem Lines: 9 Message-ID: Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII X-Trace: news.huji.ac.il 949499014 21711 132.64.169.2 (2 Feb 2000 13:43:34 GMT) X-Complaints-To: usenet@news.huji.ac.il To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Hi everybody, Does someone have an E. coli strain that is cya- (adenylate cyclase) and is lacZ+. I need it urgently! Thanks Irina From owner-proteins@hgmp.mrc.ac.uk Wed Feb 2 18:43:14 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 1152617C2B; Wed, 2 Feb 2000 18:42:36 +0000 (GMT) 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 SAA00942 for ; Wed, 2 Feb 2000 18:42:34 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id SAA00265 for proteins-list@hgmp.mrc.ac.uk; Wed, 2 Feb 2000 18:42:33 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f Message-ID: <38987AEA.5D094F22@pacbell.net> From: Leah Jaffe-Greenwood Reply-To: leahjg@pacbell.net Organization: Pangea Systems X-Mailer: Mozilla 4.6 (Macintosh; I; PPC) X-Accept-Language: en MIME-Version: 1.0 X-Newsgroups: bionet.molbio.proteins Subject: genomics web site Content-Type: text/plain; charset=us-ascii; x-mac-type="54455854"; x-mac-creator="4D4F5353" Content-Transfer-Encoding: 7bit Lines: 13 Date: Wed, 02 Feb 2000 10:43:55 -0800 X-Complaints-To: abuse@pacbell.net X-Trace: nnrp1-w.snfc21.pbi.net 949516947 63.192.134.46 (Wed, 02 Feb 2000 10:42:27 PST) To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Check out www.DoubleTwist.com, it's a new portal that uses 'automated research agents' to do different types of genetic analysis. For example, compare DNA and protein sequences, find homologs, new gene family members, longer cDNAs, etc. Eleven of their 13 research agents are free now for a limited time. The agents do the work for you...you don't have to know which databases to search or which algorithms to use, it's all programmed in. Also, they keep monitoring the databases and notify you by email if something new related to your sequence is found. Leah Jaffe-Greenwood, Ph.D. From owner-proteins@hgmp.mrc.ac.uk Wed Feb 2 21:11:22 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 78F4717C1B; Wed, 2 Feb 2000 21:11:21 +0000 (GMT) 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 VAA12677 for ; Wed, 2 Feb 2000 21:11:19 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id VAA02104 for proteins-list@hgmp.mrc.ac.uk; Wed, 2 Feb 2000 21:11:17 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Neil McKenna X-Newsgroups: bionet.molbio.proteins Subject: Re: PMSF dire straits Date: Wed, 02 Feb 2000 16:02:54 -0500 Organization: University of Ottawa, Ottawa, Canada Lines: 11 Message-ID: <38989B7E.304E8E23@ottawa.com> References: <86tjvs$81$1@bgtnsc03.worldnet.att.net> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.5 [en] (Win95; I) X-Accept-Language: en To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk bradley turner wrote: > > Hello Margaret, > > I've always prepared 1.0M stock solutions of PMSF in DMSO > and stored aliquots at -20oC. I've used them for years. Similarly, we keep ours at 100 mM in methanol at -20oC, we've had our stock since 1991, and it still works. From owner-proteins@hgmp.mrc.ac.uk Thu Feb 3 13:05:31 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 407F417AF7; Thu, 3 Feb 2000 12:58:11 +0000 (GMT) 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 MAA09511 for ; Thu, 3 Feb 2000 12:58:09 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id MAA21935 for proteins-list@hgmp.mrc.ac.uk; Thu, 3 Feb 2000 12:58:08 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: =?iso-8859-1?Q?Jes=FAs?= Sanz X-Newsgroups: bionet.molbio.proteins Subject: Phage-display at high temperature Date: Thu, 03 Feb 2000 13:48:30 +0100 Organization: CBMC-UMH Lines: 15 Message-ID: <3899791E.DF48D9FB@umh.es> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: atenea.umh.es 949582239 31361 193.147.141.10 (3 Feb 2000 12:50:39 GMT) X-Complaints-To: usenet@atenea.umh.es X-Mailer: Mozilla 4.7 [en] (Win98; I) X-Accept-Language: en To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Dear netters, I would like to carry out a selection of thermostable mutants of a protein by phage-display. Therefore, I want to select binders at high temperatures. Does anybody know what is the highest temperature that M13 can stand before being killed? Thanks a lot Jesus Sanz From owner-proteins@hgmp.mrc.ac.uk Thu Feb 3 16:25:45 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 0926E17A7C; Thu, 3 Feb 2000 16:25:41 +0000 (GMT) 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 QAA04467 for ; Thu, 3 Feb 2000 16:25:38 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id QAA24624 for proteins-list@hgmp.mrc.ac.uk; Thu, 3 Feb 2000 16:25:37 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Shawn Strande X-Newsgroups: bionet.molbio.proteins Subject: ISMB 2000: Final Call for Papers Date: Thu, 03 Feb 2000 08:21:47 -0800 Organization: San Diego SuperComputer Center at UCSD Lines: 88 Message-ID: <3899AB1B.1468D02E@sdsc.edu> Mime-Version: 1.0 Content-Type: multipart/mixed; boundary="------------08F3C91EECE391FF2483AD52" X-Mailer: Mozilla 4.61 [en] (X11; U; SunOS 5.5.1 sun4u) X-Accept-Language: en To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk This is a multi-part message in MIME format. --------------08F3C91EECE391FF2483AD52 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit --------------08F3C91EECE391FF2483AD52 Content-Type: text/plain; charset=us-ascii; name="final" Content-Transfer-Encoding: 7bit Content-Disposition: inline; filename="final" FINAL CALL FOR PAPERS The Eighth International Conference on Intelligent Systems for Molecular Biology (ISMB 2000) August 19 - August 23, 2000 San Diego, California USA http://ismb2000.sdsc.edu Submission deadlines are rapidly approaching for papers, posters, and demonstrations for the upcoming Eighth International Conference on Intelligent Systems for Molecular Biology (ISMB 2000), August 19-23, 2000, UC San Diego, California USA. Essential information is given below, with additional details available at: http://ismb2000.sdsc.edu. We look forward to your participation to help make this a successful meeting. Sincerely, Phil Bourne & Michael Gribskov Program Chairs, ISMB 2000 ------------------------ Papers ------ Deadline for receipt: February 14, 2000 Papers should be a maximum of 12 pages, single-spaced, including title, abstract, figures, tables, and bibliography. The first page should give keywords, postal and electronic mailing addresses, telephone and fax numbers. Submit papers electronically to ismb2000@sdsc.edu in either postscript or pdf format. Papers will only be accepted electronically. Complete formatting details are available at: http://www.aaai.org/Publications/Author/authorinstructions.html PAPERS NOT CONFORMING TO THESE GUIDELINES WILL BE RETURNED WITHOUT REVIEW. Tutorials --------- Closed. Posters and Software Demonstrations ----------------------------------- Abstracts due: May 31, 2000 An abstract of your poster should be submitted by May 31, 2000, including title, author(s), affiliation(s), e-mail address(es), text, and references. Poster space will be approximately 145 cm (4 ft) high by 110 (3 ft) cm wide. If you represent an academic group and wish to demonstrate software, please notify the conference organizers by sending e-mail to ismb2000@sdsc.edu as soon as possible. Key Dates --------- Meeting: Tutorial presentations: Aug 19, 2000 Paper presentations: Aug 20-23, 2000 Paper Submissions: Papers must be received by: Feb 14, 2000 Replies to authors by: Mar 20, 2000 Revised papers must be received by: Apr 10, 2000 Open Poster Submissions Abstracts must be received by: May 31, 2000 Tutorial Proposals Closed. Registration Begins: Apr. 15, 2000 Ends: June 15, 2000 --------------08F3C91EECE391FF2483AD52-- From owner-proteins@hgmp.mrc.ac.uk Thu Feb 3 20:00:21 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 89DF817A4E; Thu, 3 Feb 2000 20:00:20 +0000 (GMT) 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 UAA25913 for ; Thu, 3 Feb 2000 20:00:10 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id UAA27443 for proteins-list@hgmp.mrc.ac.uk; Thu, 3 Feb 2000 20:00:09 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) X-Newsgroups: bionet.molbio.proteins Subject: Re: Help to find software Date: Thu, 03 Feb 2000 19:51:45 GMT Organization: UW Lines: 19 Message-ID: <87cmaf$19fo$1@news.doit.wisc.edu> References: <008401bf6d40$70302080$4e8013d4@timour> Mime-Version: 1.0 Content-Type: text/plain; charset=KOI8-R Content-Transfer-Encoding: 8bit X-Trace: news.doit.wisc.edu 949607567 42488 144.92.18.149 (3 Feb 2000 19:52:47 GMT) X-Complaints-To: abuse@doit.wisc.edu X-Newsreader: News Xpress 2.01 X-No-Archive: yes To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk timour@itte.kz (Timour Ivashchenko) wrote: :Dear All! : :Can anybody tell me where to get free software which can :calculate the following parameters :based on amino acid sequences: :1. Kyte&Doolittle Hydrophobicity priofile :2. Protein secondary structure : I don't know off-hand about free software, but there are many web sites that do both. Try http://neehow.ym.edu.tw/wonderful/soft-online/ or for hydrophobicity profile http://grserv.med.jhmi.edu/~raj/MISC/hphobh.html - Dima From owner-proteins@hgmp.mrc.ac.uk Thu Feb 3 21:08:32 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 7209117A4E; Thu, 3 Feb 2000 21:08:31 +0000 (GMT) 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 VAA00639 for ; Thu, 3 Feb 2000 21:08:29 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id VAA28309 for proteins-list@hgmp.mrc.ac.uk; Thu, 3 Feb 2000 21:08:28 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Chris Larosa X-Newsgroups: bionet.molbio.proteins Subject: Re: Help to find software Date: Thu, 03 Feb 2000 03:07:22 -0600 Organization: University of Nebraska-Lincoln Lines: 25 Message-ID: <3899454A.7682C966@biocomp.unl.edu> References: <008401bf6d40$70302080$4e8013d4@timour> <87cmaf$19fo$1@news.doit.wisc.edu> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.6 [en] (Win95; I) X-Accept-Language: en To: Dima Klenchin To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Do web search for WinPep.....freeware does Kyte an Doolittle. There are so many predictions servers on web,,,, just do web search and you will find dozens..! Dima Klenchin wrote: > timour@itte.kz (Timour Ivashchenko) wrote: > :Dear All! > : > :Can anybody tell me where to get free software which can > :calculate the following parameters > :based on amino acid sequences: > :1. Kyte&Doolittle Hydrophobicity priofile > :2. Protein secondary structure > : > > I don't know off-hand about free software, but there are many web > sites that do both. Try > > http://neehow.ym.edu.tw/wonderful/soft-online/ > or for hydrophobicity profile > http://grserv.med.jhmi.edu/~raj/MISC/hphobh.html > > - Dima From owner-proteins@hgmp.mrc.ac.uk Fri Feb 4 04:09:43 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 3142017A91; Fri, 4 Feb 2000 04:09:42 +0000 (GMT) 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 EAA01326 for ; Fri, 4 Feb 2000 04:09:38 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id