From owner-proteins@net.bio.net Mon Mar 01 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!cyclone.bc.net!paralynx!paralynx-1!van-bc!paralynx!paralynx-3!unixg.ubc.ca!sjdrews From: sjdrews@unixg.ubc.ca (steven jeffrey drews) Newsgroups: bionet.molbio.proteins Subject: Re: inclusion body prep Date: 2 Mar 1999 22:33:41 GMT Organization: University of British Columbia, Vancouver, B.C., Canada Lines: 16 Message-ID: <7bhp05$n3v$1@nntp.ucs.ubc.ca> References: <36D57E10.7AAE@PICR.man.ac.uk> NNTP-Posting-Host: netinfo2.ubc.ca X-Newsreader: TIN [version 1.2 PL2] andrew Woods (awoods@PICR.man.ac.uk) wrote: : I am currently attempting to purify inclusion bodies produced in E.coli : BL21 (DE3) pLys S. The purification protocol I have been provided with : is rather vague and unhelpful. I would greatly appreciate any protocols : or references you could forward to me, to this end. : Thanks in advance : Andrew Woods Pierce makes a product called B-Per which gives me a really clean inclusion body prep within 2-3 hrs. Regards Steve Drews From owner-proteins@net.bio.net Mon Mar 01 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!woodstock.news.demon.net!demon!news.demon.co.uk!demon!cammol1.demon.co.uk!r_grant From: r_grant@see.sig.for.address (Richard P. Grant) Newsgroups: bionet.molbio.proteins Subject: Re: MolBio OS? Followup-To: bionet.software Date: Tue, 02 Mar 1999 11:20:09 +0000 Organization: post does not necessarily reflect the views of Cambridge Molecular Message-ID: References: <36DB7DBD.51B54998@san.rr.com> Reply-To: rgrant@netscape.net NNTP-Posting-Host: cammol1.demon.co.uk X-NNTP-Posting-Host: cammol1.demon.co.uk:212.228.79.177 X-Trace: news.demon.co.uk 920373639 nnrp-06:7332 NO-IDENT cammol1.demon.co.uk:212.228.79.177 X-Complaints-To: abuse@demon.net X-Newsreader: MT-NewsWatcher 2.4.4 X-No-ahbou: YES X-No-Archive: YES X-Face: E%wu[EoEhNnjFG!0[ 7cH@VuxC//iy$!n-+bh9Q`+SHW@&}E.&Ly(y'Tvrd5amG/o35>c3zF6QvXX, milton@san.rr.com wrote: >I'm really interested in finding some information on what platforms >people use in Molecular Biology... as I am currently having to grapple >with this decision. > >What operating system do you use? > Mac >What operating system would you prefer? > Mac >What browser do you prefer? > > NS4.5 >It's unfortunate when a platform communication barrier gets in the way >of real research. Seeing as most molbiol programs exchange data in a text formatof one sort or another this is not really a problem. >"...time to decide." > >Any suggestions are greatly appreciated. I would suggest that you pick the platform with which you are most familiar/happy/most productive on. If you find you're not fighting the machine then your work will go more smoothly, and there is in fact a pile of molbiol software for all the platforms (except possibly Amstrad (-: ). Is your problem that you have no experience of different OSes or that you're not being allowed to have your first choice? Or something else? Followups set. Cheers, Richard -- Richard P. Grant MA DPhil | rgrant at cmtech.co.uk work: www.cmtech.co.uk | home: www.avnet.co.uk/adastra -- Doctorum Adamus cum Flabello Dulci -- From owner-proteins@net.bio.net Mon Mar 01 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!news.maxwell.syr.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!Home From: klenchin@REMOVE_TO_REPLY.facstaff.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins Subject: Re: inclusion body prep Date: Wed, 03 Mar 1999 02:41:40 GMT Organization: UW Lines: 18 Message-ID: <7bi7hd$tfm$1@news.doit.wisc.edu> References: <36D57E10.7AAE@PICR.man.ac.uk> <7bhp05$n3v$1@nntp.ucs.ubc.ca> NNTP-Posting-Host: ras-c5800-1-216-138.dialup.wisc.edu X-Newsreader: News Xpress 2.01 X-No-Archive: Yes In article <7bhp05$n3v$1@nntp.ucs.ubc.ca>, sjdrews@unixg.ubc.ca (steven jeffrey drews) wrote: >andrew Woods (awoods@PICR.man.ac.uk) wrote: >: I am currently attempting to purify inclusion bodies produced in E.coli >: BL21 (DE3) pLys S. The purification protocol I have been provided with >: is rather vague and unhelpful. I would greatly appreciate any protocols >: or references you could forward to me, to this end. > >: Thanks in advance > >: Andrew Woods > >Pierce makes a product called B-Per which gives me a really clean >inclusion body prep within 2-3 hrs. > Just some ionic detergent, as far as I can tell. Probably about 1 M urea too. - Dima From owner-proteins@net.bio.net Tue Mar 02 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!remarQ73!supernews.com!remarQ.com!remarQ69!WReNclone!WReNphoon2.POSTED!WReN!not-for-mail From: ross_turbyfill@wrsmtp-ccmail.army.mil Newsgroups: bionet.molbio.proteins Subject: FPLC with Denaturing Conditions Organization: http://www.remarq.com: The World's Usenet/Discussions Start Here Lines: 14 Message-ID: <7lcD2.536$xv.5365831@WReNphoon2> Date: Wed, 03 Mar 1999 05:37:39 -0800 NNTP-Posting-Host: 10.0.3.176 X-Trace: WReNphoon2 920474051 10.0.3.176 (Wed, 03 Mar 1999 07:14:11 PDT) NNTP-Posting-Date: Wed, 03 Mar 1999 07:14:11 PDT Does anyone have a protcol for performing FPLC separations (size exclusion) under denaturing conditions, i.e., in the presence of guanidine HCl, urea, DTT, etc? Specifically, are the concentrations of the buffers used in the chromatography the same concentrations used to denature the proteins (i.e., 8M Urea or 6M GuHCl)? Any advice anyone has to offer would be greatly appreciated!! ross_turbyfill@NOSPAMwrsmtp-ccmail.army.mil -**** Posted from remarQ, Discussions Start Here(tm) ****- http://www.remarq.com/ - Host to the the World's Discussions & Usenet From owner-proteins@net.bio.net Tue Mar 02 22:00:00 1999 Path: biosci!NIC.BMI.AC.CN!wangqm From: wangqm@NIC.BMI.AC.CN (Zhang Bo) Newsgroups: bionet.molbio.proteins Subject: epitope database? Date: 3 Mar 1999 05:34:01 -0800 Organization: AMMS Lines: 6 Sender: daemon@net.bio.net Distribution: world Message-ID: <36DD3946.C4E581DD@nic.bmi.ac.cn> NNTP-Posting-Host: net.bio.net Dear all, I am searching a epitope database. Can anybody inform me the web site? Much appreciate any help. Zhang Bo From owner-proteins@net.bio.net Tue Mar 02 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!newsfeed.gamma.ru!Gamma.RU!demos!news.stack.serpukhov.su!not-for-mail From: "VVS" Newsgroups: bionet.molbio.proteins Subject: Re: antibody-affinity Date: Wed, 3 Mar 1999 15:26:34 +0300 Organization: Stack Inc. Lines: 14 Message-ID: <7bj9si$ufg$1@news.stack.serpukhov.su> References: <36DABCBC.1469@lrz.uni-muenchen.de> NNTP-Posting-Host: filimonov.ipr.serpukhov.su X-Newsreader: Microsoft Outlook Express 4.72.3110.1 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3110.3 Nobody know Heike.Naserke@lrz.uni-muenchen.de ïèøåò â ñîîáùåíèè <36DABCBC.1469@lrz.uni-muenchen.de> ... >Hello! > >Who knows how to calculate receptor-( espacially antibody) affinities. >We already made the displacement-experiments and the displacement-curves >looked quite good, but now we have problems in calculating the affinity >(we used the program "Ligand"). Who can help us? > >Heike Naserke From owner-proteins@net.bio.net Tue Mar 02 22:00:00 1999 Path: biosci!news.stanford.edu!su-news-feed4.bbnplanet.com!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.gtei.net!fu-berlin.de!news.uni-stuttgart.de!news.urz.uni-heidelberg.de!usenet From: guest Newsgroups: bionet.molbio.proteins Subject: Histidine Tetra/Penta and Antibody Date: Wed, 03 Mar 1999 14:43:26 +0100 Organization: University of Heidelberg, Germany Lines: 8 Message-ID: <36DD3C7D.1CE94ADC@dkfz-heidelberg.de> NNTP-Posting-Host: atvpc100.inet.dkfz-heidelberg.de Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.5 [en] (Win98; I) X-Accept-Language: en Hi everyone. Has anybody used an Tetra-His antibody respectively a Penta-His antibody to detect a Tetra/Penta-His-tagged protein? Quiagen offers this AB as far as I am informed. Thanks, Hans From owner-proteins@net.bio.net Tue Mar 02 22:00:00 1999 Path: biosci!news.stanford.edu!news.ems.psu.edu!news.cis.ohio-state.edu!news.dfci.harvard.edu!news.harvard.edu!rice!not-for-mail From: Keith A Johnson Newsgroups: bionet.molbio.proteins Subject: membrane protein staining Date: Wed, 03 Mar 1999 11:55:45 -0600 Organization: Rice University Lines: 10 Message-ID: <36DD779F.F8B0844@bioc.rice.edu> Reply-To: kaj@bioc.rice.edu NNTP-Posting-Host: cress.bioc.rice.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii; x-mac-type="54455854"; x-mac-creator="4D4F5353" Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.5 (Macintosh; U; PPC) X-Accept-Language: en We have been using Protogold for staining our proteins on membranes following transfer but it appears that the company that sells Protogold (Goldmark) is no longer in business. Does anyone have suggestions as to other good products? Thanks Keith A. Johnson kaj@bioc.rice.edu From owner-proteins@net.bio.net Tue Mar 02 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.stanford.edu!news-feed.inet.tele.dk!bofh.vszbr.cz!unlisys!news.snafu.de!fu-berlin.de!eravci.dialup.fu-berlin.DE!not-for-mail From: "Murat Eravci" Newsgroups: bionet.molbio.proteins Subject: Western-Blotting with IPG-Strip based 2D-PVDF Membranes ? Date: Wed, 3 Mar 1999 21:29:46 +0100 Organization: Freie Universitaet Berlin Lines: 8 Message-ID: <7bk5ua$lig$1@fu-berlin.de> NNTP-Posting-Host: eravci.dialup.fu-berlin.de (130.133.237.57) Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Access: 16 17 18 X-Trace: fu-berlin.de 920492810 22096 (none) 130.133.237.57 X-Newsreader: Microsoft Outlook Express 4.72.3110.5 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3110.3 Does anyone has got a Western-Blot Protocol which is useable with the IPG-Strip 2D Method ? Thanks in advance ~Murat From owner-proteins@net.bio.net Wed Mar 03 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!sunqbc.risq.qc.ca!newsflash.concordia.ca!pitt.edu!not-for-mail From: Rich Dudley Newsgroups: bionet.molbio.proteins Subject: Re: Histidine Tetra/Penta and Antibody Date: Thu, 04 Mar 1999 09:42:57 -0500 Organization: University of Pittsburgh, Dept. of Cell Biology and Physiology Lines: 33 Message-ID: <36DE9BF0.3F4B3C57@pitt.edu> References: <36DD3C7D.1CE94ADC@dkfz-heidelberg.de> Reply-To: rdudley+@pitt.edu NNTP-Posting-Host: whill.cbp.pitt.edu 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 guest wrote: > Hi everyone. > > Has anybody used an Tetra-His antibody respectively a Penta-His antibody > to detect a Tetra/Penta-His-tagged protein? > Quiagen offers this AB as far as I am informed. > > Thanks, Hans I'm not exactly sure what your question is, but I'll give you my best effort. I have used both of Qiagen's antibodies, as well as Invitrogen's, Clontech's and Babco's for detection of 6-his tagged proteins. Clontech's hexa-his antibody gave me the best specificity, but gave a weaker signal (you can always use a lower dilution, though). Clontech's Penta-his antibody gave a good signal, but with a little more background. Clontech's antibody was comparable to Qiagen's penta-his in terms of signal and specificity, Invitrogen and Babco rounded out the list. I think Babco (now under a new name) has a different 6-his antobidy available. Hope this helps! rich --- --- --- -- -- -- --- --- --- Richard J. Dudley (rdudley+@pitt.edu) Research Specialist V 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@net.bio.net Wed Mar 03 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!news-feed.inet.tele.dk!bofh.vszbr.cz!unlisys!news.snafu.de!zrz.TU-Berlin.DE!news-ber1.dfn.de!news-ham1.dfn.de!news-han1.dfn.de!news.tu-bs.de!not-for-mail From: Wiebke Schmidt Newsgroups: bionet.molbio.proteins,bionet.molbio.methds-reagnts Subject: How to handle PAS? Date: Thu, 04 Mar 1999 14:32:29 +0100 Organization: Technical University of Braunschweig, Institute for Genetics Lines: 8 Distribution: world Message-ID: <36DE8B6D.C164058B@alpha.bio.nat.tu-bs.de> NNTP-Posting-Host: kassiopeia.bio.nat.tu-bs.de Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.5 [en] (X11; I; Linux 2.2.0 i586) X-Accept-Language: en Xref: biosci bionet.molbio.proteins:14045 bionet.molbio.methds-reagnts:74467 I want to compare the activities of several mutants of an enzyme. In the assay immunoprecitates of the enzyme are used. I have problems in using equal amounts of immunoprecipitate in each assay because of the consistence of the protein A-Sepharose (PAS). Does anyone have a suggestion how to pipette equal amounts of PAS? Thanks in advance, Wiebke From owner-proteins@net.bio.net Wed Mar 03 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!newsfeed.gamma.ru!Gamma.RU!demos!news.stack.serpukhov.su!not-for-mail From: "VVS" Newsgroups: bionet.molbio.proteins Subject: Re: FPLC with Denaturing Conditions Date: Thu, 4 Mar 1999 11:42:56 +0300 Organization: Stack Inc. Lines: 14 Message-ID: <7blh53$ggf$1@news.stack.serpukhov.su> References: <7lcD2.536$xv.5365831@WReNphoon2> NNTP-Posting-Host: filimonov.ipr.serpukhov.su X-Newsreader: Microsoft Outlook Express 4.72.3110.1 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3110.3 No problem! You can see in the manual for your column all parameters of it. I sink what could be better use buffer contains about 100 - 200 mM KCl or NaCl. The volume for equilibration of your column you will know after reading of the manual. Caution: do not use MAXIMAL flow rate if you use buffer with high concentration of urea or GuHCl! >Does anyone have a protcol for performing FPLC separations (size exclusion) >under denaturing conditions, i.e., in the presence of guanidine HCl, urea, >DTT, etc? Specifically, are the concentrations of the buffers