From owner-proteins@net.bio.net Fri Nov 01 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!news.ecn.uoknor.edu!feed1.news.erols.com!howland.erols.net!www.nntp.primenet.com!nntp.primenet.com!mr.net!newshub.tc.umn.edu!fu-berlin.de!informatik.tu-muenchen.de!lrz-muenchen.de!uni-erlangen.de!winx03!wpxx02!not-for-mail From: krasel@wpxx02.toxi.uni-wuerzburg.de (Cornelius Krasel) Newsgroups: bionet.molbio.proteins Subject: Re: Protein Data Bank Resources! Date: 2 Nov 1996 18:07:02 GMT Organization: University of Wuerzburg, Germany Lines: 23 Message-ID: <55g2k6$2m@winx03.informatik.uni-wuerzburg.de> References: <55fsv5$rvr@hptemp1.cc.umr.edu> NNTP-Posting-Host: wpxx02.toxi.uni-wuerzburg.de X-Newsreader: TIN [UNIX 1.3 950824BETA PL0] Raghavender Pillutla (raghav@saucer.cc.umr.edu) wrote: > I find it very difficult to get information from the protein data bank of > brookhaven national laboratory. I find the database organisation very > complex and cannot get what I want. I would like to know if any resources > such as books, or CD-roms are available which tell me how to access > information from Protein Data Bank. If these are available, I would > appreciate if someone told me where I can find them also. You should use a WWW gateway. There are several: - the one at the PDB itself which you apparently don't like: http://www.pdb.bnl.gov/cgi-bin/browse - full-text search at the NIH: http://molbio.info.nih.gov/cgi-bin/pdb - Moose: http://db2.sdsc.edu/moose/ --Cornelius. -- /* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */ /* D-97078 Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de SP3 */ /* "Science is the game we play with God to find out what His rules are." */ From owner-proteins@net.bio.net Fri Nov 01 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!swrinde!howland.erols.net!newsfeed.internetmci.com!mr.net!news.mid.net!nntp.ksu.edu!hptemp1.cc.umr.edu!raghav From: raghav@saucer.cc.umr.edu (Raghavender Pillutla) Newsgroups: bionet.molbio.proteins Subject: Protein Data Bank Resources! Date: 2 Nov 1996 16:30:29 GMT Organization: UMR Missouri's Technological University Lines: 12 Message-ID: <55fsv5$rvr@hptemp1.cc.umr.edu> NNTP-Posting-Host: saucer.cc.umr.edu X-Newsreader: TIN [version 1.2 PL2] Hello netters, I find it very difficult to get information from the protein data bank of brookhaven national laboratory. I find the database organisation very complex and cannot get what I want. I would like to know if any resources such as books, or CD-roms are available which tell me how to access information from Protein Data Bank. If these are available, I would appreciate if someone told me where I can find them also. Thanks a lot, Raghu From owner-proteins@net.bio.net Fri Nov 01 22:00:00 1996 Path: biosci!agate!howland.erols.net!feed1.news.erols.com!arclight.uoregon.edu!dispatch.news.demon.net!demon!peacocks.demon.co.uk!user From: bill@peacocks.demon.co.uk (William Peacock) Newsgroups: bionet.molbio.proteins Subject: Diff. between intrinsic/extrinsic cell membrane proteins Followup-To: bill@peacocks.demon.co.uk Date: Sat, 02 Nov 1996 20:41:40 +0100 Lines: 9 Distribution: world Message-ID: Reply-To: bill@peacocks.demon.co.uk NNTP-Posting-Host: peacocks.demon.co.uk X-NNTP-Posting-Host: peacocks.demon.co.uk PLease could someone tell me whether proteins found within cell membranes which penetrate the membrane but do not span the entire membrane are classed as intrinsic or extrinsic proteins; reference book s seem split over this. Thanks, William Peacock From owner-proteins@net.bio.net Fri Nov 01 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!news.sgi.com!mr.net!www.nntp.primenet.com!nntp.primenet.com!feed1.news.erols.com!news.enteract.com!ix.netcom.com!news From: "The Kindly One" Newsgroups: bionet.molbio.proteins Subject: Re: Reprobing western blots Date: 3 Nov 1996 04:39:27 GMT Organization: Netcom Lines: 15 Message-ID: <01bbc941$1f8e5760$4ef61fcc@natasha> References: <327545E7.6162@lucano.uco.es> NNTP-Posting-Host: lex-ky2-14.ix.netcom.com X-NETCOM-Date: Sat Nov 02 8:39:27 PM PST 1996 X-Newsreader: Microsoft Internet News 4.70.1155 Biol. Cel. wrote in article <327545E7.6162@lucano.uco.es>... > I need to reprobe western blots with a primary antibody after > detect with ECL a firs immunodetection. How can I separate the first > antibody and reprobe the blot? > > Thank you in advance. > Read the protocol given by ECL!!! It works great for us. I think it involves using 2X tris and incubating for an hour or so at 50(or 60) degrees. That should take off the primary Ab and then you can reprobe. It's all in the ECL booklet. From owner-proteins@net.bio.net Fri Nov 01 22:00:00 1996 Path: biosci!bloom-beacon.mit.edu!eru.mt.luth.se!www.nntp.primenet.com!nntp.primenet.com!uwm.