EAA11988 for proteins-list@hgmp.mrc.ac.uk; Fri, 4 Feb 2000 04:09:36 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: sac65531@saclink.csus.edu (C. Wernicke) X-Newsgroups: bionet.molbio.proteins Subject: ApoE purification Date: Fri, 04 Feb 2000 02:00:07 GMT Organization: DiS Lines: 7 Message-ID: <389a31f4.18817481@news.csus.edu> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Newsreader: Forte Agent 1.5/32.452 To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Can anyone offer any literature suggestions containing "low tech" ApoE purification techniques? Monoclonal antibodies are *not* an option. This is an undergraduate biochemistry program at a non-research oriented university. Any suggestions much appreciated. Carl From owner-proteins@hgmp.mrc.ac.uk Fri Feb 4 04:32:59 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id BF12F17A91; Fri, 4 Feb 2000 04:32:58 +0000 (GMT) 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 EAA02356 for ; Fri, 4 Feb 2000 04:32:57 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id EAA12233 for proteins-list@hgmp.mrc.ac.uk; Fri, 4 Feb 2000 04:32:56 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: sac65531@saclink.csus.edu (C. Wernicke) X-Newsgroups: bionet.molbio.proteins Subject: ApoE Date: Fri, 04 Feb 2000 02:34:54 GMT Organization: DiS Lines: 9 Message-ID: <389d3a54.20961351@news.csus.edu> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Newsreader: Forte Agent 1.5/32.452 To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Hello, Not sure if my last note posted so I am resending. I will be working on ApoE purification from blood serum samples, source likely to be canine. Can anybody recommend any "low-tech" purification techniqes or sources? Carl From owner-proteins@hgmp.mrc.ac.uk Fri Feb 4 13:12:13 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id C9FF317B05; Fri, 4 Feb 2000 13:12:11 +0000 (GMT) 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 NAA17450 for ; Fri, 4 Feb 2000 13:12:07 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id NAA19688 for proteins-list@hgmp.mrc.ac.uk; Fri, 4 Feb 2000 13:12:06 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Keith Pitts X-Newsgroups: bionet.molbio.proteins Subject: gradigels pre-cast PAGE gels- can anyone recommend? Date: Fri, 04 Feb 2000 08:16:10 -0500 Organization: The University of Georgia Lines: 16 Message-ID: <389AD11A.4DD0F391@arches.uga.edu> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.7 [en] (Win95; I) X-Accept-Language: en To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk We are considering ordering some apparently inexpensive pre-cast SDS-PAGE gels from: www.gradigels.com (Gradigels) Has anyone had any success with their products or any particular reservations? I am mainly interested in the straight 12% gels, but may also use some gradient gels if necessary. Thanks for any comments I can get. Keith -- :-:-:-:-:-:-:-:-:-:-:-:-:-:-:-:-:-:-:-:-:-:-:-:-:-:-:-:-:-:-:-:-: Keith Pitts Research Assistant Univ. of Georgia k20man@arches.uga.edu From owner-proteins@hgmp.mrc.ac.uk Fri Feb 4 14:51:39 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 6850817B0F; Fri, 4 Feb 2000 14:51:38 +0000 (GMT) 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 OAA00326 for ; Fri, 4 Feb 2000 14:51:33 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id OAA20981 for proteins-list@hgmp.mrc.ac.uk; Fri, 4 Feb 2000 14:51:32 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: rozsa@umich.edu (frank) X-Newsgroups: bionet.molbio.proteins Subject: GST/His Tag expression woes Date: 4 Feb 2000 14:51:31 -0000 Organization: BIOSCI/MRC Human Genome Mapping Project Resource Centre Lines: 90 Message-ID: <389AE76C.CBFFCAB2@umich.edu> Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1; x-mac-type="54455854"; x-mac-creator="4D4F5353" Content-Transfer-Encoding: 8bit X-Trace: niobium.hgmp.mrc.ac.uk 949675892 20979 193.62.192.80 (4 Feb 2000 14:51:32 GMT) X-Complaints-To: news@net.bio.net X-Received: from changeofhabit.rs.itd.umich.edu (changeofhabit.rs.itd.umich.edu [141.211.144.17]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id OAA00295 for ; Fri, 4 Feb 2000 14:51:26 GMT X-Received: from umich.edu (host-88.subnet-184.med.umich.edu [141.214.184.88]) by changeofhabit.rs.itd.umich.edu (8.9.3/3.2r) with ESMTP id JAA06803 for ; Fri, 4 Feb 2000 09:51:25 -0500 (EST) X-Reply-To: rozsa@umich.edu X-Mailer: Mozilla 4.7 (Macintosh; I; PPC) X-Accept-Language: en X-To: proteins@hgmp.mrc.ac.uk To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Hello Protein gurus! I’m trying to express a human 55 kDa protein using a GST fusion approach, vector is pGEX4T3. I switched to GST (from His-Tag) since the lab down the hall had GST going well, a tried and sometimes true rationale in science! Cells are grown and induced for 3 hours, then lysozyme and sonicated, lysates are put on GST-sepharose and eluted. For quick looks sometimes I just look at lysates. Culture volumes are 1 ml, of which 25% of the lysate or GST-sepharose eluate is put in a single mini-protein gel lane. When using the GST-sepharose I grow 10 ml cultures. In all cases the vector alone gives lovely bands by immunoblotting, so I know my semi-dry blotting and anti-GST and secondary Ab is working! There are no fusion bands around 82 kDa, only a weak band that corresponds to GST vector alone. Conditions tested: E.coli BL21 Codon strain (Stratagene), E.coli Top 10 (Invitrogen) +/- 1 mM IPTG (three hour induction) +/- 1 % glucose growth at 28C vs 37C (with 1% glucose) Native (sonicated) vs Denaturing (Urea) When doing a larger prep using GST-sepharose, I’ve even tried binding the putative protein out of the media, the lysate, the insoluble pellet, no luck. Before you ask: Clone is "in frame" relative to the gst. No stop codon is introduced between gst and the ATG of the gene The native stop codons are preserved in frame. Protease Inhibitors: PMSF, benzamidine, and Inhibitor (EDTA-free) cocktail tablets. Previously I was trying to work with His-Tags constructs. The first approach was a amino-terminal His-tag. After a couple months of amibigious results and talking to people at meetings I switched the His Tag to the carboxy terminal end. It turns out that frameshift mutations occur in the coding sequence gene either due to the Taq or possibly by forcing a mutation to deactivate a “toxic” protein. The sequence of the cloned PCR product prior to cloning is OK, but only a couple of colonies arose when cloning into the pTrcHis vector. This is when I switched to GST, and used Pfu to amplify out of cDNA. Overall I am more impressed with the ability of GST-Sepharose to bind to controls than His-Tag. I have not varied the length of induction, nor the concentration of IPTG. Can anyone out there offer any suggestions as to which conditions to vary. Is this doomed to fail? Should I start looking at baculovirus or gateway technology? Thanks for listening to this drawn out, painful experience! Frank rozsa@umich.edu -- \\\\\||||||///// <( @ @ )> ------oOOo--(..)--oOOo------- Frank W. Rozsa, MPH, PhD Research Investigator: Glaucoma Genetics Dept. of Ophthalmology Kellogg Eye Center University of Michigan 1000 Wall Street Ann Arbor, MI 48105 email: rozsa@umich.edu alt: cmotdibbler@hotmail.com\ alt2: frozsa@mac.com --- From owner-proteins@hgmp.mrc.ac.uk Fri Feb 4 19:40:35 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 5CB9E17B01; Fri, 4 Feb 2000 19:40:34 +0000 (GMT) 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 TAA03504 for ; Fri, 4 Feb 2000 19:40:32 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id TAA24775 for proteins-list@hgmp.mrc.ac.uk; Fri, 4 Feb 2000 19:40:31 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: spena@upracd.upr.clu.edu (Sandra Pena) X-Newsgroups: bionet.molbio.proteins Subject: stripping nitrocellulose membrane Date: 4 Feb 2000 19:40:29 -0000 Organization: BIOSCI/MRC Human Genome Mapping Project Resource Centre Lines: 5 Message-ID: <389B2BE5.22322AA1@upracd.upr.clu.edu> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: niobium.hgmp.mrc.ac.uk 949693230 24773 193.62.192.80 (4 Feb 2000 19:40:30 GMT) X-Complaints-To: news@net.bio.net X-Received: from rrpac.upr.clu.edu (rrpac.UPR.CLU.EDU [136.145.180.246]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id TAA03491 for ; Fri, 4 Feb 2000 19:40:27 GMT X-Received: from upracd.upr.clu.edu ([136.145.178.93]) by rrpac.upr.clu.edu (8.9.3/8.9.3) with ESMTP id PAA17929 for ; Fri, 4 Feb 2000 15:39:52 -0400 (GMT-0400) X-Mailer: Mozilla 4.7 [en] (Win95; I) X-Accept-Language: en X-To: proteins@hgmp.mrc.ac.uk To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Can somebody give me a good protocol for stripping nitrocellulose membranes for immunoblots? Please send it to me at: sandra_vazquez@hotmail.com --- From owner-proteins@hgmp.mrc.ac.uk Fri Feb 4 20:29:40 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 8A08417B2C; Fri, 4 Feb 2000 20:29:39 +0000 (GMT) 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 UAA07075 for ; Fri, 4 Feb 2000 20:29:37 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id UAA25452 for proteins-list@hgmp.mrc.ac.uk; Fri, 4 Feb 2000 20:29:36 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Ilya Shamovsky X-Newsgroups: bionet.molbio.proteins Subject: Re: stripping nitrocellulose membrane Date: Fri, 04 Feb 2000 22:28:53 +0200 Organization: Technion, Israel Institute of Technology Lines: 22 Message-ID: <389B3685.245F2BA4@techunix.technion.ac.il> References: <389B2BE5.22322AA1@upracd.upr.clu.edu> Mime-Version: 1.0 Content-Type: text/plain; charset=koi8-r Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.7 [en] (Win98; I) X-Accept-Language: en To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Sandra Pena wrote: > Can somebody give me a good protocol for stripping nitrocellulose > membranes for immunoblots? > Please send it to me at: sandra_vazquez@hotmail.com > > --- Sandra, Try 50 mM glycine, pH 2.3 + 1 mM EDTA for 15-30 min followed by PBS/0.1% Tween 20 and then reprobing the membrane as usual. Works great for me. Alternatively, 30 min at 50-70 degrees C in 62.5 mM tris-Cl, pH 6.8, 100 mM beta-mercaptoethanol and 2% SDS should do the job. Hope this helps. Cheers, Ilya. From owner-proteins@hgmp.mrc.ac.uk Sat Feb 5 19:07:57 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id C1FE417A5B; Sat, 5 Feb 2000 19:07:56 +0000 (GMT) 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 TAA00363 for ; Sat, 5 Feb 2000 19:07:53 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id TAA20328 for proteins-list@hgmp.mrc.ac.uk; Sat, 5 Feb 2000 19:07:52 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: n02052000@eastmail.com X-Newsgroups: bionet.molbio.proteins Subject: Work From The Comfort Of Your Home! 