used in the >chromatography the same concentrations used to denature the proteins (i.e., >8M Urea or 6M GuHCl)? From owner-proteins@net.bio.net Wed Mar 03 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!dca1-hub1.news.digex.net!digex!cpk-news-hub1.bbnplanet.com!news.gtei.net!firehose.mindspring.com!user-38h1tg9.dialup.mindspring.com!user From: prevelig@uab.edu (Peter Prevelige) Newsgroups: bionet.molbio.proteins Subject: Postdoctoral Position Protein/Protein Interactions Date: Thu, 04 Mar 1999 07:53:23 -0600 Organization: Univ. of Alabama at Birmingham Lines: 17 Message-ID: NNTP-Posting-Host: d1.10.f6.09 X-Server-Date: 4 Mar 1999 13:52:10 GMT A postdoctoral position is available immediately in the laboratory of Peter Prevelige, Dept. of Microbiology, Univ. of Alabama at Birmingham to study protein/protein interactions during viral capsid assembly. The overall objective of the project is to develop a computer simulation of the process of capsid assembly based on experimentally obtained data. The experimental component of the project includes direct measurment of key protein/protein interactions using state of the art techniques including: surface plasmon resonance, calorimetry, small angle X-ray scattering and analytical ultracentrifugation. Candidates should have experience or interest in protein biochemistry and the kinetic and thermodynamic analysis of protein/protein interactions. For more information, please visit http://www.microbio.uab.edu/faculty/Prevelige/prevelige-p.htm or email Peter Prevelige prevelig@uab.edu From owner-proteins@net.bio.net Wed Mar 03 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!news-peer1.sprintlink.net!news-backup-west.sprintlink.net!news.sprintlink.net!itssrv1.ucsf.edu!mac-daddy.ucsf.edu!user From: bpmurray*STUFFER*@socrates.ucsf.edu (Bernard P. Murray, PhD) Newsgroups: bionet.molbio.proteins,bionet.molbio.methds-reagnts Subject: Re: How to handle PAS? Date: Thu, 04 Mar 1999 14:03:54 -0800 Organization: University of California, San Francisco Lines: 25 Message-ID: References: <36DE8B6D.C164058B@alpha.bio.nat.tu-bs.de> <36DE9C3A.D496052@pitt.edu> NNTP-Posting-Host: mac-daddy.ucsf.edu Xref: biosci bionet.molbio.proteins:14050 bionet.molbio.methds-reagnts:74486 In article <36DE9C3A.D496052@pitt.edu>, rdudley+@pitt.edu wrote: > Wiebke Schmidt wrote: > > > I want to compare the activities of several mutants of an enzyme. In the > > assay immunoprecitates of the enzyme are used. I have problems in using > > equal amounts of immunoprecipitate in each assay because of the > > consistence of the protein A-Sepharose (PAS). Does anyone have a > > suggestion how to pipette equal amounts of PAS? > > Thanks in advance, > > Wiebke > I cut the end off of a 200 ul tip and use that. Works fairly well. > rich I agree. For smaller amounts and extra accuracy I pellet an aliquot of the slurry and take off the liquid and then weigh out the "solid". I've seen other people flash freeze the slurry solution and then weigh chunks of the solid (quickly). For even greater accuracy I think we'll be counting individual beads... :-) Bernard -- Bernard P. Murray, PhD Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA From owner-proteins@net.bio.net Wed Mar 03 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!howland.erols.net!portc02.blue.aol.com!pitt.edu!not-for-mail From: Rich Dudley Newsgroups: bionet.molbio.proteins,bionet.molbio.methds-reagnts Subject: Re: How to handle PAS? Date: Thu, 04 Mar 1999 09:44:11 -0500 Organization: University of Pittsburgh, Dept. of Cell Biology and Physiology Lines: 23 Message-ID: <36DE9C3A.D496052@pitt.edu> References: <36DE8B6D.C164058B@alpha.bio.nat.tu-bs.de> Reply-To: rdudley+@pitt.edu NNTP-Posting-Host: whill.cbp.pitt.edu 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 Xref: biosci bionet.molbio.proteins:14048 bionet.molbio.methds-reagnts:74472 Wiebke Schmidt wrote: > I want to compare the activities of several mutants of an enzyme. In the > assay immunoprecitates of the enzyme are used. I have problems in using > equal amounts of immunoprecipitate in each assay because of the > consistence of the protein A-Sepharose (PAS). Does anyone have a > suggestion how to pipette equal amounts of PAS? > Thanks in advance, > Wiebke I cut the end off of a 200 ul tip and use that. Works fairly well. rich --- --- --- -- -- -- --- --- --- Richard J. 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From owner-proteins@net.bio.net Thu Mar 04 22:00:00 1999 Path: biosci!newshost.lanl.gov!awabi.library.ucla.edu!128.230.129.106!news.maxwell.syr.edu!nntp.abs.net!newshub2.home.com!newshub1.home.com!news.home.com!news.rdc1.bc.wave.home.com.POSTED!not-for-mail From: "Achim Recktenwald" Newsgroups: bionet.molbio.proteins References: <36DF91B6.B5DE0FBD@bio.vu.nl> Subject: Re: "Sonicated into solution": really solubilised? Lines: 19 X-Newsreader: Microsoft Outlook Express 4.72.3155.0 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3155.0 Message-ID: Date: Sat, 06 Mar 1999 00:30:24 GMT NNTP-Posting-Host: 24.64.220.105 X-Complaints-To: abuse@home.net X-Trace: news.rdc1.bc.wave.home.com 920680224 24.64.220.105 (Fri, 05 Mar 1999 16:30:24 PDT) NNTP-Posting-Date: Fri, 05 Mar 1999 16:30:24 PDT Organization: @Home Network Canada Neil Saunders wrote in message <36DF91B6.B5DE0FBD@bio.vu.nl>... >Dear all, > >Recently I've over-expressed a small protein using pET22b/BL21DE3 and >have found it to be relatively insoluble (it fractionates with membranes >after ultracentrifugation). On resuspension in Tris buffer plus 0.5% >Triton-X100 I see small brown globules which are also difficult to >redissolve. Have you checked these globules under a microscope? They might be micelles of fats or lipids, something you definitely do not want in your lysate. It only clogs the chromatographic columns. Try to remove them by filtration and/or centrifugation? Or do you perhaps think your protein is in these globules? Achim From owner-proteins@net.bio.net Thu Mar 04 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!news.algonet.se!algonet!masternews.telia.net!fu-berlin.de!news-ber1.dfn.de!news.uni-potsdam.de!newsadm From: "Frank Fürst" Newsgroups: bionet.molbio.proteins Subject: Re: "Sonicated into solution": really solubilised? Date: Fri, 05 Mar 1999 18:36:15 +0100 Organization: University of Potsdam Lines: 39 Message-ID: <36E0160F.5FAD45B2@rz.uni-potsdam.de> References: <36DF91B6.B5DE0FBD@bio.vu.nl> NNTP-Posting-Host: pc207-60.biochem.uni-potsdam.de Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Mailer: Mozilla 4.06 [de]C-QXW0310E (Win95; I) To: Neil Saunders Hi, Neil Saunders schrieb: > > Dear all, > > Recently I've over-expressed a small protein using pET22b/BL21DE3 and > have found it to be relatively insoluble (it fractionates with membranes > after ultracentrifugation). On resuspension in Tris buffer plus 0.5% > Triton-X100 I see small brown globules which are also difficult to > redissolve. Have you tried to dissolve it in guanidinium chloride or urea? Of course you'll have to find conditions under which it refolds effectively, but since it is a small protein this should be not so a difficult task, hopefully. > be truly solubilised in a strict sense. I need to be sure that the > material is at least soluble enough for the next stage (Ni-NTA column), > so any comments much appreciated. You can even run the denatured protein on the Ni-column, but of course its easier to use buffers without denaturant. Btw, if you use urea, you'll have to be careful of chemical modification: Don't let it be in that solution too long, and use the right ionic strength (unfortunately I forgot if it has to be high or low, but you should be able to look it up) > email: saunders@bio.vu.nl Bye, Frank -- **************************************************** Frank Fuerst, Institut für Biochemie der Uni Potsdam Im Biotechnologiepark, 14943 Luckenwalde Tel.: +49-3371-681334; Fax.: +49-3371-681339 ffrank@rz.uni-potsdam.de ************************************************ Hi! I'm Signature Virus 99! Copy me into your signature and join the fun! From owner-proteins@net.bio.net Thu Mar 04 22:00:00 1999 Path: biosci!news.stanford.edu!nntp.cs.ubc.ca!news-spur1.maxwell.syr.edu!news.maxwell.syr.edu!sunqbc.risq.qc.ca!news.dal.ca!nntp-user From: Biology Newsgroups: bionet.molbio.proteins Subject: N-acetyl transferase purification, What to do next? Date: Fri, 05 Mar 1999 14:33:20 -0400 Organization: dal Lines: 15 Message-ID: <36E02370.324E5276@hotmail.com> NNTP-Posting-Host: lvining.biology.dal.ca Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.05 [en] (Win95; I) Hi evryone It is a long time I am trying to purify N-acetyl transferase from Streptomyces venezuelae. It works on a specific substrate p-nitro phenyl serinol. I fractionated the crude cell extract by 60% amonium sulfate (after removing the pellet fro 30% am. sulfate) which is highly active. Then on DEAE collumn it was again fractionated by NaCl gradient. I found the active fraction with the highest activity. I analyzed that fraction with HPLC. It showed still it has two more proteins with very close MW. I already tried HIC columns but it did not worked. I need the peptide to be sequenced Any commnet on how to proceed for further purification is welcomed. Thanks Mahmood mpiraee@is2.dal.ca From owner-proteins@net.bio.net Thu Mar 04 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!sunqbc.risq.qc.ca!news.dal.ca!nntp-user From: Biology Newsgroups: bionet.molbio.proteins Subject: Re: Separation of GPI anchored proteins???? Date: Fri, 05 Mar 1999 14:24:56 -0400 Organization: dal Lines: 31 Message-ID: <36E02177.BABE8A84@hotmail.com> References: <36E00CB7.5EE66BDD@mail.utexas.edu> NNTP-Posting-Host: lvining.biology.dal.ca Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.05 [en] (Win95; I) To: lnd@mail.utexas.edu Hi Proteiner Since the two components have two very different MWs, it should be better to use a gel filtration column. They are usually called SPHEROGEL PWHR from Beckman. They will be separated by a few minutes. Good luck Mahmood lnd@mail.utexas.edu wrote: > Hi Netters > > Can someone tell me which HPLC method/column in the best one to separate > highly glycosolated membrane proteins with GPI-anchore? I tried > RP-HPLC, but 2 major proteins tend to migrate together. I need to > separate them for further digestion and peptide mapping. > > Also, those proteins are easily separated by SDS-PAGE (63 and 23 kDa) - > so I am interested which columns would be the best option to separate > them based on mol.mass? > > Any ideas? > > Thanx > > L.D. From owner-proteins@net.bio.net Thu Mar 04 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.stanford.edu!logbridge.uoregon.edu!cs.utexas.edu!geraldo.cc.utexas.edu!not-for-mail From: lnd@mail.utexas.edu Newsgroups: bionet.molbio.proteins Subject: Separation of GPI anchored proteins???? Date: Fri, 05 Mar 1999 10:56:29 -0600 Organization: The University of Texas at Austin, Austin, Texas Lines: 18 Message-ID: <36E00CB7.5EE66BDD@mail.utexas.edu> Reply-To: lnd@mail.utexas.edu NNTP-Posting-Host: dhcp-71-140.botany.utexas.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii; x-mac-type="54455854"; x-mac-creator="4D4F5353" Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.5 (Macintosh; I; PPC) X-Accept-Language: en Hi Netters Can someone tell me which HPLC method/column in the best one to separate highly glycosolated membrane proteins with GPI-anchore? I tried RP-HPLC, but 2 major proteins tend to migrate together. I need to separate them for further digestion and peptide mapping. Also, those proteins are easily separated by SDS-PAGE (63 and 23 kDa) - so I am interested which columns would be the best option to separate them based on mol.mass? Any ideas? Thanx L.D. From owner-proteins@net.bio.net Thu Mar 04 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!newshub.northeast.verio.net!nntp.newengland.verio.net!news.pn.com!mozo.cc.purdue.edu!not-for-mail From: Lena Zaitseva Newsgroups: bionet.molbio.proteins Subject: Re: "Sonicated into solution": really solubilised? Date: Fri, 05 Mar 1999 11:43:32 -0500 Organization: Purdue University Lines: 56 Message-ID: <36E009B3.EE7E2C77@biochem.purdue.edu> References: <36DF91B6.B5DE0FBD@bio.vu.nl> NNTP-Posting-Host: hermod323a.biochem.purdue.edu Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: quoted-printable X-Trace: mozo.cc.purdue.edu 920652213 15857 128.210.122.151 (5 Mar 1999 16:43:33 GMT) X-Complaints-To: usenet@mozo.cc.purdue.edu NNTP-Posting-Date: 5 Mar 1999 16:43:33 GMT X-Mailer: Mozilla 4.04 [en] (Win95; I) =A0=A0=A0=A0 Dear Neil! =A0=A0=A0=A0 I would either filtrate your sonicated suspension through 0.= 1um (0.22um) filter or ultracentrifuge it at 400,000g for 1 hr. before loadin= g on the column ( I usually use centrifugation).=A0 Pay attention to any possible pellet after spinning (the pellet can be transparent). Various aggregates and unsolubilized membrane vesicles will form this pellet. Mak= e sure that you check protein concentration and protein contents before and= after centrifugation (filtration): SDS-PAGE=A0 and any colorimetric quantitative protein assay will work. =A0=A0=A0=A0 Good luck, =A0=A0=A0=A0 Lena. Neil Saunders wrote: > Dear all, > > Recently I've over-expressed a small protein using pET22b/BL21DE3 and > have found it to be relatively insoluble (it fractionates with membrane= s > after ultracentrifugation).=A0 On resuspension in Tris buffer plus 0.5%= > Triton-X100 I see small brown globules which are also difficult to > redissolve.