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!daemon From: klenchin@facstaff.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins Subject: Re: Diff. between intrinsic/extrinsic cell membrane proteins Date: Sun, 03 Nov 96 01:41:13 GMT Organization: UW-Madison Lines: 13 Distribution: world Message-ID: <55gppg$1dfu@news.doit.wisc.edu> References: NNTP-Posting-Host: f181-194.net.wisc.edu Mime-Version: 1.0 Content-Type: text/plain; charset=KOI8-R Content-Transfer-Encoding: 8bit X-No-Archive: Yes In article , bill@peacocks.demon.co.uk wrote: #PLease could someone tell me whether proteins found within cell membranes #which penetrate the membrane but do not span the entire membrane are #classed as intrinsic or extrinsic proteins; reference book s seem split #over this. This is just a matter of definition not worth to think about. IMO, it's better to use "functional" definition, one possible example of which is extraction with ~ 6 M urea. "True" membrane proteins (embedded in the membrane) cannot be extracted since urea does not dissolve membrane bilyaer. - Dima From owner-proteins@net.bio.net Sat Nov 02 22:00:00 1996 Path: biosci!rutgers!news.sgi.com!howland.erols.net!vixen.cso.uiuc.edu!sdd.hp.com!night.primate.wisc.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!daemon From: klenchin@facstaff.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins Subject: Re: Q:stability of immobilized enzyme Date: Mon, 04 Nov 96 05:18:18 GMT Organization: UW-Madison Lines: 19 Message-ID: <55jqsh$k20@news.doit.wisc.edu> References: <55iiif$65e@rks1.urz.tu-dresden.de> NNTP-Posting-Host: f181-190.net.wisc.edu Mime-Version: 1.0 Content-Type: text/plain; charset=KOI8-R Content-Transfer-Encoding: 8bit X-No-Archive: Yes In article <55iiif$65e@rks1.urz.tu-dresden.de>, xn@space.wh1.tu-dresden.de wrote: #Hello # #Does anyone knows a paper or literature that deals with the stability #of an enzyme (especially glucoseoxidase (GOD EC1.1.3.4 )) after #immobilization into a polymeric surface. I wonder if the #immobilization might conservate the enzyme activity for longer time. I don't have any refs, but think such should be easy to find in > 10 yo literature when immobilized enzymes were thought to be the "next great thing". Specifically, glucoseoxidase activity, once immobilized on sepharose (no personal experience) or in the form of conjugates with Ab (that's I know for sure), is very stable. By analogy to conjugates, I'd say that sepharose could could be equilibrated with 50% glycerol or 40% ethylene glycole (I like the latter better because of lower viscosity) and stored at -20C forever. - Dima From owner-proteins@net.bio.net Sat Nov 02 22:00:00 1996 Path: biosci!agate!howland.erols.net!www.nntp.primenet.com!nntp.primenet.com!nntp.uio.no!news.nacamar.de!news-kar1.dfn.de!news-stu1.dfn.de!news-mue1.dfn.de!news-nue1.dfn.de!news-lei1.dfn.de!news.uni-leipzig.de!news1.urz.tu-dresden.de!news From: xn@space.wh1.tu-dresden.de (Anne Riedel) Newsgroups: bionet.molbio.proteins Subject: Q:stability of immobilized enzyme Date: Sun, 03 Nov 1996 16:51:30 GMT Organization: TU-Dresden Lines: 12 Message-ID: <55iiif$65e@rks1.urz.tu-dresden.de> Reply-To: xn@space.wh1.tu-dresden.de NNTP-Posting-Host: gaga408.wh1.tu-dresden.de X-Newsreader: Forte Free Agent 1.0.82 Hello Does anyone knows a paper or literature that deals with the stability of an enzyme (especially glucoseoxidase (GOD EC1.1.3.4 )) after immobilization into a polymeric surface. I wonder if the immobilization might conservate the enzyme activity for longer time. Thanks in advance Anne Please reply via e-mail From owner-proteins@net.bio.net Sat Nov 02 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!newshost.convex.com!newsgate.duke.edu!news-server.ncren.net!concert!ussun2n.glaxo.com!uswsum.glaxo.com!user From: sujoy_ghosh@glaxo.com (sujoy ghosh) Newsgroups: bionet.molbio.proteins Subject: Re: GST fusion proteins HELP Date: Tue, 22 Oct 1996 15:18:37 -0400 Organization: glaxo wellcome Lines: 18 Distribution: world Message-ID: References: <324AA269.D71@warren.med.harvard.edu> NNTP-Posting-Host: uswsum.glaxo.com In article <324AA269.D71@warren.med.harvard.edu>, berez@WARREN.MED.HARVARD.EDU wrote: > I am trying to express a