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Thank You! --- From owner-proteins@hgmp.mrc.ac.uk Sun Feb 6 01:07:04 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 3EC0C17A5E; Sun, 6 Feb 2000 01:07:03 +0000 (GMT) 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 BAA25886 for ; Sun, 6 Feb 2000 01:07:01 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id BAA03187 for proteins-list@hgmp.mrc.ac.uk; Sun, 6 Feb 2000 01:07:00 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: "Toby Skinner" X-Newsgroups: bionet.molbio.proteins Subject: Describing secondary structure Date: Sun, 6 Feb 2000 00:55:13 -0000 Organization: Customer of Planet Online Lines: 12 Message-ID: <87ihfh$1bq$1@newsg4.svr.pol.co.uk> X-Trace: newsg4.svr.pol.co.uk 949799217 1402 62.136.131.212 (6 Feb 2000 01:06:57 GMT) X-Complaints-To: abuse@theplanet.net X-Newsreader: Microsoft Outlook Express 4.72.3110.5 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3110.3 To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Hi, I'm trying to define a grammar for describing the secondary structure of a protein, so far I've trawled the net and all the various prediction sites but I cant find any that require the user to specify the seconday structure. I need a simple language which can encompass all the essential information about a proteins secondary structure. If you have any ideas on this or would like to contribute then please reply to this, I'll be sure to check back soon. Toby From owner-proteins@hgmp.mrc.ac.uk Sun Feb 6 20:20:25 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 3518917A65; Sun, 6 Feb 2000 20:20:20 +0000 (GMT) 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 UAA19262 for ; Sun, 6 Feb 2000 20:20:17 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id UAA17338 for proteins-list@hgmp.mrc.ac.uk; Sun, 6 Feb 2000 20:20:15 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: sandy_globin@my-deja.com X-Newsgroups: bionet.molbio.proteins Subject: acid phosphatase Date: Sun, 06 Feb 2000 20:12:36 GMT Organization: Deja.com - Before you buy. Lines: 12 Message-ID: <87kkji$7cs$1@nnrp1.deja.com> X-Article-Creation-Date: Sun Feb 06 20:12:36 2000 GMT X-Http-User-Agent: Mozilla/4.0 (compatible; MSIE 4.01; Windows 98) X-Http-Proxy: 1.1 x35.deja.com:80 (Squid/1.1.22) for client 203.197.192.13 X-MyDeja-Info: XMYDJUIDsandy_globin To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk hi, 1. could someone please define specific enzyme activity, and also give its formula? 2. where can i get the Km value for wheat germ acid phosphatase? thanx, sandy Sent via Deja.com http://www.deja.com/ Before you buy. From owner-proteins@hgmp.mrc.ac.uk Sun Feb 6 20:40:13 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id A98D217A65; Sun, 6 Feb 2000 20:40:12 +0000 (GMT) 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 UAA20149 for ; Sun, 6 Feb 2000 20:40:11 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id UAA17580 for proteins-list@hgmp.mrc.ac.uk; Sun, 6 Feb 2000 20:40:10 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: sandy_globin@my-deja.com X-Newsgroups: bionet.molbio.proteins Subject: Re: Describing secondary structure Date: Sun, 06 Feb 2000 20:33:57 GMT Organization: Deja.com - Before you buy. Lines: 27 Message-ID: <87klrl$88v$1@nnrp1.deja.com> References: <87ihfh$1bq$1@newsg4.svr.pol.co.uk> X-Article-Creation-Date: Sun Feb 06 20:33:57 2000 GMT X-Http-User-Agent: Mozilla/4.0 (compatible; MSIE 4.01; Windows 98) X-Http-Proxy: 1.1 x28.deja.com:80 (Squid/1.1.22) for client 203.197.192.13 X-MyDeja-Info: XMYDJUIDsandy_globin To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk > Hi, I'm trying to define a grammar for describing the secondary structure secondary structure is described in terms of spatial angles known as dihedral angles, which specify the angles between the nitrogen and carbon atoms of the backbone of the polypeptide. you can check the following site. its very useful for understanding various aspects of protein structure, right from the beginning: www.pps97.cryst.bbk.ac.uk\course\index.htm go to the "protein geometry" page, and then subsequently to the secondary structure page. also, if you can check this book out, i think you'd find it quite useful: Introduction to Protein Structure, by Branden,Carl and Tooze,John hope this helps. sandy Sent via Deja.com http://www.deja.com/ Before you buy. From owner-proteins@hgmp.mrc.ac.uk Sun Feb 6 23:15:20 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 5171F17A87; Sun, 6 Feb 2000 23:15:19 +0000 (GMT) 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 XAA07829 for ; Sun, 6 Feb 2000 23:15:17 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id XAA28080 for proteins-list@hgmp.mrc.ac.uk; Sun, 6 Feb 2000 23:15:16 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Rick Bright X-Newsgroups: bionet.molbio.proteins Subject: Re: Describing secondary structure Date: Sun, 06 Feb 2000 18:16:01 -0500 Organization: Yerkes Regional Primate Research Center; Emory University, Immunology & Molecular Pathogenesis Lines: 31 Message-ID: <389E00B1.CA9A82CF@emory.edu> References: <87ihfh$1bq$1@newsg4.svr.pol.co.uk> <87klrl$88v$1@nnrp1.deja.com> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: lendl.cc.emory.edu 949878851 11597 170.140.167.42 (6 Feb 2000 23:14:11 GMT) X-Complaints-To: abuse@emory.edu X-Mailer: Mozilla 4.7 [en] (Win95; U) X-Accept-Language: en,pdf To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Can you please post a valid URL for the information you supplied? The one listed is not functional. Thanks sandy_globin@my-deja.com wrote: > > > Hi, I'm trying to define a grammar for describing the secondary > structure > > secondary structure is described in terms of spatial angles known > as dihedral angles, which specify the angles between the nitrogen and > carbon atoms of the backbone of the polypeptide. > > you can check the following site. its very useful for understanding > various aspects of protein structure, right from the beginning: > www.pps97.cryst.bbk.ac.uk\course\index.htm > go to the "protein geometry" page, and then subsequently to the > secondary structure page. > > also, if you can check this book out, i think you'd find it quite > useful: > Introduction to Protein Structure, by Branden,Carl and Tooze,John > > hope this helps. > > sandy > > Sent via Deja.com http://www.deja.com/ > Before you buy. From owner-proteins@hgmp.mrc.ac.uk Mon Feb 7 03:00:16 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 43BE617A56; Mon, 7 Feb 2000 03:00:15 +0000 (GMT) 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 DAA19222 for ; Mon, 7 Feb 2000 03:00:13 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id DAA00778 for proteins-list@hgmp.mrc.ac.uk; Mon, 7 Feb 2000 03:00:12 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Fred Davis X-Newsgroups: bionet.molbio.proteins Subject: Re: Describing secondary structure Date: Sun, 06 Feb 2000 22:03:25 -0500 Organization: . Lines: 5 Message-ID: <389E35FD.AF280D9@netscape.net> References: <87ihfh$1bq$1@newsg4.svr.pol.co.uk> <87klrl$88v$1@nnrp1.deja.com> Reply-To: davisfp@netscape.net Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: mozo.cc.purdue.edu 949892119 4041 128.211.253.17 (7 Feb 2000 02:55:19 GMT) X-Complaints-To: usenet@mozo.cc.purdue.edu X-Mailer: Mozilla 4.7 [en] (X11; I; Linux 2.2.5-22smp i686) X-Accept-Language: en To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk The correct address is: http://pps97.cryst.bbk.ac.uk/course/index.html -fred From owner-proteins@hgmp.mrc.ac.uk Mon Feb 7 03:50:13 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 1218A17A56; Mon, 7 Feb 2000 03:50:12 +0000 (GMT) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id DAA21012 for ; Mon, 7 Feb 2000 03:50:11 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id DAA01351 for proteins-list@hgmp.mrc.ac.uk; Mon, 7 Feb 2000 03:50:09 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: sandy_globin@my-deja.com X-Newsgroups: bionet.molbio.proteins Subject: Re: Describing secondary structure Date: Mon, 07 Feb 2000 03:36:35 GMT Organization: Deja.com - Before you buy. Lines: 14 Message-ID: <87lek3$p4c$1@nnrp1.deja.com> References: <87ihfh$1bq$1@newsg4.svr.pol.co.uk> <87klrl$88v$1@nnrp1.deja.com> <389E00B1.CA9A82CF@emory.edu> X-Article-Creation-Date: Mon Feb 07 03:36:35 2000 GMT X-Http-User-Agent: Mozilla/4.0 (compatible; MSIE 4.01; Windows 98) X-Http-Proxy: 1.1 x37.deja.com:80 (Squid/1.1.22) for client 203.197.192.120 X-MyDeja-Info: XMYDJUIDsandy_globin To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk In article <389E00B1.CA9A82CF@emory.edu>, Rick Bright wrote: > Can you please post a valid URL for the information you supplied? The > one listed is not functional. > I apologise. i hope this one works. http://PPS97.cryst.bbk.ac.uk/course/index.html sandy Sent via Deja.com http://www.deja.com/ Before you buy. From owner-proteins@hgmp.mrc.ac.uk Mon Feb 7 09:22:28 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 5948517A60; Mon, 7 Feb 2000 09:22:27 +0000 (GMT) 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 JAA14133 for ; Mon, 7 Feb 2000 09:22:25 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id JAA05434 for proteins-list@hgmp.mrc.ac.uk; Mon, 7 Feb 2000 09:22:24 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Frank Fuerst X-Newsgroups: bionet.molbio.proteins Subject: Re: acid phosphatase Date: Mon, 07 Feb 2000 10:28:59 +0100 Lines: 32 Message-ID: <87m2se$7vtc$1@fu-berlin.de> References: <87kkji$7cs$1@nnrp1.deja.com> Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Trace: fu-berlin.de 949915342 262060 141.89.207.60 (16 49 955) 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 sandy_globin@my-deja.com wrote: > > 1. could someone please define specific enzyme activity, and also give > its formula? specific catalytic activity is the activity of a enzyme (as measured by an assay that is, hopefully, specific for that one enzyme) divided by the mass of protein in the sample. Thus, it is a measurement not only for the "intrinsic" activity (that could by measured by the molar activity), but also for the purity with respect to other proteins or peptides. The formula is (I'm using z as the sign for catalytic activity) z_sp(enzyme) = z(enzyme)/m(protein), where z(enzyme) = Delta_n(substrate)/Delta_t where Delta_n(substrate) is the change in amount of substance (in moles) of substrate (or product), as measured in the assay, in the time Delta_t. > 2. where can i get the Km value for wheat germ acid phosphatase? I'm sorry. Frank -- Flhacs wird im Usenet grundsätzlich alsfhc geschrieben. Schreibt man lafhsc nicht slfach, so ist das schlichtweg hclafs. Hingegen darf man rihctig ruhig rhitcgi schreiben, weil eine shcalfe Schreibweise bei irhictg nicht als shflac angesehen wird. [Hajo Pflüger in dnq] From owner-proteins@hgmp.mrc.ac.uk Mon Feb 7 15:16:23 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 2DEDD17A4E; Mon, 7 Feb 2000 15:16:22 +0000 (GMT) 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 PAA28347 for ; Mon, 7 Feb 2000 15:16:20 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id PAA10141 for proteins-list@hgmp.mrc.ac.uk; Mon, 7 Feb 2000 15:16:19 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Pavel Sova X-Newsgroups: bionet.molbio.proteins Subject: Re: stripping nitrocellulose membrane Date: Mon, 7 Feb 2000 10:16:15 -0500 Organization: Columbia University Lines: 26 Message-ID: References: <389B2BE5.22322AA1@upracd.upr.clu.edu> Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII X-Trace: newsmaster.cc.columbia.edu 949936569 907 128.59.59.136 (7 Feb 2000 15:16:09 GMT) X-Complaints-To: postmaster@columbia.edu To: Sandra Pena In-Reply-To: <389B2BE5.22322AA1@upracd.upr.clu.edu> To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk On 4 Feb 2000, Sandra Pena wrote: > Date: 4 Feb 2000 19:40:29 -0000 > From: Sandra Pena > Newsgroups: bionet.molbio.proteins > Subject: stripping nitrocellulose membrane > > Can somebody give me a good protocol for stripping nitrocellulose > membranes for immunoblots? > Please send it to me at: sandra_vazquez@hotmail.com > > --- How about this one: Incubate your membranes in 0.2 M NaOH for 5 min. Neutralize in TPBS or TTBS (Tween - PBS etc.) and block with 5% milk in ditto. I think this is a modification of protocol from Red Book, it works fine for me (might be a little rough, though). Pavel Sova, Ph.D. New York ps44@columbia.edu From owner-proteins@hgmp.mrc.ac.uk Mon Feb 7 19:02:55 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 7112917ADE; Mon, 7 Feb 2000 19:02:54 +0000 (GMT) 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 TAA25458 for ; Mon, 7 Feb 2000 19:02:52 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id TAA12998 for proteins-list@hgmp.mrc.ac.uk; Mon, 7 Feb 2000 19:02:50 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Andrea Hansen X-Newsgroups: bionet.molbio.proteins Subject: Re: Help to find software Date: 07 Feb 2000 20:02:38 +0100 Lines: 17 Message-ID: References: <008401bf6d40$70302080$4e8013d4@timour> X-Trace: fu-berlin.de 949950167 327153 134.99.202.44 (16 [4974]) X-Newsreader: Gnus v5.5/XEmacs 20.4 - "Emerald" To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Hi Timour! > Can anybody tell me where to get free software which can > calculate the following parameters > based on amino acid sequences: > 1. Kyte&Doolittle Hydrophobicity priofile > 2. Protein secondary structure Perhaps you can find some help here: http://bioinformatics.weizmann.ac.il/hydroph/ Andrea -- Institut fuer Botanik III Heinrich-Heine-Universitaet Universitaetsstr.1 email : webmaster@bioinformatik.de 40225 Duesseldorf http://www.bioinformatik.de From owner-proteins@hgmp.mrc.ac.uk Tue Feb 8 10:50:11 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id C21F017A69; Tue, 8 Feb 2000 10:50:09 +0000 (GMT) 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 KAA07655 for ; Tue, 8 Feb 2000 10:50:04 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id KAA03428 for proteins-list@hgmp.mrc.ac.uk; Tue, 8 Feb 2000 10:50:03 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: "Dr. Duncan Clark" X-Newsgroups: bionet.molbio.proteins Subject: Purification of monomeric actin Date: Tue, 8 Feb 2000 10:41:34 +0000 Organization: DNAmp Ltd. Message-ID: Reply-To: "Dr. Duncan Clark" X-NNTP-Posting-Host: genesys.demon.co.uk:158.152.17.110 X-Trace: news.demon.co.uk 950006957 nnrp-13:24635 NO-IDENT genesys.demon.co.uk:158.152.17.110 X-Complaints-To: abuse@demon.net MIME-Version: 1.0 X-Newsreader: Turnpike Integrated Version 5.00 beta 1 M Lines: 18 To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Hi Folks, Does anyone have a protocol (or reference) for purification of gram quantities of monomeric or G actin. Many thanks Duncan -- The problem with being on the cutting edge is that you occasionally get sliced from time to time.... Duncan Clark DNAmp Ltd. Tel: +44(0)1252376288 FAX: +44(0)8701640382 http://www.dnamp.com http://www.genesys.demon.co.uk From owner-proteins@hgmp.mrc.ac.uk Tue Feb 8 12:32:23 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 4247717AFB; Tue, 8 Feb 2000 12:31:40 +0000 (GMT) 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 MAA19671; Tue, 8 Feb 2000 12:31:38 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id MAA05185; Tue, 8 Feb 2000 12:31:37 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: "Richard P. Grant" X-Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: N-terminal seq/ MS reqd, UK Followup-To: bionet.molbio.proteins Date: Tue, 08 Feb 2000 12:31:34 +0000 Organization: MRC LMB Lines: 20 Message-ID: User-Agent: MT-NewsWatcher/3.0 (PPC) X-No-Archive: yes To: methods@hgmp.mrc.ac.uk, proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk (apologies for x-post) Given a known protein of ~ 20 kDa, a limiting tryptic digest gives 2 major fragments. I'm looking for a service in the UK, preferably in/near Cambridge, that could N-terminal sequence and mass-spec to identify the fragments. Our own service is out of whack for 3 weeks. recommendations?? Cheers, Richard -- Richard P. Grant MA DPhil Structural Studies Group, MRC-LMB http://www2.mrc-lmb.cam.ac.uk/personal/rpg/index.html Please reply to rpg 'at' mrc-lmb.cam.ac.uk From owner-proteins@hgmp.mrc.ac.uk Tue Feb 8 12:45:15 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 9CEF217AFB; Tue, 8 Feb 2000 12:45:13 +0000 (GMT) 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 MAA21433 for ; Tue, 8 Feb 2000 12:45:10 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id MAA05371 for proteins-list@hgmp.mrc.ac.uk; Tue, 8 Feb 2000 12:45:08 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Petri Kursula X-Newsgroups: bionet.molbio.proteins Subject: BL21(DE3) and pTrc Date: Tue, 8 Feb 2000 14:31:26 +0200 Organization: University of Oulu Lines: 17 Message-ID: Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII X-Trace: ousrvr3.oulu.fi 950013088 10400 130.231.240.13 (8 Feb 2000 12:31:28 GMT) X-Complaints-To: news@news.oulu.fi X-Sender: pkursula@sun3.oulu.fi To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Confused by all these E.coli genotypes and strains, I would like to know if anyone has used BL21(DE3) cells to produce recombinant proteins from a vector with the trc promoter, such as pTrc99a. If yes, is this expression tightly regulatable by IPTG, or do you get leaky expression? ----------------------------------------------------------------------- Petri Kursula "I am somehow less interested in the weight and University of Oulu convolutions of Einstein's brain than in the near Petri.Kursula@oulu.fi certainty that people of equal talent have lived http://start.at/MAG and died in cotton fields and sweatshops." http://cc.oulu.fi/~pkursula -- Stephen Jay Gould ----------------------------------------------------------------------- From owner-proteins@hgmp.mrc.ac.uk Tue Feb 8 14:15:33 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 4762C17B5C; Tue, 8 Feb 2000 14:15:30 +0000 (GMT) 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 OAA01875 for ; Tue, 8 Feb 2000 14:15:28 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id OAA06596 for proteins-list@hgmp.mrc.ac.uk; Tue, 8 Feb 2000 14:15:27 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: "Richard P. Grant" X-Newsgroups: bionet.molbio.proteins Subject: Re: Purification of monomeric actin Date: Tue, 08 Feb 2000 14:15:21 +0000 Organization: MRC LMB Lines: 40 Message-ID: References: User-Agent: MT-NewsWatcher/3.0 (PPC) X-No-Archive: yes To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk In article , "Dr. Duncan Clark" wrote: > Does anyone have a protocol (or reference) for purification of gram > quantities of monomeric or G actin. > Hi Duncan, Try the method at http://www.users.waitrose.com/~rkrs/thesis/2matmeth.htm#216 which is scalable. The appropriate refs are Feuer et al, 1948, Spudich & Watt, 1971 and MacLean-Fletcher & Pollard, 1980. I can dig out the full citations if you can't find these on Medline - http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed If you want 'dirty' G-actin (i.e. monomeric) then omit the S-100 step, although this *will* leave a known contaminant in there (check the MacLean ref). I used to get 80 g acetone powder from 2 rabbits, and 20 mg actin/g powder, so that's a gram and a half. :-)) Note that polymerization is a key step in the purification, but it is easy (if tedious) to obtain G-actin - just dialyse the filamentous (F) actin against 10 mM Tris-Cl pH 8, 0.2 mM ATP, 0.2 mM CaCl2, 0.2 mM DTT for two days . . . HTH, Richard -- Richard P. Grant MA DPhil Structural Studies Group, MRC-LMB http://www2.mrc-lmb.cam.ac.uk/personal/rpg/index.html Please reply to rpg 'at' mrc-lmb.cam.ac.uk From owner-proteins@hgmp.mrc.ac.uk Tue Feb 8 14:44:15 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 53EEE17B97; Tue, 8 Feb 2000 14:44:10 +0000 (GMT) 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 OAA05187; Tue, 8 Feb 2000 14:44:01 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id OAA06970; Tue, 8 Feb 2000 14:44:00 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Simon Hubbard X-Newsgroups: bionet.molec-model,bionet.jobs,bionet.jobs.offered,bionet.molbio.proteins,comp.jobs Subject: UK PhD Position in Bioinformatics at UMIST, Manchester Date: 8 Feb 2000 14:44:00 -0000 Organization: UMIST Lines: 36 Message-ID: <389FDB83.C13AB34@sjh.bi.umist.ac.uk> Reply-To: Simon.Hubbard@umist.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: niobium.hgmp.mrc.ac.uk 950021040 6968 193.62.192.80 (8 Feb 2000 14:44:00 GMT) X-Complaints-To: news@net.bio.net X-Accept-Language: en X-Auth: PGPMoose V1.1 PGP bionet.jobs.offered iQCVAwUAOKArq6CIqsWvyF5FAQENiAP+If2v4kS4rhXHIgPKwSybmkofW3eIIpy+ Xinfx9JL+d3GkjchRPmHuTtlXRdmeQssN1Cwb5HuKidqYUl3xNSFbLa2P5LZ/6H3 lS6uR4jLzWPCinS7Kjm4JSiEG6x6utz5HoLW5QgPdgFTBr6yRkeSI8ITWwA45zwV qdWQhTXlYdQ= =KXb+ To: molmodel@hgmp.mrc.ac.uk, proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk UK PhD Position in Bioinformatics at UMIST, Manchester NOVEL BIOINFORMATIC APPROACHES TO PROTEOME PROTEIN IDENTIFICATION A 3 year BBSRC/EPSRC studentship in Bioinformatics is available from October 2000 working in the group of Dr. Simon Hubbard at UMIST, Manchester. The project will entail developing and extending exisiting programs and tools for proteome protein identification from mass spectrometric data. Novel data sources will be evaluated, including non-denaturing digestions on proteins prior to analysis and the ability to "jump-species" will be investigated. Results will be exploited in a new range of software for proteome protein identification. These will then be tested using exemplary systems where mass spectrometric data is already being collected here at UMIST. Examples include studies on the Chicken muscle proteome (Beynon group) and the protein phosphorylation in developmental pathways in leukaemic cell lines (Whetton group). The ability of the tools to routinely identify likely protein matches will be assessed. For more details on the project, see http://sjh.bi.umist.ac.uk/~sjh or email Simon.Hubbard@umist.ac.uk Applicants should have a good first degree in a Science, which does not necessarily contain a biological component. Applications are particularly welcome from students with computational and/or physical backgrounds. BBSRC/EPSRC studentships are only open to UK/EU applicants. -- ____________________________________________________________________ Dr. Simon Hubbard TEL: +44 (0)161 200 8930 Lecturer FAX: +44 (0)161 236 0409 Department of Biomolecular Sciences mailto:sjh@sjh.bi.umist.ac.uk UMIST, PO Box 88, Manchester M60 1QD http://sjh.bi.umist.ac.uk/~sjh From owner-proteins@hgmp.mrc.ac.uk