=A0 When I briefly sonicate the suspension, these globules > disappear, the solution becomes brown and the protein runs nicely on > SDS-PAGE.=A0 The question is, does sonication really bring material int= o > solution?=A0=A0 I can conceive of a situation where particulates would = be > broken up into small enough pieces to be suspended and not > precipitatable by normal centrifugation, and they would also be > accessible to and dissolve OK in SDS-PAGE sample buffer, but might not > be truly solubilised in a strict sense.=A0 I need to be sure that the > material is at least soluble enough for the next stage (Ni-NTA column),= > so any comments much appreciated. > > Neil Saunders > > -- > Department of Molecular Cell Physiology, > Faculty of Biology, > Vrije Universiteit, > De Boelelaan 1087, > 1081 HV, Amsterdam > The Netherlands > > phone: +31 20 4447194 > email: saunders@bio.vu.nl > WWW: http://members.xoom.com/paracoccus/ =A0 From owner-proteins@net.bio.net Thu Mar 04 22:00:00 1999 Path: biosci!newshost.lanl.gov!awabi.library.ucla.edu!128.230.129.106!news.maxwell.syr.edu!rill.news.pipex.net!pipex!bore.news.pipex.net!pipex!not-for-mail From: Simon Brocklehurst Newsgroups: bionet.molbio.proteins Subject: Re: Protein Bonding Date: Fri, 05 Mar 1999 10:08:23 +0000 Organization: Cambridge Antibody Technology Group plc Lines: 38 Message-ID: <36DFAD17.C07D067D@camb-antibody.co.uk> References: <36D3E980.AFBDD415@tesco.net> NNTP-Posting-Host: pc106.camb-antibody.co.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.5 [en] (WinNT; I) X-Accept-Language: en To: Ali Ali wrote: > Dear All, > > My question is this: Can anyone help me in how to determine if atoms are > bonded in PDB files?? Someone recommended that I bond all atoms < 1.7A > together, but this is yielding too many bonds in comparison to the > number that RasMol generates, but RasMol uses a Voxel engine in > determining bonds from what I see. Ali, You had good advice in the first place (although, I would use 1.8 Anstroms myself) - your algorithm should work fine for proteins, but you need to bear a couple of things in mind: o The structures you're looking at need to have good stereochemistry - that is, the techniques used to build the co-ordinates in the first place should have been correctly applied. o If you are looking at an all-atom structure - that is, hydrogen atoms have been included explicitly then you need it won't work quite properly (hydrogen atoms can be closer than 1.7 Angstroms and not be covalently bonded). Thus you will need to make a special case of hydrogen atoms o Your cut-off won't recognise disulphide bonds, i.e. in PDB-speak, SG-SG bonds between CYS residues won't be identified. Use a 2.3 Angstrom cut-off in this case. Good luck with your structure viewer! -- Simon M. Brocklehurst, Ph.D. Head of Bioinformatics & Advanced IS Cambridge Antibody Technology The Science Park, Melbourn, Cambridgeshire, UK http://www.catplc.co.uk/ mailto:simon.brocklehurst@camb-antibody.co.uk From owner-proteins@net.bio.net Thu Mar 04 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!news.maxwell.syr.edu!isdnet!pasteur.fr!jussieu.fr!univ-lyon1.fr!univ-aix.fr!not-for-mail From: "cuyer" Newsgroups: bionet.molbio.proteins Subject: aaaaaa Date: 5 Mar 1999 09:42:33 GMT Organization: Universite de la Mediterranee Aix en Provence Lines: 1 Message-ID: <01be66ec$1a067b60$1c57d6c2@EJCM-POSTE08.marron.univ-mrs.fr> NNTP-Posting-Host: 194.214.87.28 Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Trace: news.univ-aix.fr 920626953 21924 194.214.87.28 (5 Mar 1999 09:42:33 GMT) X-Complaints-To: usenet@news.univ-aix.fr NNTP-Posting-Date: 5 Mar 1999 09:42:33 GMT X-Newsreader: Microsoft Internet News 4.70.1155 ceci est un essai From owner-proteins@net.bio.net Thu Mar 04 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.stanford.edu!newsfeed.concentric.net!newshub.northeast.verio.net!newspeer.monmouth.com!skynet.be!News.Amsterdam.UnisourceCS!news.unisource.nl!gate.news.unisource.nl!sun4nl!star.cs.vu.nl!bio.vu.nl!not-for-mail From: Neil Saunders Newsgroups: bionet.molbio.proteins Subject: "Sonicated into solution": really solubilised? Date: Fri, 05 Mar 1999 09:11:34 +0100 Organization: VU Biology, Amsterdam, The Netherlands Lines: 32 Message-ID: <36DF91B6.B5DE0FBD@bio.vu.nl> NNTP-Posting-Host: dyn-b150.bio.vu.nl Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: feresa.bio.vu.nl 920621425 2449 130.37.84.150 (5 Mar 1999 08:10:25 GMT) X-Complaints-To: usenet@bio.vu.nl NNTP-Posting-Date: 5 Mar 1999 08:10:25 GMT X-Mailer: Mozilla 4.04 [en] (WinNT; I) Dear all, Recently I've over-expressed a small protein using pET22b/BL21DE3 and have found it to be relatively insoluble (it fractionates with membranes after ultracentrifugation). On resuspension in Tris buffer plus 0.5% Triton-X100 I see small brown globules which are also difficult to redissolve. When I briefly sonicate the suspension, these globules disappear, the solution becomes brown and the protein runs nicely on SDS-PAGE. The question is, does sonication really bring material into solution? I can conceive of a situation where particulates would be broken up into small enough pieces to be suspended and not precipitatable by normal centrifugation, and they would also be accessible to and dissolve OK in SDS-PAGE sample buffer, but might not be truly solubilised in a strict sense. I need to be sure that the material is at least soluble enough for the next stage (Ni-NTA column), so any comments much appreciated. Neil Saunders -- Department of Molecular Cell Physiology, Faculty of Biology, Vrije Universiteit, De Boelelaan 1087, 1081 HV, Amsterdam The Netherlands phone: +31 20 4447194 email: saunders@bio.vu.nl WWW: http://members.xoom.com/paracoccus/ From owner-proteins@net.bio.net Thu Mar 04 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!news-feed.inet.tele.dk!bofh.vszbr.cz!newsgate.cistron.nl!het.net!news.belnet.be!surfnet.nl!surfnet.nl!barba.uci.kun.nl!sci.kun.nl!not-for-mail From: "Gerrit Bouw" Newsgroups: bionet.molbio.proteins,bionet.molbio.methds-reagnts Subject: Re: How to handle PAS? Date: Fri, 5 Mar 1999 00:00:52 +0100 Organization: University of Nijmegen, The Netherlands Lines: 17 Message-ID: <7bo3jb$2r2$1@wnnews.sci.kun.nl> References: <36DE8B6D.C164058B@alpha.bio.nat.tu-bs.de> NNTP-Posting-Host: gbouw.sci.kun.nl X-Priority: 3 X-MSMail-Priority: Normal X-Newsreader: Microsoft Outlook Express 5.00.0810.800 X-MimeOLE: Produced By Microsoft MimeOLE V5.00.0810.800 Xref: biosci bionet.molbio.proteins:14053 bionet.molbio.methds-reagnts:74503 Another idea is to vortex or shake well after each sample or after a few samples.... that's how I do it, works OK for me! Gerrit Wiebke Schmidt wrote in message news:36DE8B6D.C164058B@alpha.bio.nat.tu-bs.de... >I want to compare the activities of several mutants of an enzyme. In the >assay immunoprecitates of the enzyme are used. I have problems in using >equal amounts of immunoprecipitate in each assay because of the >consistence of the protein A-Sepharose (PAS). Does anyone have a >suggestion how to pipette equal amounts of PAS? >Thanks in advance, >Wiebke > From owner-proteins@net.bio.net Thu Mar 04 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!fu-berlin.de!eravci.dialup.fu-berlin.DE!not-for-mail From: "Murat Eravci" Newsgroups: bionet.molbio.proteins Subject: Cheapest Colloidal Gold Total Protein Staining Solution ? Date: Fri, 5 Mar 1999 09:22:04 +0100 Organization: Freie Universitaet Berlin Lines: 7 Message-ID: <7bo3ps$2hf$1@fu-berlin.de> NNTP-Posting-Host: eravci.dialup.fu-berlin.de (130.133.237.57) Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Access: 16 17 18 X-Trace: fu-berlin.de 920621692 2607 (none) 130.133.237.57 X-Newsreader: Microsoft Outlook Express 4.72.3110.5 X-Mimeole: Produced By Microsoft MimeOLE V4.72.3110.3 Could somebody tell me which supplier sells the cheapest Colloidal Gold Total Protein Staining Solution ? ~Murat Eravci From owner-proteins@net.bio.net Thu Mar 04 22:00:00 1999 Path: biosci!news.stanford.edu!logbridge.uoregon.edu!fu-berlin.de!eravci.dialup.fu-berlin.DE!not-for-mail From: "Murat Eravci" Newsgroups: bionet.molbio.proteins Subject: Cheaper PVDF-Membranes ? Date: Fri, 5 Mar 1999 09:20:58 +0100 Organization: Freie Universitaet Berlin Lines: 8 Message-ID: <7bo3or$2ga$1@fu-berlin.de> NNTP-Posting-Host: eravci.dialup.fu-berlin.de (130.133.237.57) Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Access: 16 17 18 X-Trace: fu-berlin.de 920621659 2570 (none) 130.133.237.57 X-Newsreader: Microsoft Outlook Express 4.72.3110.5 X-Mimeole: Produced By Microsoft MimeOLE V4.72.3110.3 Could somebody tell me where I can order the cheapest PVDF Membranes. Most Suppliers PVDF Membranes are too expensive! And there is a large variation between their prices. ~Murat Eravci From owner-proteins@net.bio.net Thu Mar 04 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!nntp.abs.net!newshub2.home.com!newshub1.home.com!news.home.com!news.rdc1.bc.wave.home.com.POSTED!not-for-mail From: "Achim Recktenwald" Newsgroups: bionet.molbio.proteins References: <36E02370.324E5276@hotmail.com> Subject: Re: N-acetyl transferase purification, What to do next? Lines: 20 X-Newsreader: Microsoft Outlook Express 4.72.3155.0 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3155.0 Message-ID: Date: Sat, 06 Mar 1999 00:26:18 GMT NNTP-Posting-Host: 24.64.220.105 X-Complaints-To: abuse@home.net X-Trace: news.rdc1.bc.wave.home.com 920679978 24.64.220.105 (Fri, 05 Mar 1999 16:26:18 PDT) NNTP-Posting-Date: Fri, 05 Mar 1999 16:26:18 PDT Organization: @Home Network Canada Biology wrote in message <36E02370.324E5276@hotmail.com>... >Hi evryone >It is a long time I am trying to purify N-acetyl transferase from >Streptomyces venezuelae. It works on a specific substrate p-nitro phenyl >serinol. I fractionated the crude cell extract by 60% amonium sulfate >(after removing the pellet fro 30% am. sulfate) which is highly active. >Then on DEAE collumn it was again fractionated by NaCl gradient. I found >the active fraction with the highest activity. I analyzed that fraction >with HPLC. It showed still it has two more proteins with very close MW. >I already tried HIC columns but it did not worked. I need the peptide to >be sequenced Could you mention, why it did not work with HIC? Which resins did you try? Achim From owner-proteins@net.bio.net Fri Mar 05 22:00:00 1999 Path: biosci!MERCHACCTSVS.COM!info From: info@MERCHACCTSVS.COM Newsgroups: bionet.molbio.proteins Subject: ADV:CREDIT CARD PROCESSING Date: 6 Mar 1999 18:16:44 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 67 Sender: daemon@net.bio.net Distribution: world Message-ID: <199903070216.SAA18928@net.bio.net> NNTP-Posting-Host: net.bio.net ********************************************************* This message is being brought to you by World Teknologies To be removed from from further mailings respond to this message with "remove" in the subject line. ********************************************************* Dear Friend, Discover how you can accept credit cards directly from your website, telephone or fax for your products and services and never need to purchase or lease expensive credit card equipment or pay a large monthly fee for online ordering capabilities or real time processing transactions. **Brand New** Phone-Charge Credit Card acceptance program allows you to accept Visa, MastercardTM, Amex and Discover any TIME,any WHERE through phone, fax or internet without the need to purchase or lease expensive credit card equipment. 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We accept Visa, Mastercard, Discover and American Express. We do not charge a surcharge on credit card orders. We also ship UPS COD -- COD charges of $5 per shipment would apply. For more information, or to place an order, e-mail -- or contact -- Genesis Technologies Inc Toll Free : 800-433-6326 Fax : 814-231-0371 E-Mail : sales@genesis-technologies.com Web Site : http://www.genesis-technologies.com ============================================================= From owner-proteins@net.bio.net Fri Mar 05 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!news.maxwell.syr.edu!cs.utexas.edu!geraldo.cc.utexas.edu!not-for-mail From: lnd@mail.utexas.edu Newsgroups: bionet.molbio.proteins Subject: Re: N-acetyl transferase purification, What to do next? Date: Sat, 06 Mar 1999 12:39:19 -0600 Organization: The University of Texas at Austin, Austin, Texas Lines: 9 Message-ID: <36E17653.3502AD85@mail.utexas.edu> References: <36E02370.324E5276@hotmail.com> Reply-To: lnd@mail.utexas.edu NNTP-Posting-Host: dhcp-71-140.botany.utexas.