leech homeobox protein in bacteria to make an > antibody. The protein is insoluble. I followed the Frangioni and > Neel paper for solubilizing using sarkosyl. I get a good amount of > the protein into the supernatant, then I add Triton to restore binding > to glutathione sepharose but I don't get a good yeild. I get back the > tiniest fraction of what is in the supernatant. I also seem to get > poor elution from the beads. Any ideas? > > Ashley Bruce What are your growth conditions? Assuming you are growing your cultures at 37 degrees, try growing them at 20 degrees for 1-3 hours. It might help. Sujoy From owner-proteins@net.bio.net Sun Nov 03 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!feed1.news.erols.com!howland.erols.net!EU.net!sun4nl!wirehub!news.euro.net!xs4all!xs4all!usenet From: heidel@xs4all.nl (Edwin Heidelberg) Newsgroups: bionet.molbio.proteins Subject: Protein properties Date: Mon, 04 Nov 1996 12:57:24 GMT Organization: XS4ALL, networking for the masses Lines: 35 Message-ID: <55kp4t$bh5@news.xs4all.nl> NNTP-Posting-Host: asn02-02.dial.xs4all.nl X-XS4ALL-Date: Mon, 04 Nov 1996 13:55:57 MET X-Newsreader: Forte Free Agent 1.0.82 Hi there, I am working for my graduation project an a flowsheeting program, which we can use to optimise downstream processes. I want to create a database with properties for proteins from which the user can pick the components in a mixture. I have to find properties like: Molecular weight Radius Diffusion Coefficient Iso Electric Points Precipitation Coeeficients (Cohn equation) Sedimentation coefficient The proteins in the database are: Catalase Alpha-amylase Beta-Galactosidase and a lot of other familiar proteins. Before spending a couple of weeks in the library finding all the properties, does anyone know if there are any links to useful databanks. Thanks in advance. Regards Edwin Heidelberg From owner-proteins@net.bio.net Sun Nov 03 22:00:00 1996 Path: biosci!daresbury!nntp-trd.UNINETT.no!online.no!sn.no!www.nntp.primenet.com!nntp.primenet.com!news.sprintlink.net!news-peer.sprintlink.net!uunet!in1.uu.net!rcogate.rco.qc.ca!altitude!usenet From: "Achim Recktenwald, PhD" Newsgroups: bionet.molbio.proteins Subject: Re: Protein Separation Date: Fri, 01 Nov 1996 15:25:15 -0500 Organization: IBEX Technologies, Inc., Biochemistry, 5485 Pare, Montreal, PQ, H4P 1P7, Canada Lines: 33 Message-ID: <327A5CAA.11A5@ibex.ca> References: <01bbc0ec$0e73b720$58fd5380@UT.cc.utexas.edu> <553gbe$meg@ecuador.earthlink.net> NNTP-Posting-Host: ibex.hip.cam.org Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0 (Macintosh; I; PPC) To: bamsden@prozyme.com Bruce Amsden wrote: > > "Brett S. Phinney" wrote: > > >I need to separate and purify two very similar membrane proteins, and have > >not been successful so far. They both have a very similar molecular weight > >and presumably shape, because they only separate on an SDS-PAGE under > >reducing conditions (unfortunately I need them non-reduced). They also seem > >to have a similar hydophobicity plot, because I have not been able to > >separate them using Reversed phase HPLC either. They do have differing > >isoelectric points and have been separated using IEF in the past, but I > >have not been able to figure out how to purify my proteins away from the > >ampolytes. > > >Does anyone have any suggestions on how I can separate and purify these? > > >Any help would be greatly appreciated > > >-- > >Brett Phinney > >Cell Research Institute > >University of Texas, Austin > > Once you've purified your protein by IEF, can't you use gel filtration > chromatography to get rid of the ampholytes? > > Bruce Amsden The latter usually never works. The stuff sticks to proteins like crazy glue. Achim From owner-proteins@net.bio.net Sun Nov 03 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.erols.net!www.nntp.primenet.com!nntp.primenet.com!news.bbnplanet.com!cpk-news-hub1.bbnplanet.com!cpk-news-feed4.bbnplanet.com!news.udel.edu!chopin.udel.edu!not-for-mail From: xiaohan@chopin.udel.edu (Xiaohan Du) Newsgroups: bionet.molbio.proteins Subject: Test only: Date: 4 Nov 1996 14:17:55 -0500 Organization: University of Delaware Lines: 2 Sender: Cash@wilmington.trust Message-ID: <55lfh3$d43@chopin.udel.edu> NNTP-Posting-Host: chopin.udel.edu Test only. Please ignore. : From owner-proteins@net.bio.net Sun Nov 03 22:00:00 1996 Path: biosci!daresbury!nntp-trd.UNINETT.no!nntp.uio.no!www.nntp.primenet.com!nntp.primenet.com!howland.erols.net!surfnet.nl!news.unisource.nl!xs4all!xs4all!usenet