Tue Feb 8 15:41:10 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 5850617A85; Tue, 8 Feb 2000 15:41:09 +0000 (GMT) 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 PAA13712 for ; Tue, 8 Feb 2000 15:41:05 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id PAA07750 for proteins-list@hgmp.mrc.ac.uk; Tue, 8 Feb 2000 15:41:04 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: "Dr. Duncan Clark" X-Newsgroups: bionet.molbio.proteins Subject: Re: Purification of monomeric actin Date: Tue, 8 Feb 2000 14:42:00 +0000 Organization: DNAmp Ltd. Message-ID: <4txweIA4sCo4EAMY@genesys.demon.co.uk> References: Reply-To: "Dr. Duncan Clark" X-NNTP-Posting-Host: genesys.demon.co.uk:158.152.17.110 X-Trace: news.demon.co.uk 950024457 nnrp-13:1468 NO-IDENT genesys.demon.co.uk:158.152.17.110 X-Complaints-To: abuse@demon.net MIME-Version: 1.0 X-Newsreader: Turnpike Integrated Version 5.00 beta 1 M Lines: 25 To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk In article , Richard P. Grant writes >Try the method at >http://www.users.waitrose.com/~rkrs/thesis/2matmeth.htm#216 > >which is scalable. Brilliant! Just what I was after. Now for the bunnies or should I juts go and get a few lb of primer beef steak :-). I don't suppose for the use I'm going to put it too that it will matter. Many thanks Duncan -- The problem with being on the cutting edge is that you occasionally get sliced from time to time.... Duncan Clark DNAmp Ltd. Tel: +44(0)1252376288 FAX: +44(0)8701640382 http://www.dnamp.com http://www.genesys.demon.co.uk From owner-proteins@hgmp.mrc.ac.uk Tue Feb 8 15:53:19 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id D355017AA6; Tue, 8 Feb 2000 15:53:17 +0000 (GMT) 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 PAA16045 for ; Tue, 8 Feb 2000 15:53:14 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id PAA07883 for proteins-list@hgmp.mrc.ac.uk; Tue, 8 Feb 2000 15:53:13 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: "Richard P. Grant" X-Newsgroups: bionet.molbio.proteins Subject: Re: Purification of monomeric actin Date: Tue, 08 Feb 2000 15:52:58 +0000 Organization: MRC LMB Lines: 33 Message-ID: References: <4txweIA4sCo4EAMY@genesys.demon.co.uk> User-Agent: MT-NewsWatcher/3.0 (PPC) X-No-Archive: yes To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk In article <4txweIA4sCo4EAMY@genesys.demon.co.uk>, "Dr. Duncan Clark" wrote: > Brilliant! Just what I was after. pleased to be of service. > Now for the bunnies or should I juts > go and get a few lb of primer beef steak :-). I don't suppose for the > use I'm going to put it too that it will matter. > Hmm, any skeletal muscle should do. Bunnies, cute, white, fluffy white New Zealand bunnies are traditional because animal houses usually have a surfeit of retired, fat, old animals, and it's good practice to take the muscle when an animal is terminally bled for antisera. It seems a waste of prime steak to make actin from it. :)) You could use beef heart[0] I guess, but then you'd get smooth muscle actin . . . R (who is intrigued - why do you want grams of G-actin?) [0] Damn. None of us here can remember what Captn Beefheart's hit was! -- Richard P. Grant MA DPhil Structural Studies Group, MRC-LMB http://www2.mrc-lmb.cam.ac.uk/personal/rpg/index.html Please reply to rpg 'at' mrc-lmb.cam.ac.uk From owner-proteins@hgmp.mrc.ac.uk Wed Feb 9 20:26:42 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id C55FC17A5C; Wed, 9 Feb 2000 20:26:41 +0000 (GMT) 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 UAA13885 for ; Wed, 9 Feb 2000 20:26:40 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id UAA07514 for proteins-list@hgmp.mrc.ac.uk; Wed, 9 Feb 2000 20:26:39 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: sandyjif@dnes.nec.co.jp X-Newsgroups: bionet.molbio.proteins Subject: Accept All Major Credit Cards!!! Date: 9 Feb 2000 20:26:38 -0000 Organization: BIOSCI/MRC Human Genome Mapping Project Resource Centre Lines: 100 Message-ID: <6284619573.qzx94726@rly-v75924.dnes.nec.co.jp> X-Trace: niobium.hgmp.mrc.ac.uk 950127998 7512 193.62.192.80 (9 Feb 2000 20:26:38 GMT) X-Complaints-To: news@net.bio.net X-Received: from yesv1.yon-e.co.jp (ns.yon-e.co.jp [210.157.249.226]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id UAA13877 for ; Wed, 9 Feb 2000 20:26:35 GMT X-Received: from 210.157.249.226 (localhost.yon-e.co.jp.0.0.127.in-addr.arpa [127.0.0.1] (may be forged)) by yesv1.yon-e.co.jp (8.9.3/3.7W) with SMTP id FAA21969; Thu, 10 Feb 2000 05:26:06 +0900 (JST) X-To: gildak24@earthlink.net X-Reply-To: sandyjif@dnes.nec.co.jp To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk To be removed, please email lynettedj@bigfoot.com and type remove in the subject line. 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Although cost is important to me, I also value customer service, and I must say I was extremely pleased with the service I received." - D.H. in California ******************************************************* ******************************************************* ****************** 95037 --- From owner-proteins@hgmp.mrc.ac.uk Thu Feb 10 00:30:19 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 2797E17A59; Thu, 10 Feb 2000 00:30:17 +0000 (GMT) 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 AAA07932; Thu, 10 Feb 2000 00:30:16 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id AAA19022; Thu, 10 Feb 2000 00:30:14 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: mlevin@is08.fas.harvard.edu (-7758,599-3231) X-Newsgroups: bionet.molbio.proteins,bionet.cellbiol,bionet.molbio.methds-reagnts Subject: how to do a membrane protein prep from Xenopus? Date: 9 Feb 2000 13:43:54 GMT Organization: Harvard University, Cambridge, MA Lines: 15 Message-ID: <87rquq$rah$1@news.fas.harvard.edu> To: cellbiol@hgmp.mrc.ac.uk, methods@hgmp.mrc.ac.uk, proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Hi All - Can someone point me to a protocol (whether on-line, or in a journal) for doing the following: I want to do a prep of membranes (both the cell membrane and vesicle membranes) from Xenopus embryos (early embryos, gastrulation stage). The idea is to then test binding of a drug (radioactively tagged) to a protein which is found in the cytoplasmic and various vesicle membranes. If anyone knows where I can get a protocol for doing this, please email me (my access to this newsgroup is sporadic) at mlevin@husc.harvard.edu. Thanks in advance! Mike Levin From owner-proteins@hgmp.mrc.ac.uk Thu Feb 10 13:40:38 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id E057917A7C; Thu, 10 Feb 2000 13:40:35 +0000 (GMT) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id NAA09076; Thu, 10 Feb 2000 13:40:27 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id NAA28440; Thu, 10 Feb 2000 13:40:26 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: "Richard J. Dudley" X-Newsgroups: bionet.molbio.proteins,bionet.cellbiol,bionet.molbio.methds-reagnts Subject: Re: how to do a membrane protein prep from Xenopus? Date: Thu, 10 Feb 2000 08:40:22 -0500 Organization: University of Pittsburgh, Dept. of Cell Biology and Physiology Lines: 31 Message-ID: <38A2BFC6.F6C70552@pitt.edu> References: <87rquq$rah$1@news.fas.harvard.edu> Reply-To: rdudley@pitt.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.7 [en] (Win98; I) X-Accept-Language: en To: cellbiol@hgmp.mrc.ac.uk, methods@hgmp.mrc.ac.uk, proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk There's a volume of Methods in Enzymology about Xenopus oocytes that has fractionation methods. There's also a Methods in Cell Biology about Xenopus. Try your library. rich "-7758,599-3231" wrote: > Hi All - > > Can someone point me to a protocol (whether on-line, or in a > journal) for doing the following: I want to do a prep of membranes (both > the cell membrane and vesicle membranes) from Xenopus embryos (early > embryos, gastrulation stage). The idea is to then test binding of a drug > (radioactively tagged) to a protein which is found in the cytoplasmic and > various vesicle membranes. If anyone knows where I can get a protocol for > doing this, please email me (my access to this newsgroup is sporadic) at > mlevin@husc.harvard.edu. Thanks in advance! > > Mike Levin -- Richard J. Dudley (rdudley+@pitt.edu) Research Specialist V Cystic Fibrosis Research Center Dept. of Cell Biology and Physiology University of Pittsburgh http://www.cbp.pitt.edu ---> search BIONET archives at http://www.bio.net <--- From owner-proteins@hgmp.mrc.ac.uk Thu Feb 10 22:41:39 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id AB2B317A88; Thu, 10 Feb 2000 22:41:38 +0000 (GMT) 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 WAA10283 for ; Thu, 10 Feb 2000 22:41:37 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id WAA13854 for proteins-list@hgmp.mrc.ac.uk; Thu, 10 Feb 2000 22:41:36 GMT 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: adenine-free media for cAMP?? Date: Thu, 10 Feb 2000 17:43:14 -0500 Organization: University of Pennsylvania Lines: 23 Message-ID: To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk I want to start up some simple assays for cAMP levels in cultured cells. The approach I am going to take is to metabolically label the cells with [3H]-adenine, and then measure the [3H]-cAMP that is produced from drug treatments in the assay several hours later. Ideally, I would like the use cell culture medium that is deficient in adenine in order to increase the amount of hot adenine that is taken up. However, when I look over the list of ingredients in D-MEM, I see that adenine isn't one of them. Therefore, either the cells are making their own nucleotides, or their primary source of adenine must be from the FBS that we conventionally add to the D-MEM. Does anyone do these kind of assays, and do you typically remove the serum when you load the cells with hot adenine? If I can avoid serum-starving the cells, I would prefer to do so. John hines@pharm.med.upenn.edu From owner-proteins@hgmp.mrc.ac.uk Fri Feb 11 15:11:20 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 066B117A5B; Fri, 11 Feb 2000 15:11:18 +0000 (GMT) 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 PAA29480 for ; Fri, 11 Feb 2000 15:11:16 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id PAA26146 for proteins-list@hgmp.mrc.ac.uk; Fri, 11 Feb 2000 15:11:11 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Martijn van Duijn X-Newsgroups: bionet.molbio.proteins Subject: Coomassie stain on Western Blots Date: Fri, 11 Feb 2000 16:03:41 +0100 Organization: University of Leiden The Netherlands Lines: 15 Message-ID: <38A424CD.3F675531@dds.nl> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: highway.leidenuniv.nl 950281520 6564 132.229.11.59 (11 Feb 2000 15:05:20 GMT) X-Complaints-To: usenet@highway.leidenuniv.nl X-Mailer: Mozilla 4.7 [en] (Win98; I) X-Accept-Language: en To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Hi, Just something I always wondered about. When I transfer proteins from a gel to a (pvdf) membrane, they stick to the membrane. In a subsequent stain, coomassie binds to the protein, and I get nice blue bands. No problems so far. But why don't I get a completely blue membrane after a blocking step with a protein mix like non-fat milk? I would think that the milk protein would both stick to the membrane, and be stained by Coomassie. Any insights? Thanks, Martijn From owner-proteins@hgmp.mrc.ac.uk Fri Feb 11 21:00:20 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 7EBC817B66; Fri, 11 Feb 2000 21:00:19 +0000 (GMT) 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 VAA01176 for ; Fri, 11 Feb 2000 21:00:15 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id VAA00450 for proteins-list@hgmp.mrc.ac.uk; Fri, 11 Feb 2000 21:00:13 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Cornelius Krasel X-Newsgroups: bionet.molbio.proteins Subject: Re: Coomassie stain on Western Blots Date: Fri, 11 Feb 2000 21:51:15 +0100 Organization: =?ISO-8859-1?Q?Universit=E4t_W=FCrzburg?