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii; x-mac-type="54455854"; x-mac-creator="4D4F5353" Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.5 (Macintosh; I; PPC) X-Accept-Language: en If your protein is well soluble in water, I mean if that's a cytosolic one, so why do not you run that 2-prot fraction on gradient SDS-Gel and cut band for further in gel digestion/peptide mapping or sequenscing? You can, once, extract all bands separately and test enzymic activity...so later will know which one should be digested. L.D. From owner-proteins@net.bio.net Sat Mar 06 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!oleane!pasteur.fr!not-for-mail From: Alain Kohl Newsgroups: bionet.molbio.proteins Subject: ribosomal proteins Date: Sun, 07 Mar 1999 18:10:46 +0200 Organization: institut pasteur Lines: 10 Message-ID: <36E2A51A.15FD@pasteur.fr> Reply-To: kohla@pasteur.fr NNTP-Posting-Host: 55w1-1.dyn.pasteur.fr Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Trace: desdemone.pasteur.fr 920826280 28358 157.99.104.74 (7 Mar 1999 17:04:40 GMT) X-Complaints-To: usenet@pasteur.fr NNTP-Posting-Date: 7 Mar 1999 17:04:40 GMT X-Mailer: Mozilla 3.01 [fr] (Macintosh; I; PPC) Hello, does anybody know where i could find or buy antibodies against ribosomal proteins (PO etc)? Thanks for your help! Alain Alain Kohl Unité des Arbovirus Institut Pasteur Paris, France From owner-proteins@net.bio.net Sun Mar 07 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!newsfeed.cwix.com!158.43.192.17!rill.news.pipex.net!pipex!server1.netnews.ja.net!hgmp.mrc.ac.uk!montana.ebi.ac.uk!user From: ouzounis@ebi.ac.uk (Christos Ouzounis) Newsgroups: bionet.molbio.proteins Subject: Discovery Notes @ Bioinformatics Date: Mon, 08 Mar 1999 15:14:53 +0000 Organization: The European Bioinformatics Institute Lines: 24 Message-ID: NNTP-Posting-Host: montana.ebi.ac.uk X-Trace: niobium.hgmp.mrc.ac.uk 920906029 813 193.62.199.18 (8 Mar 1999 15:13:49 GMT) X-Complaints-To: news@hgmp.mrc.ac.uk NNTP-Posting-Date: 8 Mar 1999 15:13:49 GMT THE BIOINFORMATICS JOURNAL The editors of Bioinformatics have now introduced a new category of paper to the journal called "Discovery Notes". This is intended for the reporting of biologically interesting discoveries using computational techniques. Topics may include sequence motif detection, structural similarities, gene structure prediction, comparative genomics, metabolic pathways and other aspects of computational molecular biology. The new category complements the original papers, reviews and applications notes that are presently published. The description of the analysis can be up to 2 pages long (1000 words) including one/two figures. An abstract is not required. Sequences must be freely available in the database and the results of the analyses should not have been published elsewhere. The paper will be peer-reviewed. For more information see: http://www.oup.co.uk/bioinformatics From the BIOINFORMATICS office at the EBI From owner-proteins@net.bio.net Sun Mar 07 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!news.algonet.se!algonet!masternews.telia.net!News.Amsterdam.UnisourceCS!news.unisource.nl!gate.news.unisource.nl!sun4nl!star.cs.vu.nl!bio.vu.nl!not-for-mail From: Neil Saunders Newsgroups: bionet.molbio.proteins Subject: Re: "Sonicated into solution": really solubilised? Date: Mon, 08 Mar 1999 08:45:04 +0100 Organization: VU Biology, Amsterdam, The Netherlands Lines: 23 Message-ID: <36E38000.C099C99A@bio.vu.nl> References: <36DF91B6.B5DE0FBD@bio.vu.nl> <36E009B3.EE7E2C77@biochem.purdue.edu> NNTP-Posting-Host: dyn-b150.bio.vu.nl Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: feresa.bio.vu.nl 920879034 22256 130.37.84.150 (8 Mar 1999 07:43:54 GMT) X-Complaints-To: usenet@bio.vu.nl NNTP-Posting-Date: 8 Mar 1999 07:43:54 GMT X-Mailer: Mozilla 4.04 [en] (WinNT; I) > Thanks to all who replied. I figure the brown material does contain a large part of my protein, and will be looking into ways of increasing the soluble portion before trying denaturants. The filtration/ultracentrifugation advice is also a good plan, I think. Neil Saunders -- Department of Molecular Cell Physiology, Faculty of Biology, Vrije Universiteit, De Boelelaan 1087, 1081 HV, Amsterdam The Netherlands phone: +31 20 4447194 email: saunders@bio.vu.nl WWW: http://members.xoom.com/paracoccus/ From owner-proteins@net.bio.net Sun Mar 07 22:00:00 1999 Path: biosci!biosci!not-for-mail From: Pier Carlo Montecucchi <100143.765@CompuServe.COM> Newsgroups: bionet.molbio.proteins, Subject: Biotech Products Date: 8 Mar 1999 08:47:56 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 11 Sender: daemon@net.bio.net Distribution: world Message-ID: <7c0uvs$jpo@net.bio.net> NNTP-Posting-Host: net.bio.net Do you need outsourcing services in biotech areas? We can assist you. Contact: MonteGen - A Bureau for Biotechnology Email: innovations@montegen.com (1) 305-776-2241 internationally --  From owner-proteins@net.bio.net Sun Mar 07 22:00:00 1999 From: "SciMedWeb" Newsgroups: bionet.molbio.proteins Subject: SciMedWeb news Date: Mon, 8 Mar 1999 20:44:58 +0100 Organization: EUnet Belgium, Leuven, Belgium Lines: 11 Message-ID: <7c19te$a1n$16@news3.Belgium.EU.net> NNTP-Posting-Host: adial051.liege.eunet.be X-Newsreader: Microsoft Outlook Express 4.71.1712.3 X-MimeOLE: Produced By Microsoft MimeOLE V4.71.1712.3 Path: biosci!news.ic.sunysb.edu!news-nysernet-16.sprintlink.net!news-east1.sprintlink.net!-program!news-peer-europe.sprintlink.net!news.sprintlink.net!Sprint!news-feed.inet.tele.dk!bofh.vszbr.cz!news.belnet.be!news.tvd.be!news0.Belgium.EU.net!newsr.Belgium.EU.net!not-for-mail SciMedWeb, molecular aspects of breast cancer: http://www.geocities.com/HotSprings/Spa/3430/intro1.htm Breast cancer metastasis - Papers Nov-Dec 1998: http://www.geocities.com/HotSprings/Spa/3430/1998_6.htm Estrogen receptors and breast cancer - Papers Nov-Dec 1998: http://www.geocities.com/HotSprings/Spa/3430/er1998_6.htm Breast cancer and gene therapy - Papers 1998: http://www.geocities.com/HotSprings/Spa/3430/bcther98.htm From owner-proteins@net.bio.net Mon Mar 08 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!news.maxwell.syr.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!Home From: klenchin@REMOVE_TO_REPLY.facstaff.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins Subject: Re: N-acetyl transferase purification, What to do next? Date: Tue, 09 Mar 1999 15:39:16 GMT Organization: UW Lines: 31 Message-ID: <7c3fbe$c5a$1@news.doit.wisc.edu> References: <36E02370.324E5276@hotmail.com> NNTP-Posting-Host: ras-c5800-1-216-246.dialup.wisc.edu X-Newsreader: News Xpress 2.01 X-No-Archive: Yes In article <36E02370.324E5276@hotmail.com>, Biology wrote: >Hi evryone >It is a long time I am trying to purify N-acetyl transferase from >Streptomyces venezuelae. It works on a specific substrate p-nitro phenyl >serinol. I fractionated the crude cell extract by 60% amonium sulfate >(after removing the pellet fro 30% am. sulfate) which is highly active. >Then on DEAE collumn it was again fractionated by NaCl gradient. I found >the active fraction with the highest activity. I analyzed that fraction >with HPLC. It showed still it has two more proteins with very close MW. >I already tried HIC columns but it did not worked. I need the peptide to >be sequenced >Any commnet on how to proceed for further purification is welcomed. > It is a standard practice to do higher resolution ion exchange after initial crude purification. If you have an access to FPLC or HPLC, do chromatography on Mono Q - it's a different chemistry and has very high resolution. It is likely to help. Alternatively, try hydroxyl apatite chromatography. (I'd recommend ceramic HA from Bio-Rad). There is no reason high resolution HIC should not work too. Just play with conditions... Finally, high resolution chromatofocusing (FPLC/MonoP) virtually gurantees you a nice separation if you are lucky and your ptotein does not precipitate during it. Hope this helps, Dima From owner-proteins@net.bio.net Mon Mar 08 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!news.maxwell.syr.edu!news-was.dfn.de!news-kar1.dfn.de!hades.rz.uni-sb.de!hades.rz.uni-sb.de From: "Gernot T. John" Newsgroups: bionet.molbio.proteins Subject: storing penicillin-amidase Date: Tue, 09 Mar 1999 11:59:36 +0100 Organization: University of Saarland, Computing Center, Germany. Lines: 11 Message-ID: <36E4FF17.6B7989BB@rz.uni-sb.de> NNTP-Posting-Host: pb16eh04.microbiol.uni-sb.de Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Mailer: Mozilla 4.06 [en] (Win95; I) Hi! today we discovered an old solution of penicillin-amidase from E.coli (EC 3.5.1.11) in our fridge. It was stored there for about 4 months at -4°C. (I don't know who put it there!!!) At this temperature it is liquid. The supplier specifies that the enzyme is to stored below -18°C, where the solution is freezed. Does anybody know whether this solution is still OK? gernot From owner-proteins@net.bio.net Mon Mar 08 22:00:00 1999 Path: biosci!newshost.lanl.gov!awabi.library.ucla.edu!208.134.241.18!newsfeed.cwix.com!198.138.0.5!newshub.northeast.verio.net!newsserver.jvnc.net!192.54.35.50!gmd.de!news.ruhr-uni-bochum.de!not-for-mail From: Arnd Richardt Newsgroups: bionet.molbio.proteins Subject: cloning-problem Date: Tue, 09 Mar 1999 12:45:07 +0100 Organization: Ruhr-Universitaet Bochum, Rechenzentrum Lines: 19 Message-ID: <36E509C3.B503B533@ruhr-uni-bochum.de> NNTP-Posting-Host: mikroskop.orch.ruhr-uni-bochum.de Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Trace: sun579.rz.ruhr-uni-bochum.de 920980062 29680 134.147.66.40 (9 Mar 1999 11:47:42 GMT) NNTP-Posting-Date: 9 Mar 1999 11:47:42 GMT X-Mailer: Mozilla 4.06 [de] (Win95; I) I´ve tried to ligate a 6kb-fragment into a Gal4-Transformation-Vektor (p221-4; 12kb long), but failed several times. Does anybody have a protocol or reference for such a cloning problem ?? I hope, it´s not impossible Thanks -- Arnd Richardt Arndt.Richardt@ruhr-uni-bochum.de Ruhr-Universität Bochum Fakultät Chemie Molekulare Zellbiochemie Gebäude NC 5 / 171 44780 Bochum From owner-proteins@net.bio.net Mon Mar 08 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!newsfeed.gamma.ru!Gamma.RU!demos!news.stack.serpukhov.su!not-for-mail From: "VVS" Newsgroups: bionet.molbio.proteins Subject: Re: cloning-problem Date: Tue, 9 Mar 1999 18:35:01 +0300 Organization: Stack Inc. Lines: 11 Message-ID: <7c3f4f$8pa$1@news.stack.serpukhov.su> References: <36E509C3.B503B533@ruhr-uni-bochum.de> NNTP-Posting-Host: filimonov.ipr.serpukhov.su X-Newsreader: Microsoft Outlook Express 4.72.3110.1 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3110.3 By my meance you use too little vector for such large fragment. May be you could use cosmid or lambda phage... See Sambrook, Maniatis and Freech (it may be mistakely writing) manual. >I´ve tried to ligate a 6kb-fragment into a Gal4-Transformation-Vektor >(p221-4; 12kb long), but failed several times. Does anybody have a >protocol or reference for such a cloning problem ?? I hope, it´s not >impossible From owner-proteins@net.bio.net Mon Mar 08 22:00:00 1999 Path: biosci!PILOT.MSU.EDU!yanh From: yanh@PILOT.MSU.EDU (Honggao Yan) Newsgroups: bionet.molbio.proteins Subject: Protein NMR Postdoctoral Associate Date: 9 Mar 1999 08:40:59 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 39 Sender: daemon@net.bio.net Distribution: world Message-ID: <36E54C96.1A8FDAAD@pilot.msu.edu> NNTP-Posting-Host: net.bio.net A postdoctoral position is available to study the structures and dynamics of the enzymes in the shikimate and folate biosynthetic pathways. Since the shikimate and folate biosynthetic pathways are unique to microbial and plant worlds, the two pathways are ideal targets for development of antimicrobial agents and herbicides. The long-term goals of the projects are to elucidate the structure-function relationships of these proteins that account for their catalytic mechanisms and to design specific inhibitors that may become new antimicrobial agents. A variety of techniques is employed in these projects, including recombinant DNA, biochemical, and biophysical methods. The main structural technique is multidimensional NMR spectroscopy. These projects are of great biomedical significance, especially in light of rapidly increasing antibiotic resistance in recent years that has rendered the current antibiotics ineffective for treating many microbial infections and caused a worldwide health care crisis. Our laboratory is well equipped for protein NMR spectroscopy. Instrumentation includes three computer workstations (one from SGI and two from Sun) and 500 MHz and 600 MHz Varian NMR spectrometers. The 600 MHz Varian Inova NMR spectrometer is equipped with four rf channels and tri-axis pulsed field gradients and is dedicated to biomolecular NMR studies. The initial appointment for the position will be two years and is renewable upon mutual agreement. The applicants should have experience with multidimensional NMR spectroscopy. If interested, please send a CV along with telephone numbers and email addresses of three references to the address below. Email responses are encouraged. Dr. Honggao Yan Department of Biochemistry Michigan State University East Lansing, MI 48824 Tel: (517) 353-8786 Fax: (517) 353-9334 Email: yanh@pilot.msu.edu From owner-proteins@net.bio.net Mon Mar 08 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!netnews.com!newsfeed.wirehub.nl!surfnet.nl!barba.uci.kun.nl!sci.kun.nl!not-for-mail From: "Gerrit Bouw" Newsgroups: bionet.molbio.proteins Subject: pCB6 Date: Tue, 9 Mar 1999 19:47:35 +0100 Organization: University of Nijmegen, The Netherlands Lines: 26 Message-ID: <7c3qb7$aj8$1@wnnews.sci.kun.nl> NNTP-Posting-Host: gbouw.sci.kun.nl X-Priority: 3 X-MSMail-Priority: Normal X-Newsreader: Microsoft Outlook Express 5.00.0810.800 X-MimeOLE: Produced By Microsoft MimeOLE V5.00.0810.800 Dear everyone, I just got the cloning vector pCB6, but I do not have the sequence of this vector at all and could not find it yet on the web... I hope somebody can provide me with the sequence, thanks in advance! Gerrit -- Gerrit Bouw Dept. Animal Physiology Nijmegen University Room 304A Toernooiveld 1 6525 ED Nijmegen, The Netherlands Tel: +31 (0)24 3652729 Fax: +31 (0)24 3652714 E-mail: gbouw@sci.kun.nl Private: van den Havestraat 83 6521 JP Nijmegen The Netherlands Tel: +31 (0)24 3601055 From owner-proteins@net.bio.net Mon Mar 08 