From: tvink@xs4all.nl (Tom Vink) Newsgroups: bionet.molbio.proteins Subject: Re: immobilized neuraminidase Date: Mon, 04 Nov 1996 11:07:31 GMT Organization: XS4ALL, networking for the masses Lines: 30 Message-ID: <55kis0$8se@news.xs4all.nl> References: NNTP-Posting-Host: mas03-02.dial.xs4all.nl X-XS4ALL-Date: Mon, 04 Nov 1996 12:08:48 MET X-Newsreader: Forte Free Agent 1.0.82 hornig@bc.biol.ethz.ch (Ruediger Hornig) wrote: >Hello everybody, >for removing sialic acid residues from the outside of intact cells, I >would like to use neuraminidase. Does anybody know who is supplying it >immobilized on beads or does anybody think of a different way to remove >the enzyme from the reaction mixture ? >thanks in advance for your help >Rudi >---------------------------------------------------------------------- > _/_/_/_/_/_/_/_/_/ _/ ETH Zurich > _/ _/ _/ _/ Biochemie II > _/_/_/ _/ _/_/_/_/ Rüdiger Hornig (hornig@bc.biol.ethz.ch) > _/ _/ _/ _/ Universitaetstr. 16, ETH Zentrum >_/_/_/_/ _/ _/ _/ CH-8092 Zurich (Switzerland) > Phone: +41 1 632 3007 FAX: +41 1 632 12 69 > http://www.bc.biol.ethz.ch/BiochemistryII/BioII.html >---------------------------------------------------------------------- Dear Rudi, You could inactivate the Neuraminidase by adding an inhibitor after the treatment of the cells. From owner-proteins@net.bio.net Sun Nov 03 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!news.sgi.com!esiee.fr!jussieu.fr!univ-lyon1.fr!in2p3.fr!swidir.switch.ch!scsing.switch.ch!elna.ethz.ch!bclutz2.ethz.ch!user From: hornig@bc.biol.ethz.ch (Ruediger Hornig) Newsgroups: bionet.molbio.proteins Subject: immobilized neuraminidase Date: Mon, 04 Nov 1996 09:44:26 +0100 Organization: ETH Zurich Lines: 21 Message-ID: NNTP-Posting-Host: bclutz2.ethz.ch Hello everybody, for removing sialic acid residues from the outside of intact cells, I would like to use neuraminidase. Does anybody know who is supplying it immobilized on beads or does anybody think of a different way to remove the enzyme from the reaction mixture ? thanks in advance for your help Rudi ---------------------------------------------------------------------- _/_/_/_/_/_/_/_/_/ _/ ETH Zurich _/ _/ _/ _/ Biochemie II _/_/_/ _/ _/_/_/_/ Rüdiger Hornig (hornig@bc.biol.ethz.ch) _/ _/ _/ _/ Universitaetstr. 16, ETH Zentrum _/_/_/_/ _/ _/ _/ CH-8092 Zurich (Switzerland) Phone: +41 1 632 3007 FAX: +41 1 632 12 69 http://www.bc.biol.ethz.ch/BiochemistryII/BioII.html ---------------------------------------------------------------------- From owner-proteins@net.bio.net Sun Nov 03 22:00:00 1996 Path: biosci!daresbury!nntp-trd.UNINETT.no!nntp.uio.no!www.nntp.primenet.com!nntp.primenet.com!howland.erols.net!cam-news-hub1.bbnplanet.com!news.bbnplanet.com!cpk-news-hub1.bbnplanet.com!cpk-news-feed4.bbnplanet.com!tulane.edu!rs5.tcs.tulane.edu!not-for-mail From: jayuziuk@rs5.tcs.tulane.edu (Jeffrey Yuziuk) Newsgroups: bionet.molbio.proteins Subject: Re: immobilized neuraminidase Date: 4 Nov 1996 15:23:17 GMT Organization: Tulane University Lines: 8 Message-ID: <55l1p5$eno@rs10.tcs.tulane.edu> References: NNTP-Posting-Host: rs5.tcs.tulane.edu X-Newsreader: TIN [UNIX 1.3 950824BETA PL0] Dear Rudi, You can make the immobilized neuraminidase yourself. It is quite easy. Just follow the standard methods for affinity column preparation, but of course, use a method/matrix that doesn't inactivate your enzyme. A student in my lab did it with no problem and it worked great! Jeff From owner-proteins@net.bio.net Sun Nov 03 22:00:00 1996 Message-ID: <327E00C0.418A@uni-konstanz.de> Date: Mon, 04 Nov 1996 15:42:08 +0100 From: Bernd Langkau Reply-To: bernd.langkau@uni-konstanz.de Organization: University of Constance X-Mailer: Mozilla 3.0Gold (Win95; I) MIME-Version: 1.0 Newsgroups: bionet.molbio.hiv,bionet.molbio.methds-reagnts,bionet.molbio.proteins,bionet.molbio.yeast CC: Bernd.Langkau, Thomas.Seebacher Subject: Molbio-Software (Macintosh) Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit NNTP-Posting-Host: toprope.verwaltung.uni-konstanz.de Lines: 7 Path: biosci!daresbury!nntp-trd.UNINETT.no!due.unit.no!nntp.uio.no!www.nntp.primenet.com!nntp.primenet.com!mr.net!newshub.tc.umn.edu!fu-berlin.de!news.belwue.de!news.uni-konstanz.de!toprope.verwaltung.uni-konstanz.de Xref: biosci bionet.molbio.hiv:2624 bionet.molbio.methds-reagnts:51140 bionet.molbio.proteins:9194 bionet.molbio.yeast:6070 A new program for the evaluation of gel images is available from http://www.uni-konstanz.de/tt/software/mwmacro.html or from ftp://ftp.uni-konstanz.de/pub/local/Biologie/MW-Macro.sea.bin. The new shareware program (50.