= Lines: 20 Message-ID: <3os188.aaa.ln@wpxx02.toxi.uni-wuerzburg.de> References: <38A424CD.3F675531@dds.nl> X-Trace: fu-berlin.de 950302805 710727 132.187.168.2 (16 [16673]) User-Agent: tin/pre-1.4-19990805 ("Preacher Man") (UNIX) (Linux/2.2.13 (i686)) To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Martijn van Duijn wrote: > Just something I always wondered about. > When I transfer proteins from a gel to a (pvdf) membrane, they stick to > the membrane. In a subsequent stain, coomassie binds to the protein, and > I get nice blue bands. No problems so far. > But why don't I get a completely blue membrane after a blocking step > with a protein mix like non-fat milk? I would think that the milk > protein would both stick to the membrane, and be stained by Coomassie. I don't know, but we have observed the same when staining blots with Ponceau S *after* doing a full western blot. I suppose that the amount of milk protein actually binding to the membrane must be rather negligible (sp?). --Cornelius. -- /* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */ /* D-97078 Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de SP4 */ /* "Science is the game we play with God to find out what His rules are." */ From owner-proteins@hgmp.mrc.ac.uk Fri Feb 11 22:30:26 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id C705317B78; Fri, 11 Feb 2000 22:30:25 +0000 (GMT) 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 WAA00056; Fri, 11 Feb 2000 22:30:24 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id WAA09981; Fri, 11 Feb 2000 22:30:19 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) X-Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Source for plasmid expressing tagged Protein A? Date: Fri, 11 Feb 2000 22:27:16 GMT Organization: UW Lines: 26 Message-ID: <8822e1$ikck$1@news.doit.wisc.edu> Mime-Version: 1.0 Content-Type: text/plain; charset=KOI8-R Content-Transfer-Encoding: 8bit X-Trace: news.doit.wisc.edu 950308097 610708 144.92.18.149 (11 Feb 2000 22:28:17 GMT) X-Complaints-To: abuse@doit.wisc.edu X-Newsreader: News Xpress 2.01 X-No-Archive: yes To: methods@hgmp.mrc.ac.uk, proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Greeting all, We go through tremendous quantities of Protein A linked Sepharose in the lab. It is expensive ($50/ml). I've heard that protein A is easily expressed in E.coli to huge amounts with retention of binding activity. If we could get such a vector, we could purify enough protein and immobilize it to the Sepharose very cheaply (with periodate oxydation method, it is basically a price of Sepharose CL-4B, protein not counting). 50 mg/liter culture --> 2 x 5 l preps --> 5 mg/ml Sepharose --> 100 ml sorbent which can be stored in 50% ethylene glycol at -20C essentially forever. Sounds like a three days worth of work. Could make a good exercize for some undergraduate student wanting to get his/her hands on some basic biochemical techniques. I've only seen Pharmacia vector but it needs to be modified as it does not have a tag or a stop codon. Pharmacia itself sells rProteinA Sepharose, so I figure this or a similar construct must be in someone's hands. An info on Protein G expression would also be helpful. Thanks for any hints, - Dima From owner-proteins@hgmp.mrc.ac.uk Fri Feb 11 23:33:29 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 5070517B74; Fri, 11 Feb 2000 23:33:27 +0000 (GMT) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id XAA21139; Fri, 11 Feb 2000 23:33:25 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id XAA10747; Fri, 11 Feb 2000 23:33:24 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Chris Larosa X-Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: Source for plasmid expressing tagged Protein A? Date: Sat, 12 Feb 2000 05:33:28 -0600 Organization: University of Nebraska-Lincoln Lines: 33 Message-ID: <38A54508.38187EEC@biocomp.unl.edu> References: <8822e1$ikck$1@news.doit.wisc.edu> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.6 [en] (Win95; I) X-Accept-Language: en To: Dima Klenchin To: methods@hgmp.mrc.ac.uk, proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Perhaps you can simply pcr it out of S. A. designing primers based on the published sequence, and ligate into an appropriote vector.... Sounds like an extra week of work. Dima Klenchin wrote: > Greeting all, > > We go through tremendous quantities of Protein A linked Sepharose > in the lab. It is expensive ($50/ml). > > I've heard that protein A is easily expressed in E.coli to huge amounts > with retention of binding activity. If we could get such a vector, > we could purify enough protein and immobilize it to the Sepharose > very cheaply (with periodate oxydation method, it is basically a price > of Sepharose CL-4B, protein not counting). > > 50 mg/liter culture --> 2 x 5 l preps --> 5 mg/ml Sepharose --> 100 ml > sorbent which can be stored in 50% ethylene glycol at -20C essentially > forever. Sounds like a three days worth of work. > > Could make a good exercize for some undergraduate student wanting > to get his/her hands on some basic biochemical techniques. > > I've only seen Pharmacia vector but it needs to be modified as it does > not have a tag or a stop codon. Pharmacia itself sells rProteinA > Sepharose, so I figure this or a similar construct must be in someone's > hands. An info on Protein G expression would also be helpful. > > Thanks for any hints, > > - Dima From owner-proteins@hgmp.mrc.ac.uk Sat Feb 12 01:22:39 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 45E1417A74; Sat, 12 Feb 2000 01:22:37 +0000 (GMT) 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 BAA27147 for ; Sat, 12 Feb 2000 01:22:35 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id BAA12089 for proteins-list@hgmp.mrc.ac.uk; Sat, 12 Feb 2000 01:22:34 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: PGegen@UKans.nolospamare.edu (Dr. Peter Gegenheimer) X-Newsgroups: bionet.molbio.proteins Subject: Re: BL21(DE3) and pTrc Date: 12 Feb 2000 01:22:26 GMT Organization: Univ. Kansas (Molecular Biosciences) Lines: 21 Message-ID: <7opiGDf98QgB-pn2-rkjJuVlqh7TL@rnaworld.bio.ukans.edu> References: Mime-Version: 1.0 Content-Type: text/plain; charset="iso-8859-1" X-Newsreader: ProNews/2 Version 1.50á1/02 To: proteins@hgmp.mrc.ac.uk Content-Transfer-Encoding: 8bit X-MIME-Autoconverted: from quoted-printable to 8bit by hgmp.mrc.ac.uk id BAA27147 Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk On Tue, 8 Feb 2000 12:31:26, Petri Kursula wrote: ð Confused by all these E.coli genotypes and strains, I would like to know ð if anyone has used BL21(DE3) cells to produce recombinant proteins from a ð vector with the trc promoter, such as pTrc99a. If yes, is this expression ð tightly regulatable by IPTG, or do you get leaky expression? BL21(DE3) contains the phage T7 RNA polymerase under lac repression; it's used to express genes cloned behind a T7 promoter. You cannot transform it with a plasmid containing the lac repressor; that induces the T7 polymerase gene and the cells stop growing. o----------------------------------------------------------------------o | Dr. Peter Gegenheimer | Vox: 785-864-3939 FAX: 785-864-5321 | | Department of | PGegen@UKans.nospam.edu | | Molecular Biosciences | http://rnaworld.bio.ukans.edu/ | | University of Kansas |"When you have excluded the impossible, | | 2045 Haworth Hall | whatever remains, however improbable, | | Lawrence KS 66045-2106 | must be the truth." S. Holmes | o_____________________________|________________________________________o From owner-proteins@hgmp.mrc.ac.uk Sat Feb 12 04:42:55 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id B305117A74; Sat, 12 Feb 2000 04:42:54 +0000 (GMT) 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 EAA04475 for ; Sat, 12 Feb 2000 04:42:53 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id EAA14378 for proteins-list@hgmp.mrc.ac.uk; Sat, 12 Feb 2000 04:42:51 GMT 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: BL21(DE3) and pTrc Date: 12 Feb 2000 04:42:50 -0000 Organization: BIOSCI/MRC Human Genome Mapping Project Resource Centre Lines: 8 Message-ID: Mime-Version: 1.0 Content-Type: text/plain; charset="US-ASCII" Content-Transfer-Encoding: 7bit X-Trace: niobium.hgmp.mrc.ac.uk 950330570 14375 193.62.192.80 (12 Feb 2000 04:42:50 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 EAA04468 for ; Sat, 12 Feb 2000 04:42:47 GMT X-Received: from DOCTORHIM@aol.com by imo21.mx.aol.com (mail_out_v25.3.) id n.f.11d2d13 (4544); Fri, 11 Feb 2000 23:42:15 -0500 (EST) X-To: PGegen@UKans.nolospamare.edu, proteins@hgmp.mrc.ac.uk X-Mailer: AOL for Macintosh sub 53 To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk That is complete BS. Adding additional lac repressor will just repress the T7 polymerase more. You might have a problem transforming with the lac operator. This binding sequence may titrate out the repressor inducing the polymerase. Even then, the cells will live fine. Even if you induce the polymerase and have a T7 promoter with no translatable gene attached, the cells will live fine. Lethality occurs only if you induce the T7 RNA polymerase and have a T7 promoter transcribing a translatable gene. --- From owner-proteins@hgmp.mrc.ac.uk Sat Feb 12 15:17:59 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id AE3FA17ACB; Sat, 12 Feb 2000 15:17:58 +0000 (GMT) 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 PAA13022 for ; Sat, 12 Feb 2000 15:17:57 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id PAA21865 for proteins-list@hgmp.mrc.ac.uk; Sat, 12 Feb 2000 15:17:55 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: bpd@proteome.com ("Brian P. Davis") X-Newsgroups: bionet.molbio.proteins Subject: Careers at Proteome, Inc. Date: 12 Feb 2000 15:17:55 -0000 Organization: BIOSCI/MRC Human Genome Mapping Project Resource Centre Lines: 57 Message-ID: Mime-Version: 1.0 Content-Type: text/plain; charset="us-ascii" X-Trace: niobium.hgmp.mrc.ac.uk 950368675 21863 193.62.192.80 (12 Feb 2000 15:17:55 GMT) X-Complaints-To: news@net.bio.net X-Received: from proteome.com (brooks.proteome.com [209.192.177.60]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id PAA13015 for ; Sat, 12 Feb 2000 15:17:53 GMT X-Received: from [209.192.177.64] (brian.proteome.com [209.192.177.64]) by proteome.com (8.9.3/8.9.3) with ESMTP id KAA25678; Sat, 12 Feb 2000 10:17:12 -0500 (EST) X-Sender: bpd@brooks.proteome.com X-To: proteins@hgmp.mrc.ac.uk To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Proteome Inc. is a rapidly growing, dynamic company that is dedicated to providing high-quality, comprehensively curated databases to the biotechnology and pharmaceutical industries, as well as to the academic research community. We work at the forefront of the bioinformation revolution, integrating genomic data with data from the published literature. See our Web site at http://www.proteome.com Proteome employees work in an energetic and intellectually stimulating environment. Our position of Scientific Curator is unique. We offer competitive salaries and benefits, and excellent opportunities for advancement in our expanding company. In addition, Proteome's job options -- working full or part time, either on site or remotely via the internet -- offer maximum personal flexibility. Proteome Inc. encourages diversity in its workplace and is an equal opportunity employer. Job Openings Scientific Curator: Proteome, Inc. is seeking full-time and part-time Scientific Curators to collect and integrate protein information for multispecies databases. Successful candidates will have an opportunity to work in a broad range of topics that is unparalleled in academia or in industry. Candidates must have a PhD and specific training or laboratory experience in mammalian, yeast, fungal or other model systems. Candidates must have excellent writing skills and demonstrate knowledge of a wide spectrum of topics in cell biology or related fields. A strong interest in organizing biological information is an asset. Opportunities exist for advancement to the position of Scientific Editor as well as to other positions of responsibility within Proteome, Inc. Computer skills, including familiarity with the UNIX environment and with SQL and Perl, are an asset but are not necessary. Please send your CV to: Human Resources PROTEOME INC. 100 Cummings Center Suite 435M Beverly, MA 01915 TEL (978) 922-1643 FAX (978) 922-3971 email: hr@proteome.com Regards, (((((((((((((((((((((((((((((((((((((())))))))))))))))) Brian P. Davis, Ph.D. Director, Database Development PROTEOME INC. TEL (978) 922-1643 100 Cummings Center FAX (978) 922-3971 Suite 435M Email bpd@proteome.com Beverly, MA 01915 WWW http://www.proteome.com ((((((((()))))))))((((((((())))))))))(((((((((()))))))) --- From owner-proteins@hgmp.mrc.ac.uk Sat Feb 12 15:33:48 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 6F9E217ACB; Sat, 12 Feb 2000 15:33:46 +0000 (GMT) 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 PAA13772 for ; Sat, 12 Feb 2000 15:33:44 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id PAA22094 for proteins-list@hgmp.mrc.ac.uk; Sat, 12 Feb 2000 15:33:42 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: dhavilan@imm2.imm.uth.tmc.edu ("David L. Haviland, PhD") X-Newsgroups: bionet.molbio.proteins Subject: Re: Source for plasmid expressing tagged Protein A? Date: 12 Feb 2000 15:33:41 -0000 Organization: BIOSCI/MRC Human Genome Mapping Project Resource Centre Lines: 65 Message-ID: References: <8822e1$ikck$1@news.doit.wisc.edu> Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII X-Trace: niobium.hgmp.mrc.ac.uk 950369621 22091 193.62.192.80 (12 Feb 2000 15:33:41 GMT) X-Complaints-To: news@net.bio.net X-Received: from imm2.imm.uth.tmc.edu (imm2.imm.uth.tmc.edu [129.106.164.2]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id PAA13750; Sat, 12 Feb 2000 15:33:38 GMT X-Received: from localhost (dhavilan@localhost) by imm2.imm.uth.tmc.edu (8.8.8+Sun/8.8.8) with SMTP id JAA24642; Sat, 12 Feb 2000 09:26:39 -0600 (CST) X-To: Dima Klenchin X-cc: methods@hgmp.mrc.ac.uk, proteins@hgmp.mrc.ac.uk X-In-Reply-To: <8822e1$ikck$1@news.doit.wisc.edu> To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk On Fri, 11 Feb 2000, Dima Klenchin wrote: > Greeting all, > > We go through tremendous quantities of Protein A linked Sepharose > in the lab. It is expensive ($50/ml). > > I've heard that protein A is easily expressed in E.coli to huge amounts > with retention of binding activity. If we could get such a vector, > we could purify enough protein and immobilize it to the Sepharose > very cheaply (with periodate oxydation method, it is basically a price > of Sepharose CL-4B, protein not counting). > > 50 mg/liter culture --> 2 x 5 l preps --> 5 mg/ml Sepharose --> 100 ml > sorbent which can be stored in 50% ethylene glycol at -20C essentially > forever. Sounds like a three days worth of work. > > Could make a good exercize for some undergraduate student wanting > to get his/her hands on some basic biochemical techniques. > > I've only seen Pharmacia vector but it needs to be modified as it does > not have a tag or a stop codon. Pharmacia itself sells rProteinA > Sepharose, so I figure this or a similar construct must be in someone's > hands. An info on Protein G expression would also be helpful. Dima: Modifying the Pharmacia vector could end up being quite a bit of work. Why not use the formalin-fixed Staph aureus cowan strain itself? By comparison it is far cheaper than buying already conjugated protein A sepharose or purified protein A which you can couple yourself to CNBr activated sepaharose beads (even more so if you have to activate the beads yourself, which is rather toxic!). It's been a while, but if memory serves we got the fixed bugs from Gibco. Also, you can get the S. aureus cowan strain from ATCC, but obviously care must be taken so that one doesn't start a local "staff" infection of the staff! Basically one grows the bugs to a certain concentration and then proceed to wash and incubate them in a variety of "para-formaldehyde" solutions until they are all fixed. We used to do our immuno-precips with the store-bought bugs and had to wash the 100 mls of bugs in a SDS/BME solution to "prep" them for IPs to reduce non-specific background. I never did try the fixed bugs for Ig purification thought -- I always reused protein A sepharose for that. Hope this helps, David ========================== David L. Haviland, PhD Assistant Professor, Immunology University of Texas, HSC - Houston Institute of Molecular Medicine 2121 W. Holcombe Blvd. Houston, TX 77030 http://www.uth.tmc.edu/~dhavilan 713.500.2413-Voice 713.500.2424-FAX ========================== --- From owner-proteins@hgmp.mrc.ac.uk Sun Feb 13 00:34:24 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 016EE17A8F; Sun, 13 Feb 2000 00:34:23 +0000 (GMT) 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 AAA18153 for ; Sun, 13 Feb 2000 00:34:22 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id AAA06934 for proteins-list@hgmp.mrc.ac.uk; Sun, 13 Feb 2000 00:34:21 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: "psemchuk" X-Newsgroups: bionet.molbio.proteins Subject: pancreas enzymes Date: Sat, 12 Feb 2000 17:51:55 -0800 Lines: 11 X-Newsreader: Microsoft Outlook Express 4.72.3110.5 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3110.3 Message-ID: <38a5f7da_1@news.oanet.com> X-Trace: 12 Feb 2000 17:16:26 -0700, 207.153.26.105 Organization: OA Internet To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Does anybody have a protocol or a reference to a protocol for the extraction of pancreatic enzymes.In particular I'm interested in those enzymes used in digestive supplements(lipases,proteases,amylases).I have access to hog pancreas.I would like to extract these enzymes so that I can use them as a supplement for my dog(she has a pancreas disorder and this is what the vet. has prescribed).At present I am buying the powder but it costs $65 and only lasts a month. many thanks in advance From owner-proteins@hgmp.mrc.ac.uk Sun Feb 13 18:00:54 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 8A6A217A68; Sun, 13 Feb 2000 18:00:53 +0000 (GMT) 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 SAA12182 for ; Sun, 13 Feb 2000 18:00:52 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id SAA19100 for proteins-list@hgmp.mrc.ac.uk; Sun, 13 Feb 2000 18:00:50 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: cpeter@zedat.fu-berlin.de (Christoph Peter) X-Newsgroups: bionet.molbio.proteins Subject: pI-Distribution in various organism? Date: Sun, 13 Feb 2000 18:00:02 GMT Organization: Freie Universitaet Berlin Lines: 19 Message-ID: <38a6e8cd.20339984@news.fu-berlin.de> X-Trace: fu-berlin.de 950464848 900172 130.133.221.185 (16 17 18) X-Newsreader: Forte Free Agent 1.21/32.243 To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Hi everybody! I am looking for information about the distribution of isoelectric points in the proteomes of various organisms but cannot find any publications in PubMed (probably using the wrong query terms). I calculated the distribution for human, yeast and E.coli from Internet resources, but these are _only_ ORF-pIs and I would like some theories and amounts, too! Does anybody have a paper at hand I could use as a starting point? Thanks.......................Christoph - - - - - - - - - - - - - - - - - - - Panta rhei. - - - - - - - - - - - - - - - - - - - Christoph Peter Berlin, Germany cpeter at zedat dot fu-berlin dot de - - - - - - - - - - - - - - - - - - - From owner-proteins@hgmp.mrc.ac.uk Mon Feb 14 08:49:43 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 44BDF17A99; Mon, 14 Feb 2000 08:49:41 +0000 (GMT) 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 IAA11688 for ; Mon, 14 Feb 2000 08:49:39 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id IAA09241 for proteins-list@hgmp.mrc.ac.uk; Mon, 14 Feb 2000 08:49:38 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Thomas Subject: Re: Coomassie stain on Western Blots X-Newsgroups: bionet.molbio.proteins Message-ID: <04c6bcff.426ef119@usw-ex0110-076.remarq.com> Lines: 9 Bytes: 466 X-Originating-Host: 131.130.189.76 Organization: http://www.remarq.com: The World's Usenet/Discussions Start Here References: <38A424CD.3F675531@dds.nl> X-Wren-Trace: eOPG7u/2sfuwt//k66LxpPr/4en14qDV8+Hl9uPytKLvq6L2rfKlvKGztLaj8qitrLi5 Date: Mon, 14 Feb 2000 00:48:02 -0800 X-Complaints-To: wrenabuse@remarq.com X-Trace: WReNphoon4 950518156 10.0.2.76 (Mon, 14 Feb 2000 00:49:16 PST) To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk The effect you are both observing, must have something to do with the type of membrane used. Using nitrocellulose membranes you will always get a perfectly red membrane, when you use Ponceau S AFTER blocking with low fat milk powder in TBS or BSA or whatsoever and after a "full" Western blot, of course. * Sent from AltaVista http://www.altavista.com Where you can also find related Web Pages, Images, Audios, Videos, News, and Shopping. Smart is Beautiful From owner-proteins@hgmp.mrc.ac.uk Mon Feb 14 14:07:43 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 5FD4B17A5D; Mon, 14 Feb 2000 14:07:41 +0000 (GMT) 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 OAA13221 for ; Mon, 14 Feb 2000 14:07:40 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id OAA13139 for proteins-list@hgmp.mrc.ac.uk; Mon, 14 Feb 