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!newsfeed.cwix.com!193.174.75.110!news-was.dfn.de!news-kar1.dfn.de!hades.rz.uni-sb.de!hades.rz.uni-sb.de From: "Gernot T. John" Newsgroups: bionet.molbio.proteins Subject: question to penicillin amidase stability Date: Tue, 09 Mar 1999 15:01:58 +0100 Organization: University of Saarland, Computing Center, Germany. Lines: 16 Message-ID: <36E529D5.B3BE9BC@rz.uni-sb.de> NNTP-Posting-Host: pb16eh04.microbiol.uni-sb.de Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Mailer: Mozilla 4.06 [en] (Win95; I) My first message was bounced. I don't know why. and again... Hi! today we discovered an old solution of penicillin-amidase from E.coli (EC 3.5.1.11) in our fridge. It was stored there for about 4 months at -4°C. (I don't know who put it there!!!) At this temperature it is liquid. The supplier specifies that the enzyme is to stored below -18°C, where the solution is freezed. Does anybody know whether this solution is still OK? gernot g.john@rz.uni-sb.de From owner-proteins@net.bio.net Mon Mar 08 22:00:00 1999 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newspump.monmouth.com!newspeer.monmouth.com!dca1-hub1.news.digex.net!digex!dca1-nnrp1.news.digex.net.POSTED!not-for-mail From: enwgyr@capitalinks.com Newsgroups: bionet.molbio.proteins Subject: FREE Horoscope - NOT ANY SPAM! 7219 Lines: 3 Message-ID: Date: Wed, 10 Mar 1999 05:07:36 GMT NNTP-Posting-Host: 207.111.39.7 X-Complaints-To: abuse@digex.net X-Trace: dca1-nnrp1.news.digex.net 921042456 207.111.39.7 (Wed, 10 Mar 1999 00:07:36 EDT) NNTP-Posting-Date: Wed, 10 Mar 1999 00:07:36 EDT Organization: DIGEX, Inc. - Beltsville, MD - http://www.digex.net Daily horoscopes free of chearge - just thought you might like to know.. http://www.capitalinks.com and press the DAILY HOROSCOPE BUTTON - not much else to say - this is really the only freebie horoscope on the net that I know of tpvjxcngcqyofjvecysrddklrcytf From owner-proteins@net.bio.net Mon Mar 08 22:00:00 1999 Path: biosci!newshost.lanl.gov!awabi.library.ucla.edu!128.230.129.106!news.maxwell.syr.edu!cyclone.bc.net!rover.ucs.ualberta.ca!gallin2.biology.ualberta.ca!wgallin From: wgallin@gpu.srv.ualberta.ca (Warren Gallin) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: Protein immobilization on Sepharose using periodate activation (Was: Purifying Antibodies using affinity support) Date: Wed, 10 Mar 99 03:55:44 GMT Organization: University of Alberta Lines: 17 Message-ID: References: <7c3oi7$rhj$1@mserv2.dl.ac.uk> <7c45t5$rt2$1@news.doit.wisc.edu> NNTP-Posting-Host: gallin2.biology.ualberta.ca X-Newsreader: VersaTerm Link v1.1.4 Xref: biosci bionet.molbio.methds-reagnts:74614 bionet.molbio.proteins:14084 In Article <7c45t5$rt2$1@news.doit.wisc.edu>, klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) wrote: >1. Wash Sepharose CL-4B (not cross-linked version DOES NOT >work!) with 10-20 volumes of pure water on glass filter. I checked with Dima, and he clarified something for me. He means to say that you must use the CL-4B, which is cross-linked, because plain 4B, which is not cross linked, will not work with this activation method. Thanks Dima. Warren Gallin Department of Biological Sciences University of Alberta Edmonton, Alberta T6G 2E9 Canada wgallin@gpu.srv.ualberta.ca From owner-proteins@net.bio.net Mon Mar 08 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!news.maxwell.syr.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!default From: klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Protein immobilization on Sepharose using periodate activation (Was: Purifying Antibodies using affinity support) Date: Tue, 09 Mar 1999 22:05:15 GMT Organization: UW-Madison Lines: 78 Message-ID: <7c45t5$rt2$1@news.doit.wisc.edu> References: <7c3oi7$rhj$1@mserv2.dl.ac.uk> NNTP-Posting-Host: martinlab.biochem.wisc.edu Mime-Version: 1.0 Content-Type: text/plain; charset=KOI8-R Content-Transfer-Encoding: 8bit X-Newsreader: News Xpress 2.01 Xref: biosci bionet.molbio.methds-reagnts:74611 bionet.molbio.proteins:14083 In article <7c3oi7$rhj$1@mserv2.dl.ac.uk>, wolfgang.schechinger@med.uni-tuebingen.de wrote: :Dima, : :Suppose the periodate protocol is oxidizing cis diols into :bis aldehydes and reduce the imines formed after coupling with :cyanoborohydride to amines. And clening leftover CHOs with boranate. :But this procedure above probably might kill any active enzyme. Is :there a way to prevent this? :You remember the exact protocol by chance? Here you go. I found the old file and tried to make it look like a protocol. Dima ******************************************************************* PROTEIN IMMOBILIZATION ON CROSS-LINKED SEPHAROSE USING PERIODATE ACTIVATION 1. Wash Sepharose CL-4B (not cross-linked version DOES NOT work!) with 10-20 volumes of pure water on glass filter. 2. Mix one volume of packed Sepharose with two volumes of 20 mg/ml sodium periodate (NaJO4) in water, incubate for two hours at room temperature on rotator. 3. Wash with 5 volumes of water, then at least 3 volumes of 100 mM sodium borate, pH 9.0-9.4 (pH is dictated by your protein stability; at 9.4 the coupling proceeds more efficiently, but still quite OK at 9.0) on glass filter. (This gives you activated Sepharose; at this point it is quite stable and can be stored at +4 for at least one month). 4. To one volume of packed Sepharose, add 1-3 volumes of protein solution at 2-5 mg/ml in the buffer with pH 9.0-9.4 that is free of amines. (For example, one volume of 5 mg/ml IgG in 200 mM sodium borate or bicarbonate, pH 9.4). Incubate on rotator at rt for three hours or in cold room overnight. 5. Sediment, collect supernatant, save, wash 2X with two volumes of 100 mM sodium borate pH 8.0 by resuspension/ sedimentation, collect washes. (Determine coupling efficiency after measuring protein concentration in original super and washes; usually around 80% bound). 6. To the Sepharose, add equal volume of FRESHLY prepared 3 mg/ml sodium borohydride (NaBH4) in 100 mM sodium borate, pH 8.0, incubate on rotator for 5 min, wash repeatedly with sodiun borate pH 8.0 until pH in the super is ~ 8.0 (use pH paper). 7. For the paranoid: remove non-specifically bound protein and block residual reactive groups by washing 3X with 2 volumes of 100 mM ethanolamine, 0.5 M NaCl, pH 8.0 8. Wash with buffer of your choice, add azide to 0.01% and store at +4C. Alternatively, add equal volume of ethylene glycol or glycerol, allow to mix throughly on rotator and store at -20C. ***** Sorry, no reference; I know for a fact that the person who developed this method somewhere around 1985 is Dr. V.V.Sinitsyn, then in the Institute of Experimental Cardiology, USSR Academy of Medical Sciences, Moscow; I could not find any published reference to this method and the above is what I used personally and can attest to be working. PLEASE, if using the method, find some way to acknowledge the author and the source. I am afraid he is no longer working in science (thanks to the revolutionary reforms in Russia that left no way for most scientists to make a living). I am leaving "X-No-Archive: yes" out to get this post archived in Dejanews, so I imagine referencing the post by providing URL would be OK. ***** -- Dima Klenchin From owner-proteins@net.bio.net Mon Mar 08 22:00:00 1999 Path: biosci!newshost.lanl.gov!awabi.library.ucla.edu!128.230.129.106!news.maxwell.syr.edu!isdnet!news-raspail.gip.net!news.gsl.net!gip.net!newsfeed.ecrc.net!news.net.uni-c.dk!not-for-mail From: "S.W. Rasmussen" Newsgroups: bionet.molbio.proteins Subject: Dnatools, revision 340 Date: Tue, 09 Mar 1999 22:02:16 +0000 Organization: UNI-C Lines: 14 Message-ID: <36E59A68.8A936655@crc.dk> NNTP-Posting-Host: 130.226.184.128 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: news.net.uni-c.dk 921013249 28242 130.226.184.128 (9 Mar 1999 21:00:49 GMT) X-Complaints-To: usenet@news.net.uni-c.dk NNTP-Posting-Date: 9 Mar 1999 21:00:49 GMT X-Mailer: Mozilla 4.5 [en] (WinNT; I) X-Accept-Language: en New revision of DNATools is available at http://www.crc.dk/phys/A01B04_dnatools.htm Regards Soeren -- Soeren W. Rasmussen, Dr. scient. Department of Physiology Carlsberg Laboratory Copenhagen, Denmark swr@crc.dk, http://www.crc.dk/phys From owner-proteins@net.bio.net Mon Mar 08 22:00:00 1999 Path: biosci!newshost.lanl.gov!awabi.library.ucla.edu!208.134.241.18!newsfeed.cwix.com!193.174.75.110!news-was.dfn.de!news-kar1.dfn.de!hades.rz.uni-sb.de!hades.rz.uni-sb.de From: "Michael (2)" Newsgroups: bionet.molbio.proteins Subject: storage of penicillinamidase Date: Tue, 09 Mar 1999 19:46:28 +0100 Organization: Technische Biochemie Lines: 55 Message-ID: <36E56C83.58E6B7DC@mx.uni-saarland.de> NNTP-Posting-Host: pb16eh18.microbiol.uni-sb.de Mime-Version: 1.0 Content-Type: multipart/mixed; boundary="------------317F5D45D0890C005B639588" X-Mailer: Mozilla 4.5 [en] (Win95; I) X-Accept-Language: en This is a multi-part message in MIME format. --------------317F5D45D0890C005B639588 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit Hi! today we discovered an old solution of penicillin-amidase from E.coli (EC 3.5.1.11) in our fridge. It was stored there for about 4 months at -4°C. (I don't know who put it there!!!) At this temperature it is liquid. The supplier specifies that the enzyme is to stored below -18°C, where the solution is freezed. Does anybody know whether this solution is still OK? -- ========================================= Dipl.-Biol. Michael HANS Department of Biochemical Engineering Saarland University P.O. BOX 151150 D-66041 Saarbruecken Germany phone : ++49 681 302 3545 fax : ++49 681 302 4572 email : m.hans@mx.uni-saarland.de --------------317F5D45D0890C005B639588 Content-Type: text/x-vcard; charset=us-ascii; name="m.hans.vcf" Content-Transfer-Encoding: 7bit Content-Description: Card for Michael (2) Content-Disposition: attachment; filename="m.hans.vcf" begin:vcard n:Hans;Michael tel;fax:++49 681 302 4572 tel;work:++49 681 302 3545 x-mozilla-html:FALSE org:Saarland University;Department of Biochemical Enginering adr:;;P.O. BOX 151150;D-66041 Saarbruecken;Germany;; version:2.1 email;internet:m.hans@mx.uni-saarland.de title:Dipl.-Biol. fn:Michael Hans end:vcard --------------317F5D45D0890C005B639588-- From owner-proteins@net.bio.net Tue Mar 09 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!news-feed.inet.tele.dk!bofh.vszbr.cz!news.belnet.be!surfnet.nl!surfnet.nl!eur.nl!news.fgg.eur.nl!usenet From: Edward van Harskamp Newsgroups: bionet.molbio.proteins Subject: gene formula? Date: Wed, 10 Mar 1999 12:39:32 +0100 Organization: Erasmus Universiteit Rotterdam Lines: 6 Message-ID: <36E659F2.8FBE511A@hotmail.com> NNTP-Posting-Host: gateway.azr.nl Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.07 [en] (Win98; I) What is the general unique molecular structure of a gene? If you have an answer to this question, please, mail this answer to EF_v_Harskamp@Hotmail.com From owner-proteins@net.bio.net Tue Mar 09 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!news-peer1.sprintlink.net!news.sprintlink.net!cs.utexas.edu!geraldo.cc.utexas.edu!not-for-mail From: lnd@mail.utexas.edu Newsgroups: bionet.molbio.proteins Subject: To solubilize transferred proteins..HOW?? Date: Wed, 10 Mar 1999 11:11:32 -0600 Organization: The University of Texas at Austin, Austin, Texas Lines: 21 Message-ID: <36E6A7BC.65B5F9E2@mail.utexas.edu> Reply-To: lnd@mail.utexas.edu NNTP-Posting-Host: dhcp-71-140.botany.utexas.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii; x-mac-type="54455854"; x-mac-creator="4D4F5353" Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.5 (Macintosh; I; PPC) X-Accept-Language: en Hi Netters I have a question here...... Can anyone tell me...if it's possible .... PRACTICALLY....... to get proteins into solution after being transferred on membrane (NC, PVDF....whatever..)? Say....to increase protein purity and concentration. I never did that...but ...who knows..maybe you or your friends did? Say...to chop membrane with certain protein band, add SDS/tween/urea/TX....detergents in buffer....boil....incubate etc.etc.? Any suggestions would be highly appreciated. Regards Levan Darjania Austin, TX From owner-proteins@net.bio.net Tue Mar 09 22:00:00 1999 Path: biosci!CC.USU.EDU!arsphys From: arsphys@CC.USU.EDU (Phil Harrison) Newsgroups: bionet.molbio.proteins Subject: Re: more questions... Date: 10 Mar 1999 09:15:31 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 29 Sender: daemon@net.bio.net Distribution: world Message-ID: <3.0.1.32.19990310100432.006dc7a0@cc.usu.edu> References: <7c658d$fve$1@news.doit.wisc.edu> NNTP-Posting-Host: net.bio.net At 04:06 PM 3/10/99 +0000, you wrote: >:Hello, >: >:I wonder if you could help me with these questions. > [snip several questions on protein synthesis] >You might want to grab some popular book(s) on biochemistry/molecular >biology. I am not aware of any partucularly good example, but I guess >James Watson has written something on this as well. :-) > > - Dima > By now these topics are covered quite thoroughly in basic Biology textbooks, even to a discussion of the enzymes invovled. One example is Biology: Concepts and Applications by Cecie Starr. good luck Phil From owner-proteins@net.bio.net Tue Mar 09 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!news.maxwell.syr.