-DM) calculates molecular weights, pI-values and band intensities from scanned gel images. The program requires Macintosh computers and NIH-Image software. From owner-proteins@net.bio.net Mon Nov 04 22:00:00 1996 Path: biosci!CS.Arizona.EDU!news.Arizona.EDU!hamblin.math.byu.edu!sol.ctr.columbia.edu!news.uoregon.edu!news.ironhorse.com!newshub.csu.net!www.nntp.primenet.com!nntp.primenet.com!news.bbnplanet.com!cam-news-hub1.bbnplanet.com!uunet!in3.uu.net!netnews.worldnet.att.net!newsadm From: clkstanley@postoffice.worldnet.att.net Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: Best way to clean cuvettes? Date: 5 Nov 1996 04:56:00 GMT Organization: AT&T WorldNet Services Lines: 4 Message-ID: <55mhd0$gpk@mtinsc01-mgt.ops.worldnet.att.net> References: <3263EF78.3D54@topaz.microbio.uab.edu> <542agq$p33@winx03.informatik.uni-wuerzburg.de> <54kr3l$82f@info-server.surrey.ac.uk> <54n9td$1n3@sun0.urz.uni-heidelberg.de> <55d4lk$aed@info-server.surrey.ac.uk> NNTP-Posting-Host: 165.238.84.241 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22ATT (Windows; U; 16bit) Xref: biosci bionet.molbio.methds-reagnts:51172 bionet.molbio.proteins:9199 I have used both quartz and disposable, and quatrz bears the bell away as far as reproducibility. Call me old-fashioned, but I would trust a result from a quartz cuvette ove a result from a plastic one anyday. From owner-proteins@net.bio.net Mon Nov 04 22:00:00 1996 Path: biosci!CS.Arizona.EDU!news.Arizona.EDU!hamblin.math.byu.edu!acs2.byu.edu!news.cuny.edu!news.sprintlink.net!news-hub.sprintlink.net!news.sprintlink.net!news-peer.sprintlink.net!arclight.uoregon.edu!usenet.eel.ufl.edu!news.alt.net!newspost1.alt.net!usenet From: belcom@public.bta.net.cn Newsgroups: bionet.molbio.proteins Subject: Need help urgently !!! Date: Tue, 05 Nov 1996 16:37:40 GMT Organization: Altopia Corp. - Affordable Usenet Access - http://www.alt.net Lines: 24 Message-ID: <327f6cfc.6641315@news.alt.net> Reply-To: belcom@public.bta.net.cn X-Newsreader: Forte Agent .99d/32.182 >HELP! HELP! HELP! > >One Chinese lady (49 years old) has been found a special disease which >could not get medicine and treatment in China. This disease happened one in >millions of people. The doctor's dianosis is as following: > >1) Tongue Biopsy: Amyloidosis > >2) Kidney Biopsy: Mild Mesangial proliferation GN > >3) Past history: > > a. Thyroidectomy due to hyperthyroidism for 8 years > > b. Post-operation of carpal tunnel syndrome for 1 year > >We need your help on finding new methods and new medicine to treat this > disease. >Thank you very much. Please help urgently! > >E-mail address: belcom@public.bta.net.cn > From owner-proteins@net.bio.net Mon Nov 04 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.erols.net!news.sprintlink.net!news-peer.sprintlink.net!uunet!in1.uu.net!amgen!usenet From: John Philo Newsgroups: bionet.molbio.proteins Subject: Re: basic molarity/curie question Date: Tue, 05 Nov 1996 13:37:37 -0800 Organization: Amgen Inc. Lines: 25 Message-ID: <327FB3A1.69E1@amgen.com> References: Reply-To: jphilo@amgen.com Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (Win95; I) To: Otter Otter wrote: > > I am trying to figure out the molarity of my reaction. I am using > tritiated s-adenosyl-methionine which has a specific activity of 55.1 > Ci/mmol. I am using 2 micro Ci in my reactions. How many micro moles is > that? Since I am trying to learn how to perform this calculation, can you > please show me how you come up with the molarity. To get the total number of moles you divide the number of curies by the specific activity. To get things in the units you want, first convert the specific activity from 55.1 Ci/mmol to .0551 Ci/micromol and the amount you are using to 2E-6 Ci. Then you have (2E-6 Ci)/(.0551 Ci/micromol) = 3.63E-5 micromol = 3.63E-11 mol To come up with the molarity, you must divide the number of moles (not micromoles) by the volume in which this activity is dispersed, in liters. If, for example, your sample is 100 microliters then the molarity is (3.63E-11 mol) / (1E-4 liter) = 3.63E-7 molar 'Hope this helps. John Philo, Protein Chemistry Amgen Inc., Thousand Oaks, CA jphilo@amgen.com *** Disclaimer: These are the opinions of the poster not Amgen Inc.