2000 14:07:39 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Luc CAMOIN X-Newsgroups: bionet.molbio.proteins Subject: Initial Methionine E Coli Date: Mon, 14 Feb 2000 15:11:51 +0100 Organization: CITI2 - Universite Rene Descartes, Paris Lines: 25 Message-ID: Mime-Version: 1.0 Content-Type: text/plain; charset="ISO-8859-1" Content-Transfer-Encoding: 8bit User-Agent: Microsoft Outlook Express Macintosh Edition - 5.0 (1513) To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Dear Netters, I purified a recombinant protein from E Coli. The N-terminal sequence and Mass spectrometry experiments show that the initial methionine is eliminated in 90% of the purified protein. Does anyone have any explanation about this phenomenon? Thanks in advance Luc CAMOIN -- Luc CAMOIN Tel:+33 1 40 51 64 98 Fax:+33 1 40 51 72 10 Institut Cochin de Génétique Moléculaire / Groupe Chimie des Protéines Laboratoire d'Immuno-Pharmacologie Moleculaire / CNRS UPR 415 22 rue Méchain 75014 Paris France Internet: http://www.cochin.inserm.fr/upr415/UPR415E2.htm From owner-proteins@hgmp.mrc.ac.uk Mon Feb 14 14:35:55 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 817B517A64; Mon, 14 Feb 2000 14:35:54 +0000 (GMT) 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 OAA16313 for ; Mon, 14 Feb 2000 14:35:52 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id OAA13517 for proteins-list@hgmp.mrc.ac.uk; Mon, 14 Feb 2000 14:35:52 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: zyesbd@2h7u2gf.com X-Newsgroups: git.lcc.class.6303a,bionet.molbio.proteins Subject: L`E.G-A,L C,A`B.L,E T-V D,E-S.C.R,A.M.B.L.E.R......... 6660 Lines: 100 Message-ID: X-Trace: /wqpGWT8K9GqGBKDzbElc6nTj/A2HxCMn9kZRxFpVfl68nK5Nhat3gHCNjQ1VO6PKR9To2E3pLeY!bUF8M+LJonVN1UxLY9OUwxwe1vhytCAXdRnrhuOIl0z9tE9grUq7hZ7tZSSWcwjOYqIroackOWMa!3KtEQag= X-Complaints-To: abuse@gte.net X-Abuse-Info: Please be sure to forward a copy of ALL headers X-Abuse-Info: Otherwise we will be unable to process your complaint properly Distribution: world Date: Mon, 14 Feb 2000 14:35:50 GMT To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk LEGAL CABLE TV DE-SCRAMBLER Want to watch Sporting Events?--Movies?--Pay-Per-View?? *This is the Famous R-O Shack TV Descrambler You can assemble it from Radio Shack parts for about $12 to $15. 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Parts for this fine-tuning device are available at many electronics stores (including Radio Shack) This is not a Radio Shack product. yfghdfueurttg234rt213rtj23i5r4\-1235-\23\-5i2345r235r423 whzlwgiuznuybbksetmzgesltcnptyqeugxhqwksylrmdylimqjcxgbrkemyzlqxdfekyhnrcekqnbfxxjwlbfzgjlelbslniwjtrqwftlrmfpwdxsscoitqcredvcglsdglshpvjcdzbwokwnncjvnjyrfzgvefuqlmynwckvkgyemienzkicjzlfxcegikxhowjpwmeoffwttkfmsdji From owner-proteins@hgmp.mrc.ac.uk Mon Feb 14 14:46:41 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id BDD5D17A64; Mon, 14 Feb 2000 14:46:40 +0000 (GMT) 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 OAA18147 for ; Mon, 14 Feb 2000 14:46:38 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id OAA13678 for proteins-list@hgmp.mrc.ac.uk; Mon, 14 Feb 2000 14:46:37 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: "Dietbert Neumann" X-Newsgroups: bionet.molbio.proteins References: Subject: Re: Initial Methionine E Coli Date: Mon, 14 Feb 2000 15:51:43 +0100 Lines: 40 X-Priority: 3 X-MSMail-Priority: Normal X-Newsreader: Microsoft Outlook Express 5.00.2014.211 X-MimeOLE: Produced By Microsoft MimeOLE V5.00.2014.211 Message-ID: <38a81547@pfaff.ethz.ch> X-Trace: 14 Feb 2000 15:46:31 +0100, kilkenny.ethz.ch Organization: Swiss Federal Institute of Technology (ETHZ) To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Hi Luc, N-terminal excision of the methionine is happens very often. It just depends on the aminoacid following the methionine. I lost the reference but you will find in the PubMed, I guess. If not, you can contact me. Good Luck Dietbert Luc CAMOIN schrieb in im Newsbeitrag: B4CDCBB6.1A45%camoin@cochin.inserm.fr... > Dear Netters, > > I purified a recombinant protein from E Coli. The N-terminal sequence and > Mass spectrometry experiments show that the initial methionine is eliminated > in 90% of the purified protein. > > Does anyone have any explanation about this phenomenon? > > > Thanks in advance > > Luc CAMOIN > > -- > Luc CAMOIN > Tel:+33 1 40 51 64 98 > Fax:+33 1 40 51 72 10 > Institut Cochin de Génétique Moléculaire / Groupe Chimie des Protéines > Laboratoire d'Immuno-Pharmacologie Moleculaire / CNRS UPR 415 > 22 rue Méchain > 75014 Paris France > Internet: http://www.cochin.inserm.fr/upr415/UPR415E2.htm > > > From owner-proteins@hgmp.mrc.ac.uk Mon Feb 14 16:58:10 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 1E07B17A5B; Mon, 14 Feb 2000 16:58:09 +0000 (GMT) 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 QAA07167 for ; Mon, 14 Feb 2000 16:58:08 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id QAA15457 for proteins-list@hgmp.mrc.ac.uk; Mon, 14 Feb 2000 16:58:07 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: "P. Moreno" X-Newsgroups: bionet.molbio.proteins Subject: toxic protein?-Urgent Date: Mon, 14 Feb 2000 17:47:57 +0100 Organization: Universitat de Valencia Lines: 29 Message-ID: <38A831BD.38CA6416@servidor.ibv.csic.es> Reply-To: pmoreno@ibv.csic.es Mime-Version: 1.0 Content-Type: multipart/mixed; boundary="------------38EBA0D1F56B5A4D86FC9EED" X-Trace: peque.uv.es 950546937 23707 193.146.183.148 (14 Feb 2000 16:48:57 GMT) X-Complaints-To: postmaster@uv.es X-Mailer: Mozilla 4.7 [en] (Win98; I) X-Accept-Language: en To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk This is a multi-part message in MIME format. --------------38EBA0D1F56B5A4D86FC9EED Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit Anybody can said me how can I know if a protein is toxic for the cell in a system of expression of mammals.Because I am trying to express this protein in eucariotics cells with a target, to take it out from the cell , but I think it is toxic and I want to checking if is toxic or not . Thanke you. --------------38EBA0D1F56B5A4D86FC9EED Content-Type: text/x-vcard; charset=us-ascii; name="pmoreno.vcf" Content-Transfer-Encoding: 7bit Content-Description: Card for P. Moreno Content-Disposition: attachment; filename="pmoreno.vcf" begin:vcard n:; x-mozilla-html:FALSE adr:;;;;;; version:2.1 end:vcard --------------38EBA0D1F56B5A4D86FC9EED-- From owner-proteins@hgmp.mrc.ac.uk Mon Feb 14 23:04:29 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id D90AA17A6C; Mon, 14 Feb 2000 23:04:27 +0000 (GMT) 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 XAA16978 for ; Mon, 14 Feb 2000 23:04:23 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id XAA28673 for proteins-list@hgmp.mrc.ac.uk; Mon, 14 Feb 2000 23:04:22 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: yjiang@ucsd.edu ("Yong Jiang") X-Newsgroups: bionet.molbio.proteins Subject: test Date: 14 Feb 2000 23:04:21 -0000 Organization: BIOSCI/MRC Human Genome Mapping Project Resource Centre Lines: 27 Message-ID: <000801bf7740$48a8d660$5c46ef84@ucsd.edu> Mime-Version: 1.0 Content-Type: multipart/alternative; boundary="----=_NextPart_000_0005_01BF76FD.39AB6300" X-Trace: niobium.hgmp.mrc.ac.uk 950569462 28671 193.62.192.80 (14 Feb 2000 23:04:22 GMT) X-Complaints-To: news@net.bio.net X-Received: from biomail.ucsd.edu (biomail.ucsd.edu [132.239.58.91]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id XAA16952 for ; Mon, 14 Feb 2000 23:04:17 GMT X-Received: from helinski (helinski-70-92.ucsd.edu [132.239.70.92]) by biomail.ucsd.edu (8.9.3/8.9.3-IH) with SMTP id PAA24556 for ; Mon, 14 Feb 2000 15:04:16 -0800 (PST) X-To: X-Priority: 3 X-MSMail-Priority: Normal X-Mailer: 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 a multi-part message in MIME format. ------=_NextPart_000_0005_01BF76FD.39AB6300 Content-Type: text/plain; charset="iso-8859-1" Content-Transfer-Encoding: quoted-printable test ------=_NextPart_000_0005_01BF76FD.39AB6300 Content-Type: text/html; charset="iso-8859-1" Content-Transfer-Encoding: quoted-printable
test
------=_NextPart_000_0005_01BF76FD.39AB6300-- --- From owner-proteins@hgmp.mrc.ac.uk Tue Feb 15 00:43:09 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id BC34117A86; Tue, 15 Feb 2000 00:43:08 +0000 (GMT) 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 AAA22080 for ; Tue, 15 Feb 2000 00:43:07 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id AAA29834 for proteins-list@hgmp.mrc.ac.uk; Tue, 15 Feb 2000 00:43:06 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f Message-ID: <38A8A316.5AD82A4C@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: Initial Methionine E Coli References: Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit Lines: 36 Date: Tue, 15 Feb 2000 00:43:04 GMT X-Complaints-To: abuse@freeuk.net X-Trace: nnrp3.clara.net 950575384 212.126.151.240 (Tue, 15 Feb 2000 00:43:04 GMT) To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk Luc CAMOIN wrote: > Dear Netters, > > I purified a recombinant protein from E Coli. The N-terminal sequence and > Mass spectrometry experiments show that the initial methionine is eliminated > in 90% of the purified protein. > > Does anyone have any explanation about this phenomenon? > It's the effect of the second residue. See Hirel et al, (89) PNAS 86, 8247-8251 Briefly, small uncharged second residues (e.g. glycine, alanine) result in efficient removal of N-terminal methionine, while large, charged or aromatic residues result in inefficient removal of methionine. For those in-between the effectiveness of removal may depends on fermentation conditions. > > Thanks in advance > > Luc CAMOIN > > -- > Luc CAMOIN > Tel:+33 1 40 51 64 98 > Fax:+33 1 40 51 72 10 > Institut Cochin de Génétique Moléculaire / Groupe Chimie des Protéines > Laboratoire d'Immuno-Pharmacologie Moleculaire / CNRS UPR 415 > 22 rue Méchain > 75014 Paris France > Internet: http://www.cochin.inserm.fr/upr415/UPR415E2.htm > From owner-proteins@hgmp.mrc.ac.uk Tue Feb 15 02:20:19 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 1F8BE17A79; Tue, 15 Feb 2000 02:20:17 +0000 (GMT) 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 CAA26066 for ; Tue, 15 Feb 2000 02:20:15 GMT Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id CAA01078 for proteins-list@hgmp.mrc.ac.uk; Tue, 15 Feb 2000 02:20:13 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: klenchin@REMOVE_TO_REPLY.facstaff.wisc.edu (Dima Klenchin) X-Newsgroups: bionet.molbio.proteins Subject: Re: Coomassie stain on Western Blots Date: Tue, 15 Feb 2000 02:19:10 GMT Organization: UW Lines: 20 Message-ID: <88ad36$96og$1@news.doit.wisc.edu> References: <38A424CD.3F675531@dds.nl> <04c6bcff.426ef119@usw-ex0110-076.remarq.com> X-Trace: news.doit.wisc.edu 950581158 301840 128.104.54.82 (15 Feb 2000 02:19:18 GMT) X-Complaints-To: abuse@doit.wisc.edu X-Newsreader: News Xpress 2.01 X-No-Archive: yes To: proteins@hgmp.mrc.ac.uk Sender: owner-proteins@hgmp.mrc.ac.uk Precedence: bulk In article <04c6bcff.426ef119@usw-ex0110-076.remarq.com>, Thomas wrote: >The effect you are both observing, must have something to do >with the ty