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!default From: klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins Subject: Re: more questions... Date: Wed, 10 Mar 1999 16:06:35 GMT Organization: UW-Madison Lines: 47 Message-ID: <7c658d$fve$1@news.doit.wisc.edu> References: <36E65D2A.C9AF5EB7@hotmail.com> NNTP-Posting-Host: martinlab.biochem.wisc.edu Mime-Version: 1.0 Content-Type: text/plain; charset=KOI8-R Content-Transfer-Encoding: 8bit X-Newsreader: News Xpress 2.01 X-No-Archive: yes :Hello, : :I wonder if you could help me with these questions. They seem to indicate you are not working in this field, so the possible answers are very general. :What is the reason for a specific mRNA molecule(coming from a specific :gene or genes) to come to expression in a specific cel and not in a :different? How does this work on a molecule level? The gene might have a specific region that needs to be recognized (bind; form complex with) by a specific protein in order to allow RNA synthesis. If such protein exists in a specific cell only, then you get specific pattern of expression. :How does the translation of mRNA into a protein precisely work on a :structural molecular level? mRNA enters in complex with proteinous molecular machine called ribosome and catalyzes protein synthesis according to exact composition (nucleotide sequence) of the RNA. :How does the gene proces of transcription and after that translation :work on a structural molecular level? Compex array of proteins interacting with DNA (gene) ultimately allows enzyme called RNA polymerase catalyze RNA syntesis complementary to the gene sequence, RNA gets transported out of the nucleus into cytoplasm where it meets ribosome + other types of RNA bearing aminoacids and together they make a protein. :How does the entire translation of mRNA molecule to the eventually 3D :molecular protein structure work? 3D protein structure is determined by 2D protein sequence due to the physical/repulsion of aminoacid residues composing the protein in specific order. Other proteins frequently aid in this process of protein "folding". The physics of the process is so compex that as of now unambigous prediction of 3D based on known 2D is generally impossible. You might want to grab some popular book(s) on biochemistry/molecular biology. I am not aware of any partucularly good example, but I guess James Watson has written something on this as well. :-) - Dima From owner-proteins@net.bio.net Tue Mar 09 22:00:00 1999 Path: biosci!MAIL.ESPN.GO.COM!espn-mail From: espn-mail@MAIL.ESPN.GO.COM Newsgroups: bionet.molbio.proteins Subject: SPECIAL OFFER FROM ESPN.com Date: 10 Mar 1999 08:13:33 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 41 Sender: daemon@net.bio.net Distribution: world Message-ID: <095f93403160a39MRELAY0@mrelay0.starwave.com> Reply-To: espn-mail-responses@starwave.com NNTP-Posting-Host: net.bio.net Hey Sports Fanatic…..Pop Quiz: 1. What was Shooty Babitt's on base percentage against lefties in domes on Tuesday nights when it was raining and 43 degrees outside in the 6th inning? 2. What is Dennis Rodman's field goal percentage in road games in February while wearing his team's alternate 3rd jersey and blue hair? 3. 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Get the best ESPN has to offer - become an ESPN Insider today! http://trans.espn.go.com/cgi/ad/trans.pl?id=1799 The editors, ESPN Insider PS---Hurry! This special discount is good only if you sign up by March 18, 1999. From owner-proteins@net.bio.net Tue Mar 09 22:00:00 1999 Path: biosci!pravda.ucr.edu!awabi.library.ucla.edu!128.230.129.106!news.maxwell.syr.edu!howland.erols.net!newsfeed.wirehub.nl!surfnet.nl!eur.nl!news.fgg.eur.nl!usenet From: Edward van Harskamp Newsgroups: bionet.molbio.proteins Subject: more questions... Date: Wed, 10 Mar 1999 12:53:16 +0100 Organization: Erasmus Universiteit Rotterdam Lines: 22 Message-ID: <36E65D2A.C9AF5EB7@hotmail.com> NNTP-Posting-Host: gateway.azr.nl Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.07 [en] (Win98; I) Hello, I wonder if you could help me with these questions. What is the reason for a specific mRNA molecule(coming from a specific gene or genes) to come to expression in a specific cel and not in a different? How does this work on a molecule level? How does the translation of mRNA into a protein precisely work on a structural molecular level? How does the gene proces of transcription and after that translation work on a structural molecular level? How does the entire translation of mRNA molecule to the even-tually 3D molecular protein structure work? If you have an answer to one or more of these questions, please, mail me (EF_v_Harskamp@hotmail.com). Thanks! From owner-proteins@net.bio.net Tue Mar 09 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!news-peer1.sprintlink.net!news-in-east1.sprintlink.net!news.sprintlink.net!itssrv1.ucsf.edu!macmac-2.ucsf.edu!user From: bpmurray*STUFFER*@socrates.ucsf.edu (Bernard P. Murray, PhD) Newsgroups: bionet.molbio.proteins Subject: Re: To solubilize transferred proteins..HOW?? Date: Wed, 10 Mar 1999 20:01:40 -0700 Organization: University of California, San Francisco Lines: 30 Message-ID: References: <36E6A7BC.65B5F9E2@mail.utexas.edu> NNTP-Posting-Host: macmac-2.ucsf.edu In article <36E6A7BC.65B5F9E2@mail.utexas.edu>, lnd@mail.utexas.edu wrote: > Hi Netters > I have a question here...... > Can anyone tell me...if it's possible .... PRACTICALLY....... to get > proteins into solution after being transferred on membrane (NC, > PVDF....whatever..)? Say....to increase protein purity and > concentration. > I never did that...but ...who knows..maybe you or your friends did? > Say...to chop membrane with certain protein band, add > SDS/tween/urea/TX....detergents in buffer....boil....incubate etc.etc.? > Any suggestions would be highly appreciated. > Regards > Levan Darjania > Austin, TX Someone can correct me if I'm wrong but I believe that nitrocellulose is soluble in DMSO. Depending on in just what condition you want the protein afterwards this, or another solvent, may be suitable. I once looked into this for electrophoretic purificatiion for immunisation but later found the literature on immunising with actual pieces of membrane (or PAGE gel). The major problem is that you don't get very much protein by this technique. Good luck, Bernard -- Bernard P. Murray, PhD Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA From owner-proteins@net.bio.net Tue Mar 09 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!intgwpad.nntp.telstra.net!news1.optus.net.au!optus!news.usyd.edu.au!unsw.edu.au!NewsWatcher!user From: c.schmitz-peiffer@garvan.unsw.edu.au (Carsten Schmitz-Peiffer) Newsgroups: bionet.molbio.proteins,bionet.cellbiol Subject: Research Assistant (Temporary position) Date: Thu, 11 Mar 1999 12:19:08 +1100 Organization: Garvan Institute of Medical Research Lines: 29 Message-ID: NNTP-Posting-Host: 129.94.136.62 Keywords: Research Assistant position Xref: biosci bionet.molbio.proteins:14093 bionet.cellbiol:11455 Research Assistant (Temporary Position) Cell Signalling Group Garvan Institute of Medical Research Sydney, Australia A temporary position (6-8 months) is available to work with Carsten Schmitz-Peiffer on the molecular mechanisms underlying skeletal muscle insulin resistance. The project is concerned with analysis of insulin signalling pathways in cultured cell lines using a variety of biochemical and molecular biological approaches. Applicants should possess a degree (preferably to Honours level) in biochemistry or molecular biology and ideally have some laboratory experience in applying these techniques. Remuneration will be commensurate with experience and in accordance with the Garvan's own competitive pay scales. For more information, please visit http://www.garvan.unsw.edu.au/hremploy.html or email Carsten Schmitz-Peiffer c.schmitz-peiffer@garvan.unsw.edu.au -- Carsten Schmitz-Peiffer, PhD Cell Signalling Group Garvan Institute of Medical Research 384, Victoria Street, Sydney NSW 2010 Australia From owner-proteins@net.bio.net Wed Mar 10 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.stanford.edu!newsfeed.concentric.net!newspeer1.nac.net!news.maxwell.syr.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!default From: klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: Protein immobilization on Sepharose using periodate activation (Was: Purifying Antibodies using affinity support) Date: Thu, 11 Mar 1999 23:34:57 GMT Organization: UW-Madison Lines: 97 Message-ID: <7c9jsm$v0k$1@news.doit.wisc.edu> References: <7c3oi7$rhj$1@mserv2.dl.ac.uk> <7c45t5$rt2$1@news.doit.wisc.edu> <36e81f1e.0@carrera.intergate.ca> NNTP-Posting-Host: martinlab.biochem.wisc.edu Mime-Version: 1.0 Content-Type: text/plain; charset=KOI8-R Content-Transfer-Encoding: 8bit X-Newsreader: News Xpress 2.01 X-No-Archive: yes Xref: biosci bionet.molbio.methds-reagnts:74684 bionet.molbio.proteins:14099 : :Here I am telling you that Periodate method is one of the most :inefficient coupling procedures, particularly for the protein sample :with low concentration. Did you try "my" protocol? :-) How can you tell? But, yes, the coupling efficiency is highly dependent on protein concentration. For most proteins, around 2-3 mg/ml (final after 1:1 dilution with sepharose suspension) is optimal. Exactly because of it's "unefficiency", it is the most gentle, preserving protein activity MUCH better than most others. :I am suggesting some of the direct, one-step and simple but highly :efficient activated matrices comercialy available. None of them is more one-step than periodate. That is, if you don't consider activation and buying a "non-step". Plain CL Sepharose is dirt cheap (because it it sitting unusable for decades in every cold room without exception), pre-activated matrix is very expensive. :1. Activated ECH Agarose. Available from both Peirce and Sigma. :Providing 6-carbon spacer and an active NHS ester as leaving group and :form stable amide linkage with your amine containing protein. :2. Cynogen Bromide activated Sepharose. One of the most efficient :matrix available and provide one carbon spacer but has very bad :reputation for leaking and ionic exchange effects. :3. A novel activated matrix developed by 3M and sold through Peirce :Chemical. The matrix contains a active lactone, which is readily form a :stable amide with primary amines of the protein. Disavantage: hyarolysed :lactone create a free carboxyl=ionic effect; low flow rate of its :acrylamide polymer matrix. :4. Activated thiol Agarose. Available from Sigma. It forms -S-S- :bond with free thiol (cysteine residue) of the protein effiently with :high yield (the matrix is not sensitive to water as others). :Disavantage: you need to reduce your protein with DTT first before :coupling. :5. Iodoactate activated Agarose. Available from Peirce. High yield :and stable for free thiol containing protein. I listed these myself in this very same thread previously :-) I only did not list thiol-based because, in my opinion, they are unsuitable for any serious application involving crude material. Oxyrane activated Sepharose (Sigma, I thinlk), is also worth consideration :The disavantage of Periodate activated Agarose: :need high pH (>pH9) to :obtain a good high yield and efficient; 9.0 is good enough. Almost never a problem for most proteins (unlike pH < 5 which kills a lot of them). :Almost no good for protein less :than 0.1 mg/mL. True. I wouldn't even try it with 0.5 mg/ml. To various extent, the same is true for all listed above. :The formation of Shif-base is not reversible unless :furthewr reduced to secondary amine (harmful to lots of proteins). ??? Did you read my post? The reduction is indeed used, creating irreversible linkage. No, I don't think borohydrid used as described is so harmful. I know from my own experience IgG and IgM sorbents prepared in this way perform much better than others, and I was told that several immobilized enzymes had higher activity too. :Oxidation damage the matrix and create leaking problems. There is no any kind of "damage" I am aware of under this procedure. Any serious leaking was not observed. In fact, it was _much_ less than for NHS and cyanogene-based sorbents. :Beside, I have to warn the one who put the first question: affinity :purification of antibody using whole protein will create lots of :problems and limits the application of the antibodies because of the :leaking of antigen, WHICH CONTAINS MULTI-EPIOPES. Unlike eptope-based :affinity chromatography, the leaking antigen will interfere the :interpretation of your results. While this can be a concern for industrial applications, it has no importance at all in research. First, the amount leaking is very small in all reasonable cases, second, all intelligent people should do controls anyway (mock or non immune pturification over the same column). For what it is worth... I only speak from my own experience. Nothing is "the best" in all cases, and every particular application plus common sense and knowledge dictates the choice of sorbent. Regards, - Dima From owner-proteins@net.bio.net Wed Mar 10 22:00:00 1999 Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: Protein immobilization on Sepharose using periodate activation (Was: Purifying Antibodies using affinity support) From: immune@intergate.bc.ca (immunechem) Reply-To: immune@intergate.bc.ca Organization: Internet Gateway X-Newsreader: WinVN 0.99.7 References: <7c3oi7$rhj$1@mserv2.dl.ac.uk> <7c45t5$rt2$1@news.doit.wisc.edu> MIME-Version: 1.0 Content-Type: Text/Plain; charset=US-ASCII NNTP-Posting-Host: pm46s45.intergate.bc.ca Message-ID: <36e81f1e.0@carrera.intergate.ca> Date: 11 Mar 99 19:53:02 GMT Lines: 56 Path: biosci!news.stanford.edu!newsfeed.stanford.edu!logbridge.uoregon.edu!sea-feed.news.verio.net!feed.news.verio.net!news-peer-west.sprintlink.net!news.sprintlink.net!news.vphos.net!carrera.intergate.ca!pm46s45.intergate.bc.ca Xref: biosci bionet.molbio.methds-reagnts:74671 bionet.molbio.proteins:14098 Here I am telling you that Periodate method is one of the most inefficient coupling procedures, particularly for the protein sample with low concentration. I am suggesting some of the direct, one-step and simple but highly efficient activated matrices comercialy available. 