*** From owner-proteins@net.bio.net Mon Nov 04 22:00:00 1996 Path: biosci!biosci!not-for-mail From: David Stepp Newsgroups: bionet.biology.cardiovascular,bionet.metabolic-reg,bionet.molbio.ageing,bionet.molbio.evolution,bionet.molbio.proteins,bionet.molecules.peptides,bionet.neuroscience Subject: Physiologic Application of Molecular Biology Date: 5 Nov 1996 16:38:08 -0800 Organization: University of Wisconsin, Madison Lines: 24 Sender: biohelp@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Xref: biosci bionet.biology.cardiovascular:1263 bionet.metabolic-reg:869 bionet.molbio.ageing:3035 bionet.molbio.evolution:5287 bionet.molbio.proteins:9202 bionet.molecules.peptides:494 bionet.neuroscience:16569 Many of us spend our days (and nights!) cloning, expressing and/or knocking out genes that interest us. Often times, however, we are too deep in the trees and lose sight of what our gene's function is in the context of a whole animal or even at the organ level. I would be interested to know what level of enthusiasm there is out there for studying the physiology of your favorite gene. If you are overexpressing a transcription factor that regulates cardiac gene expression, for example, what effect does this have on cardiac performance, blood pressure etc.? Granting agencies are asking for more than molecular biology these days; they want relevance to a physiological or pathophysiological process. Within this context, would you be interested in learning physiology techniques (blood pressure measurements, cardiac and smooth contractility, shear stress, electrophysiology etc) in a 2-3 week course as a means of broadening your knowledge base and enhancing your experimental research?? Please forward your thoughts and comments to jmiano@post.its.mcw.edu. Thanks in advance! Joe Miano & David W. Stepp From owner-proteins@net.bio.net Mon Nov 04 22:00:00 1996 Path: biosci!CS.Arizona.EDU!news.Arizona.EDU!hamblin.math.byu.edu!acs2.byu.edu!news.cuny.edu!news.sprintlink.net!news-pull.sprintlink.net!news.sprintlink.net!news-pen-16.sprintlink.net!news.up.net!news.mtu.edu!msunews!otterpop From: otterpop@pilot.msu.edu (Otter) Newsgroups: bionet.molbio.proteins Subject: basic molarity/curie question Date: Mon, 04 Nov 1996 22:48:48 -0500 Organization: Michigan State University Lines: 9 Message-ID: NNTP-Posting-Host: pm166-04.dialip.mich.net Mime-Version: 1.0 Content-Type: text/plain; charset=ISO-8859-1 Content-Transfer-Encoding: 8bit X-MSUnetID: otterpop X-Newsreader: Yet Another NewsWatcher 2.3.1 I am trying to figure out the molarity of my reaction. I am using tritiated s-adenosyl-methionine which has a specific activity of 55.1 Ci/mmol. I am using 2 micro Ci in my reactions. How many micro moles is that? Since I am trying to learn how to perform this calculation, can you please show me how you come up with the molarity. Thanks for your time and help, Otter From owner-proteins@net.bio.net Mon Nov 04 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.erols.net!news.sprintlink.net!news-peer.sprintlink.net!uunet!in3.uu.net!newstf01.news.aol.com!audrey01.news.aol.com!not-for-mail From: danter1@aol.com Newsgroups: bionet.molbio.proteins Subject: Tech/Postdoc Bethesda, MD Date: 6 Nov 1996 02:59:26 GMT Organization: America Online, Inc. (1-800-827-6364) (1.10) Lines: 87 Sender: news@aol.com Message-ID: <19961106030200.WAA16744@ladder01.news.aol.com> NNTP-Posting-Host: ladder01.news-fddi.aol.com I would like to recruit one highly motivated individual to be the first person to work with me in my new research lab as either a technician, postdoc or research assistant. You can have a BS, MS or Ph.D. and the salary will be commensurate with your experience and what I think you can bring to my lab. The salary range is from $22K to $32K and includes benefits i.e. health insurance, you must be a US citizen, and must be willing to work in the Bethesda, MD area, and must be willing to start by 1 Feb. 1997 or sooner. Funding is available for two years and possibly longer. A first-time postdoc would be expected to write postdoctoral fellowships. The perfect individual for this job would have to be competent in molecular biology i.e. in designing and constructing plasmids, making and purifying fusion proteins from bacteria, PCR, site directed mutagenesis and random mutagenesis methods, doing plasmid preps, etc. He/she would also have experience running protein and agarose gels, western blotting, working SAFELY with radioisotopes, and performing immunofluorescence experiments. Experience with EM a plus. He/she would have experience working with Saccharomyces cerevisiae