1. Activated ECH Agarose. Available from both Peirce and Sigma. Providing 6-carbon spacer and an active NHS ester as leaving group and form stable amide linkage with your amine containing protein. 2. Cynogen Bromide activated Sepharose. One of the most efficient matrix available and provide one carbon spacer but has very bad reputation for leaking and ionic exchange effects. 3. A novel activated matrix developed by 3M and sold through Peirce Chemical. The matrix contains a active lactone, which is readily form a stable amide with primary amines of the protein. Disavantage: hyarolysed lactone create a free carboxyl=ionic effect; low flow rate of its acrylamide polymer matrix. 4. Activated thiol Agarose. Available from Sigma. It forms -S-S- bond with free thiol (cysteine residue) of the protein effiently with high yield (the matrix is not sensitive to water as others). Disavantage: you need to reduce your protein with DTT first before coupling. 5. Iodoactate activated Agarose. Available from Peirce. High yield and stable for free thiol containing protein. The disavantage of Periodate activated Agarose: need high pH (>pH9) to obtain a good high yield and efficient; Almost no good for protein less than 0.1 mg/mL. The formation of Shif-base is not reversible unless furthewr reduced to secondary amine (harmful to lots of proteins). Oxidation damage the matrix and create leaking problems. Beside, I have to warn the one who put the first question: affinity purification of antibody using whole protein will create lots of problems and limits the application of the antibodies because of the leaking of antigen, WHICH CONTAINS MULTI-EPIOPES. Unlike eptope-based affinity chromatography, the leaking antigen will interfere the interpretation of your results. >I checked with Dima, and he clarified something for me. He means to say >that you must use the CL-4B, which is cross-linked, because plain 4B, which >is not cross linked, will not work with this activation method. > >Thanks Dima. > >Warren Gallin >Department of Biological Sciences >University of Alberta >Edmonton, Alberta T6G 2E9 >Canada >wgallin@gpu.srv.ualberta.ca From owner-proteins@net.bio.net Wed Mar 10 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.stanford.edu!news-feed.inet.tele.dk!bofh.vszbr.cz!nntp.news.xara.net!xara.net!server5.netnews.ja.net!daresbury!not-for-mail From: Miriam Hirshberg Newsgroups: bionet.molbio.proteins Subject: Pdb-viewer Date: 11 Mar 1999 12:57:58 -0000 Organization: Daresbury Laboratory, Warrington, U.K. Lines: 22 Message-ID: <7c8ekm$ht3$1@mserv2.dl.ac.uk> Reply-To: m.hirshberg@mole.bio.cam.ac.uk NNTP-Posting-Host: mserv2.dl.ac.uk Original-To: wangqm@nic.bmi.ac.cn, proteins@dl.ac.uk content-length: 771 Return-Path: try http://www.expasy.ch/spdbv/mainpage.html Miri >Dear all: >Have you ever used Pdb-viewer, a useful tool viewing protein structure. >Can anybody tell me where I can get the detailed help(like detailed user >manual). I think help file of the program are so simple that I can >understand. >Much appreciate any help ====================================================================== Miriam (Miri) Hirshberg e-mail: m.hirshberg@mole.bio.cam.ac.uk Department of Biochemistry Tel : (+44) 01223 766018 University of Cambridge Tel : (+44) 01223 333600 (switch board) 80 Tennis Court Road Fax : (+44) 01223 766002 Old Addenbrooke's Site Cambridge CB2 1GA, UK ===================================================================== From owner-proteins@net.bio.net Wed Mar 10 22:00:00 1999 Path: biosci!NIC.BMI.AC.CN!wangqm From: wangqm@NIC.BMI.AC.CN (Zhang Bo) Newsgroups: bionet.molbio.proteins Subject: Pdb-viewer? Date: 11 Mar 1999 04:30:16 -0800 Organization: AMMS Lines: 18 Sender: daemon@net.bio.net Distribution: world Message-ID: <36E7B640.E4BE8347@nic.bmi.ac.cn> NNTP-Posting-Host: net.bio.net Dear all: Have you ever used Pdb-viewer, a useful tool viewing protein structure. Can anybody tell me where I can get the detailed help(like detailed user manual). I think help file of the program are so simple that I can understand. Much appreciate any help -- Zhang Bo Department of Experimental Hematology Institute of Radiation Medicine 27# Taiping Road Beijing 100850 P.R.China wangqm@nic.bmi.ac.cn From owner-proteins@net.bio.net Wed Mar 10 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!btnet-peer!btnet!dispose.news.demon.net!demon!news.demon.co.uk!demon!genesys.demon.co.uk!Duncan From: "Dr. Duncan Clark" Newsgroups: bionet.molbio.proteins Subject: Re: To solubilize transferred proteins..HOW?? Date: Thu, 11 Mar 1999 09:26:22 +0000 Organization: DNAmp Ltd. Message-ID: References: <36E6A7BC.65B5F9E2@mail.utexas.edu> Reply-To: "Dr. Duncan Clark" NNTP-Posting-Host: genesys.demon.co.uk X-NNTP-Posting-Host: genesys.demon.co.uk:158.152.17.110 X-Trace: news.demon.co.uk 921147349 nnrp-01:23242 NO-IDENT genesys.demon.co.uk:158.152.17.110 X-Complaints-To: abuse@demon.net MIME-Version: 1.0 X-Newsreader: Turnpike (32) Version 4.01 Lines: 18 In article , Bernard P. Murray, PhD writes >Someone can correct me if I'm wrong but I believe that >nitrocellulose I think methoxyethanol will also dissolve nitrocellulose. 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@net.bio.net Thu Mar 11 22:00:00 1999 Path: biosci!newshost.lanl.gov!awabi.library.ucla.edu!128.230.129.106!news.maxwell.syr.edu!btnet-peer!btnet!dispose.news.demon.net!demon!news.demon.co.uk!demon!genesys.demon.co.uk!Duncan From: "Dr. Duncan Clark" Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: Protein immobilization on Sepharose using periodate activation (Was: Purifying Antibodies using affinity support) Date: Fri, 12 Mar 1999 09:18:28 +0000 Organization: DNAmp Ltd. Message-ID: References: <7c3oi7$rhj$1@mserv2.dl.ac.uk> <7c45t5$rt2$1@news.doit.wisc.edu> <36e81f1e.0@carrera.intergate.ca> Reply-To: "Dr. Duncan Clark" NNTP-Posting-Host: genesys.demon.co.uk X-NNTP-Posting-Host: genesys.demon.co.uk:158.152.17.110 X-Trace: news.demon.co.uk 921231307 nnrp-01:27864 NO-IDENT genesys.demon.co.uk:158.152.17.110 X-Complaints-To: abuse@demon.net MIME-Version: 1.0 X-Newsreader: Turnpike (32) Version 4.01 Lines: 29 Xref: biosci bionet.molbio.methds-reagnts:74704 bionet.molbio.proteins:14101 In article <36e81f1e.0@carrera.intergate.ca>, immunechem writes >Here I am telling you that Periodate method is one of the most >inefficient coupling procedures, particularly for the protein sample >with low concentration. Have a read of pp46-48 of Affinity Chromatography, A Practical Approach, IRL press. Although a protein sample of low concentration is not advisable, this method does give affinity columns with very low non- specific binding properties. It may not be the best method around but it does work OK. This book is a good practical guide to affinity chromatography. If you can get hold of a copy, LKB used to have a book from Reactifs IBF called Ultrogel, Magnogel and Trisacryl. Practical guide for use in affinity chromatography and related techniques. It has nice practical instruction section for all sorts of activation and subsequent coupling methods. 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@net.bio.net Thu Mar 11 22:00:00 1999 Path: biosci!NMSU.EDU!hroychow From: hroychow@NMSU.EDU ("Hiranya S. Roychowdhury") Newsgroups: bionet.molbio.proteins Subject: Re: Western blotting Date: 12 Mar 1999 13:40:56 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 25 Sender: daemon@net.bio.net Distribution: world Message-ID: <199903122135.OAA131016@nestor.NMSU.Edu> NNTP-Posting-Host: net.bio.net > >UAB Person wrote: > >> I am new to the Western blotting game, and am having some trouble with >> consistancy from blot to blot. Can anyone recommend a good Western >> blotting lab manual with methods, trouble-shooting, recipes etc. We are >> attempting to measure GAP-43 in cell culture lysates with a commercial >> antibody. >> >> Thanks in advance. >> >> John Moore > Very good manuals are avilable from: S&S and Hoefer. Ask them to send these. Dr. Hiranya Sankar Roychowdhury GENE LAB/ EPPWS New Mexico State University Las Cruces, NM 88003 Ph. (505) 646-5785 hroychow@nmsu.edu From owner-proteins@net.bio.net Thu Mar 11 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!sunqbc.risq.qc.ca!news.dal.ca!nntp-user From: Biology Newsgroups: bionet.molbio.proteins Subject: Re: Western blotting Date: Fri, 12 Mar 1999 15:59:06 -0400 Organization: dal Lines: 17 Message-ID: <36E9720A.A04527AF@hotmail.com> References: <7cb9t4$efn@maze.dpo.uab.edu> NNTP-Posting-Host: lvining.biology.dal.ca Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.05 [en] (Win95; I) To: UAB Person hi see Bi-Rad manual UAB Person wrote: > I am new to the Western blotting game, and am having some trouble with > consistancy from blot to blot. Can anyone recommend a good Western > blotting lab manual with methods, trouble-shooting, recipes etc. We are > attempting to measure GAP-43 in cell culture lysates with a commercial > antibody. > > Thanks in advance. > > John Moore From owner-proteins@net.bio.net Thu Mar 11 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!newshub.northeast.verio.net!newspeer.monmouth.com!falcon.america.net!news.mindspring.net!130.207.7.245.MISMATCH!cc.gatech.edu!finch!cronkite.cc.uga.edu!cronkite.cc.uga.edu From: Mark Warner Newsgroups: bionet.molbio.proteins Subject: Re: Western blotting Date: Fri, 12 Mar 1999 11:13:31 -0500 Organization: University of Georgia Lines: 24 Message-ID: <36E93D2B.64FB@dogwood.botany.uga.edu> References: <7cb9t4$efn@maze.dpo.uab.edu> NNTP-Posting-Host: schmidt.genetics.uga.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0 (Win95; I) UAB Person wrote: > > I am new to the Western blotting game, and am having some trouble with > consistancy from blot to blot. Can anyone recommend a good Western > blotting lab manual with methods, trouble-shooting, recipes etc. We are > attempting to measure GAP-43 in cell culture lysates with a commercial > antibody. > > Thanks in advance. > > John Moore John, Just about any protein methods book will have one section dedicated to blotting. I have also found some usefull information from the equipment suppliers (ie. Biorad has some tech bulletins of general use) and you can also get some good advice from the manufacturer of you membrane material. Most have good tech support departments that are more than happy to supply you with advice. I would suggest you look for a good methods book too since you will learn the ins and outs of the method along with the pitfalls associated with your protein, buffer choice etc. Good luck. Mark From owner-proteins@net.bio.net Thu Mar 11 22:00:00 1999 Path: biosci!news.stanford.edu!su-news-feed4.bbnplanet.com!su-news-hub1.bbnplanet.com!atl-news-feed1.bbnplanet.com!news.gtei.net!maze.dpo.uab.edu!usenet From: uab@uabdpo (UAB Person) Newsgroups: bionet.molbio.proteins Subject: Western blotting Date: 12 Mar 1999 14:55:32 GMT Organization: UAB Lines: 10 Message-ID: <7cb9t4$efn@maze.dpo.uab.edu> NNTP-Posting-Host: 138.26.136.134 Mime-Version: 1.0 X-Newsreader: WinVN 0.93.11 I am new to the Western blotting game, and am having some trouble with consistancy from blot to blot. Can anyone recommend a good Western blotting lab manual with methods, trouble-shooting, recipes etc. We are attempting to measure GAP-43 in cell culture lysates with a commercial antibody. Thanks in advance. John Moore From owner-proteins@net.bio.net Thu Mar 11 22:00:00 1999 Path: biosci!RULLF2.MEDFAC.LEIDENUNIV.NL!westbroek From: westbroek@RULLF2.MEDFAC.LEIDENUNIV.NL ("irene") Newsgroups: bionet.molbio.proteins Subject: (none) Date: 12 Mar 1999 06:31:08 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 64 Sender: daemon@net.bio.net Distribution: world Message-ID: <003901be6c95$e464f0e0$650be584@MedFac.LeidenUniv.NL> NNTP-Posting-Host: net.bio.net This is a multi-part message in MIME format. ------=_NextPart_000_0036_01BE6C9E.4607C720 Content-Type: text/plain; charset="iso-8859-1" Content-Transfer-Encoding: quoted-printable WANTED: chicken (bird) protein expression system! We have got an antibody, but we do not know to which protein it binds. = We would like to screen an existing expression system with our antibody. = Do you have such a system, or know anyone that has one, please contact: Irene Westbroek at I.Westbroek@mcb.medfac.leidenuniv.nl Thanks for your attention and help!! drs. I. Westbroek Molecular Cell Biology LUMC, Leiden University Wassenaarseweg 72 2333 AL Leiden Tel: ++31 71-5276401 Fax: ++31 71-5276437 E-mail: I.Westbroek@mcb.medfac.leidenuniv.nl ------=_NextPart_000_0036_01BE6C9E.4607C720 Content-Type: text/html; charset="iso-8859-1" Content-Transfer-Encoding: quoted-printable
WANTED: chicken (bird) protein expression = system!
We have got an antibody, but we do not know to which protein it = binds. We=20 would like to screen an existing expression system with our antibody. Do = you=20 have such a system, or know anyone that has one, please contact:
Irene Westbroek at
I.Westbroek@mcb.medf= ac.leidenuniv.nl
Thanks for your attention and help!!
 
drs. I. Westbroek
Molecular Cell Biology
LUMC, Leiden=20 University
Wassenaarseweg 72
2333 AL Leiden
 
Tel: ++31 71-5276401
Fax: ++31 71-5276437
 
E-mail: I.Westbroek@mcb.medf= ac.leidenuniv.nl
------=_NextPart_000_0036_01BE6C9E.4607C720-- From owner-proteins@net.bio.net Thu Mar 11 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!news.maxwell.syr.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!Home From: klenchin@REMOVE_TO_REPLY.facstaff.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins Subject: Re: Membrane protein isolation?? Date: Sat, 13 Mar 1999 02:21:39 GMT Organization: UW Lines: 31 Message-ID: <7cci3r$stm$1@news.doit.wisc.edu> References: <7cc2al$b7m@ds2.acs.ucalgary.ca> NNTP-Posting-Host: ras-c5800-1-216-173.dialup.wisc.edu X-Newsreader: News Xpress 2.01 X-No-Archive: Yes In article <7cc2al$b7m@ds2.acs.ucalgary.ca>, Neal Melvin wrote: >This is a multi-part message in MIME format. >--------------49995297F1BAE9FBC066F06F >Content-Type: text/plain; charset=us-ascii >Content-Transfer-Encoding: 7bit Could you please not post MIME stuff, and use plain text instead? >Can anyone recommend a good starting protocol for the isolation of >membrane proteins from molluscan (or any neurons to begin with) neurons? All protein purification is alike. That is, it is very different, highly empirical, and unpredictable. Unlike nucleic acids, there are no ready-to-use recipies. Almost everyhting depends on your protein and application. The list of things to ask that helps to sort out countless possibilities is HUGE. Please provide as much background info as possible, only then a reasonable advice can be tried. Partial list in no particular order: Amount of source material, abundance of protein, method of detecion, aim of puirification, equipment available, MW of the protein, biological activity, homologes in other tissues/species, etc, etc. Without additional info, the only advice possible sounds as fiollows: Solubilize membranes (well-washed under stringent conditions that still keep the protein insoluble) in cheapest detergent that preserves desired properties of your protein and apply usual fractionation techniques such as precipitation, various column chromatographies, and electophoresis to purify your protein. - See how pointless it is? - Dima From owner-proteins@net.bio.net Thu Mar 11 22:00:00 1999 Path: biosci!MAIL.BOTANY.UBC.CA!dkro From: dkro@MAIL.BOTANY.UBC.CA (Dae Kyun Ro) Newsgroups: bionet.molbio.proteins Subject: osmotic lysis Date: 12 Mar 1999 23:34:55 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 20 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Hi, protein netters. I want to rupture my cells (insect cells such as sf9, ld, hi-5) as gentle as possible. Osmotic lysis seems to be the way to go, but I don't know how I can design the lysis buffer. I looked up some papers about the osmotic shock, and I have found a variety of different lysis buffers. I don't want my cells suddenly ruptured. It would be ideal that the cells gradually take up water in certain period of time and broken open smoothly at some point. Gentle lysis is critical in my experiment because I want to analyze the multienzyme complex on ER. The nature of protein-protein interaction may be very weak. Can anybody tell me good reference or idea? Thank you in advance. ********************************** Dae Kyun Ro 439, 6335 Thunderbird Cres. Vancouver, BC Canada V6T 2G9 From owner-proteins@net.bio.net Thu Mar 11 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!cyclone.bc.net!rover.ucs.ualberta.ca!news.ucalgary.ca!news From: Neal Melvin Newsgroups: bionet.molbio.proteins Subject: Membrane protein isolation?? Date: Fri, 12 Mar 1999 14:50:33 -0700 Organization: The University of Calgary Lines: 32 Message-ID: <7cc2al$b7m@ds2.acs.ucalgary.ca> NNTP-Posting-Host: @hmr176.meds.ucalgary.ca Mime-Version: 1.0 Content-Type: multipart/mixed; boundary="------------49995297F1BAE9FBC066F06F" X-Mailer: Mozilla 4.06 [en] (Win98; I) This is a multi-part message in MIME format. --------------49995297F1BAE9FBC066F06F Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit Can anyone recommend a good starting protocol for the isolation of membrane proteins from molluscan (or any neurons to begin with) neurons? --------------49995297F1BAE9FBC066F06F Content-Type: text/x-vcard; charset=us-ascii; name="vcard.vcf" Content-Transfer-Encoding: 7bit Content-Description: Card for Neal Melvin Content-Disposition: attachment; filename="vcard.vcf" begin: vcard fn: Neal Melvin n: Melvin;Neal org: University of Calgary email;internet: nrmelvin@acs.ucalgary.ca title: Department of Neuroscience note: 3330 Hospital Drive, NW, Calgary, Alberta, T2N 4N1 x-mozilla-cpt: ;0 x-mozilla-html: FALSE version: 2.1 end: vcard --------------49995297F1BAE9FBC066F06F-- From owner-proteins@net.bio.net Fri Mar 12 22:00:00 1999 Path: biosci!agate!not-for-mail From: lhom@OCF.Berkeley.EDU (Louis Hom) Newsgroups: bionet.molbio.proteins Subject: wacky thought Date: 13 Mar 1999 20:50:31 GMT Organization: Univ. of California Berkeley Open Computing Facility Lines: 21 Message-ID: <7cej2n$24j$1@agate.berkeley.edu> NNTP-Posting-Host: apocalypse.ocf.berkeley.edu There are sometimes weird reversible cause-effect relationships between phenomena -- at the moment the only one that comes to mind is the Peltier effect (say you heat one end of an electrical conductor and chill the other; the electrons at the hot end wind up with a higher electrical potential. conversely, you can apply a voltage across certain semiconductor n/p couples and get hot on one side and cold on the other (useful for cooling Pentiums)). If anyone has a more broadly known example of this, I'd welcome the help. Maybe fluorescence and the photoelectric effect? But my real question/idea is, I wonder if there's anything that we measure or postulate in proteins (say, CD or helix end-effects) that could actually be turned around to influence the behavior (folding, whatever) of the proteins. -- ______________________________________________________________________________ Lou Hom >K'93 lhom@ocf.berkeley.edu http://www.ocf.berkeley.edu/~lhom/ From owner-proteins@net.bio.net Fri Mar 12 22:00:00 1999 Path: biosci!internet!biosci!not-for-mail From: biohelp (BIOSCI Administrator) Newsgroups: bionet.molbio.proteins Subject: BIOSCI/bionet miniFAQ & Fundraiser Date: 13 Mar 1999 02:00:22 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 233 Sender: daemon@net.bio.net Distribution: world Message-ID: <199903131000.CAA12549@net.bio.net> NNTP-Posting-Host: net.bio.net (LAST REVISION: 30-JUL-95) This BIOSCI "miniFAQ" is designed to answer the questions that come up the *most frequently*. The main BIOSCI FAQ (Frequently Asked Questions) is accessible on the World Wide Web at URL http://www.bio.net/. If you can not find an answer to your question in this or other documentation, the BIOSCI technical support staff answers e-mail queries sent to biosci-help@net.bio.net We can only answer questions about the use of the newsgroups and mailing lists. We unfortunately do not have the staff to do Internet information searches or answer scientific questions. Please post those to the appropriate BIOSCI/bionet newsgroups. Contents: -------- 0) BIOSCI NEEDS YOUR SUPPORT!! 1) Using the WWW to access the BIOSCI/bionet newsgroups. 2) What to do about "spams," i.e., junk mail, ads, etc. 3) Examples of subscribing and unsubscribing to the mailing lists. 4) The BIOSCI user address and research interest directory. 0) BIOSCI NEEDS YOUR SUPPORT!! ------------------------------ BIOSCI's government funding has been expended, and we are now operating solely from advertising revenue that we have raised from our Web site at http://www.bio.net/. We need just a few minutes of your time to help us serve you. You can do two important things which will take very little time for you individually and will immensely help us continue to help you. First, please use our WWW system at http://www.bio.net/ to access the archives. You can post or reply to messages via your Web browser as described in item #1 below. Your usage helps attract sponsors. If you contact any of our sponsors, please be sure to thank them for supporting BIOSCI. It is critical for them to get this feedback if they are to continue their sponsorship for the long term. Second, if you work for a company or organization that provides products or services of interest to the biology community, please pass this message on to your marketing or marketing communications department or other appropriate group. Please ask them to help support BIOSCI by sponsoring our Web site and explain the uses and benefits of the system to the biology community. If they are interested, they can then contact us for further information at our tech support address, biosci-help@net.bio.net. 1) Using the WWW to access the BIOSCI/bionet newsgroups. -------------------------------------------------------- As of 10 December 1995, all BIOSCI/bionet full newsgroups are accessible through the World Wide Web (WWW) at URL http://www.bio.net. One can read and reply publicly or privately to both recent postings and archived messages through one's Web browser if it is configured properly to send e-mail. Each newsgroup is equipped with its own WAIS index. The main BIOSCI home page also has access to the BIO-JOURNALS Table of Contents database WAIS index and the BIOSCI user address database described in another item further below. 2) What to do about "spams," i.e., junk mail, ads, etc. ------------------------------------------------------- BIOSCI is a set of parallel USENET newsgroups (the "bionet" groups), mailing lists, and a hypermail archive at URL http://www.bio.net/. The same postings are distributed on all media (except for a small number of mailing-list-only groups at net.bio.net). Unfortunately it is becoming a despicable practice on the Internet (by a few people out to make a fast buck) to do automated mass postings to thousands of newsgroups and mailing lists. These attempts to grab free advertising are refered to as "spams" in the usual, somewhat boneheaded, net terminology. USENET is more susceptible to this practice, and many spams originate on the USENET groups and then are passed on to the mailing lists. However, spammers also get lists of mailing addresses and hit these too, so neither medium is immune. What should you do personally if you get junk mail? --------------------------------------------------- Just delete it and move on without reading it further. Filing a protest is becoming increasingly useless because spammers are often disguising the addresses where the messages are sent from. Unless you really understand Internet mail systems, your attempt at protest by sending replies to the message will often end up being sent to the address of an innocent person that the spammer is victimizing. What can BIOSCI/bionet do to protect its newsgroups? ---------------------------------------------------- The only solution currently available is to moderate the newsgroup. If this newsgroup is already moderated, then you are in good shape. Moderation protects the USENET distribution from about 95% of the spams that are being sent to date and protects the mailing lists completely. Moderation means, however, that someone has to take the time to review each message before it goes out. We have set up software here that simply allows the moderator to forward to an address at net.bio.net messages that (s)he wishes to have distributed. This takes no more time than that needed to read the message and pass it on, say about 1 min. per message. Most newsgroups currently have a discussion leader who is responsible for their newsgroup. The discussions leaders and their e-mail addresses are listed in the BIOSCI Information Sheet which is available on the Web at http://www.bio.net/. If a newsgroup is being hit with too many junk postings, please contact the discussion leader for that group and see if there is interest in moderating the group. Please do not assume that by simply posting a complaint to the newsgroup itself, anyone on the BIOSCI staff will act on your complaint. With close to 100 newsgroups to run, the BIOSCI staff has to rely on the discussion leaders of each newsgroup to report problems directly to us at biosci-help@net.bio.net. We will moderate any of our newsgroups if the discussion leader tells us that the readership of the group wishes to do so and if a moderator is willing to do the work. For most BIOSCI/bionet groups, this entails only a few minutes of work each day. Moderating a newsgroup will resolve probably 95% of the junk postings on the USENET distribution. Unfortunately there are easy ways for determined spammers to override the moderation mechanism on USENET, but we can protect our e-mail subscribers from unwanted postings if the newsgroup is moderated. You can also access our newsgroups over the WWW at URL http://www.bio.net. While this Web interface will not stop spammers from trying to post to the groups, this will give you yet another way, besides using USENET news, to keep the junk out of your personal mail files. For those of you with local USENET news systems, the Web interface will also give you faster access to new newsgroups and recent postings. 3) Examples of subscribing and unsubscribing to the mailing lists. ------------------------------------------------------------------ PLEASE NOTE: The BIOSCI management does NOT act on subscription/unsubscription requests that are posted improperly to the newsgroups and mailing lists. People who do this only bother everyone on the lists to no avail. Please be sure to follow the proper procedures below. Gory details are in the BIOSCI Information sheets on the Web at http://www.bio.net. Below we give an example utilizing the METHODS-AND-REAGENTS list at both of our two BIOSCI sites: Users in the Americas and Pacific Rim countries who use the BIOSCI ------------------------------------------------------------------ node at computer net.bio.net: ---------------------------- A) Determine the "listname" which is the <=8 character mail address ^^^^^^^^^^^^^ for the group. These can be found in the BIOSCI Info. Sheet. For the METHODS-AND-REAGENTS group the mailing address is methods@net.bio.net. The listname is the portion of the address to the left of the @ sign, i.e., "methods". The listname is used with the "subscribe" and "unsubscribe" commands illustrated below. B) Mail all commands in the body of a mail message addressed to biosci-server@net.bio.net. Do NOT send commands to the newsgroup posting addresses! Leave the Subject: line blank, any text on it will be ignored. C) In the body of your message put one or more of the following commands with an "end" command on the last line, e.g., subscribe methods unsubscribe methods end Do NOT put your e-mail address or other text on these lines. The server only allows you to cancel your subscription if the address on your mail header matches the address on our mailing list. Please ask for help at biosci-help@net.bio.net if your address has changed, e.g., if you know you are on the list but the server tells you that you are not a member. Users in Europe, Africa, and Central Asia who use the BIOSCI node at -------------------------------------------------------------------- computer daresbury.ac.uk (also known as dl.ac.uk): ------------------------------------------------- To subscribe and unsubscribe to/from the BIOSCI lists, you need to specify the full USENET newsgroup name with "bionet-news." prepended. The USENET newsgroup names are listed in the BIOSCI Information sheet on the Web at http://www.bio.net/. For the METHODS-AND-REAGENTS list the USENET newsgroup name is bionet.molbio.methds-reagnts, thus the appropriate commands are sub bionet-news.bionet.molbio.methds-reagnts unsub bionet-news.bionet.molbio.methds-reagnts These commands are included in a message addressed to mxt@dl.ac.uk, NOT to the newsgroup mailing addresses. As usual, include the text in the bod