i.e. growing strains in liquid and on solid medium, doing gene disruptions, tetrad dissection, and experience with 2-hybrid or other genetic screening methods would be a plus. You have to be willing to work with rabbits and mice (these will not be your co-workers and the lab doesn't have mice, I think?). The fusion proteins will be used to make polyclonal antibodies in rabbits and I will also want to "raise" ascites from monoclonal hybridoma cell lines in mice. The person would also be familiar with the Macintosh OS and be able to learn Filemaker Pro, Nisuswriter, DNAStar software, Photoshop, Canvas, and Cricketgraph. The candidate need only have a subset of these skills and no one is expected to have all of them. One project which you may work on involves defining the function of a new protein required for exocytosis in Saccharomyces cerevisiae. If you work on this project, you would be expected to epitope tag the protein for immunoprecipitation experiments and for immunofluorescence localization, make temperature sensitive (or other conditional mutant) alleles of this gene, and look for genetic interactions between your mutants and other late-acting sec mutants. You would make fusion proteins to make polyclonal antibodies in rabbits and do genetic screens, either 2-hybrid or suppression screens of your mutants, to look for interacting gene products. A postdoc would be encouraged to develop his own project consistent with the goals of my lab. I am Daniel TerBush and I have been recently appointed Assistant Professor in the Department of Biochemistry at the F. Edward Hebert School of Medicine at the Uniformed Services University of the Health Sciences in the Bethesda, MD. My appointment started 1 November 1996. If you respond, I will send you a follow-up E-mail with more information about USUHS. I did my Ph.D. work in Dr. Ronald Holz's lab at the University of Michigan studying exocytosis in bovine adrenal chromaffin cells. I have recently completed a postdoc in Peter Novick's laboratory at Yale University where I have identified a multiprotein complex required for exocytosis in yeast (TerBush and Novick, 1995, July, JCB 130: 299-312). To respond to this advertisement, send a cover letter and C.V. to danter1@aol.com. In the cover letter please confirm in the first paragraph that you are a US citizen (unfortunately I can't fund a non-US citizen), that working in the Bethesda, MD area is OK and that you can start by 1 Feb. 1997. Tell me in a general way about your work experience and cover anything that isn't necessarily easy to fit into a C.V. (For instance, I started a Macintosh computer repair company as a graduate student to make extra money but this is not something that fits nicely on a scientific C.V.). Also, tell me what level of education you have BS, MS or Ph.D. and the type of position you are looking for. Depending on the number of responses I receive, I may not reply right away but I will try to respond to all applications. You will have to be willing to come to me to be interviewed since I do not have funding to pay for your visit. Please keep this in mind when if you respond to this listing. Finally, I just wanted to comment on why the "position title" is not more focused, i.e. postdoc only, or technician only, etc. Since you are the first person I am hiring, I would like someone with a lot of technical skill i.e. a postdoc or experienced technician. But I am willing to consider someone less experienced with a burning desire to learn and do research and willing to commit at least two years of time to my lab. If you are applying as a postdoc, currently I do not have a technician. I expect to have additional funding in April which will allow me to hire one, but until then (or if the funding falls through) you will have to be willing to share with me and any graduate students the "lab chores". I have the attitude that if something needs to get done then I'll do it myself and postdoctoral candidates and research assistants should share this attitude. If you are applying as a technician, you will be getting stuck with a lot of the "lab chores" as part of your job responsibilities. However, as money becomes available I'll try to hire a lab aide to make solutions and pour plates to give you more time to do science. From owner-proteins@net.bio.net Tue Nov 05 22:00:00 1996 Path: biosci!botany.uq.edu.au!J.Marcus From: J.Marcus@botany.uq.edu.au ("Marcus, Dr J.") Newsgroups: bionet.molbio.proteins Subject: Re: Sensitive peptide assay? Date: 6 Nov 1996 14:37:27 -0800 Organization: Dept of Botany, Univ of Qld Lines: 55 Sender: daemon@net.bio.net Distribution: world Message-ID: Reply-To: Marcus@tpp.uq.edu.au NNTP-Posting-Host: net.bio.net Hi Cornelius, You wrote: > I wonder whether there is a sensitive peptide assay being > able to detect between 10 pmol and 1 nmol of a peptide. I > was thinking about UV detection at 214 nm but I think that > the extinction coefficient is not high enough (when I > assume that it's 1000 per peptide bond -- which is just a > wild guess -- I would be able to detect 1 nmol / 100 ul). > > Any ideas? Do you need to know the concentration or just find out if your peptide is there? Assuming you want to quantitate, here are some thougths that may or may not be useful... 1) A very good method for accurate quantitation is acid hydrolysis followed by amino acid analysis. 2) Perhaps a less time consuming way would be to run your peptide on an RP-HPLC column and look at 214nm absorbance. 100 microgram/ml concentration should give an absorbance of around 1 at 214nm. Peaks of 0.01 Absorbance should easily be detectable; that translates to 1 microgram/ml concentration. If your protein peak is about 0.5 ml in volume then you would have 0.5 micrograms of protein. So, if your protein is say 10,000 Da you would be detecting 50 pmols of protein. If your protein was 100,000 Da your would be detecting 5 pmol. Not bad if you ask me. The nice thing here is that you can get your protein back. 3) If you have enough sample and your don't mind using some of it up you can do a micro BCA assay. Pierce sell the reagents but the mix is easy to make yourself. I hope that is useful. Sincerely, John Marcus _________________________________________________________ John Marcus Marcus@tpp.uq.edu.au (Dr J.Marcus) Cooperative Research Centre for Tropical Plant Pathology 5th Level John Hines Building University of Queensland St. Lucia, QLD 4072 AUSTRALIA Fax: 61-7-3365-4771 Phone: 61-7-3365-4764 From owner-proteins@net.bio.net Tue Nov 05 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.erols.net!www.nntp.primenet.com!nntp.primenet.com!dispatch.news.demon.net!demon!sally.ogs.co.uk From: Sally Prime Newsgroups: bionet.molbio.proteins Subject: Re: 2D PAGE quantitation software Date: Wed, 06 Nov 1996 19:05:57 +0000 Organization: Oxford Glycosciences Lines: 16 Message-ID: <3280E195.58F6@ogs.co.uk> References: <3277E4E7.3E5B@durham.ac.uk> Reply-To: sallyp@ogs.co.uk NNTP-Posting-Host: sally.ogs.co.uk X-NNTP-Posting-Host: sally.ogs.co.uk X-Mailer: Mozilla 3.0 (Win95; I) MIME-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit > Is anyone aware of any cheap, or better still free software for > quantifying and comparing 2D PAGE protein patterns? WWW address, E-mail > number or other contact address would be useful. I am not aware of any cheap 2D PAGE quantitation and matching software, possibly due to the fact that it is complex and expensive software to produce. For a serious package at lowish cost, try the Phoretix 2D package, which runs on NT. -- -------------------------------------------- Sally Prime Ph.D., sallyp@ogs.co.uk Oxford Glycosciences, T (+44) 1235 543225 Unit 4 Hitching Court, F (+44) 1235 554701 Blacklands Way, Abingdon, Oxon, OX14 1RG From owner-proteins@net.bio.net Tue Nov 05 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.erols.net!newspump.sol.net!news-peer.gsl.net!news.gsl.net!news-lond.gsl.net!news.gsl.net!netcom.net.uk!nntpfeed.doc.ic.ac.uk!sunsite.doc.ic.ac.uk!sunews!suma3!sapgdfel From: Ian Goodfellow Newsgroups: bionet.molbio.proteins Subject: Re: Convert protein sequence to DNA Date: Mon, 4 Nov 1996 11:52:05 +0000 Organization: University of Reading, U.K. Lines: 27 Message-ID: References: <98E7055D3A@medlib2.kku.ac.th> NNTP-Posting-Host: suma3-e2.reading.ac.uk Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII To: SURASAK WONGRATANACHEEWIN In-Reply-To: <98E7055D3A@medlib2.kku.ac.th> On 23 Oct 1996, SURASAK WONGRATANACHEEWIN wrote: > Hi Netters, > > Anybody there can tell me how to convert the protein sequences > into correct DNA sequences (DNA of pseudomonas)? > > Thanks for help. > > Surasak HI, the ideal program is Backtranslate from the GCG packages or is it egcg - anyway one of them is ideal. You can make a codon usage chart from Pseudo. if there isn't one available and the program will use that to predict the most likely sequence. If you want any more details - just email me directly Regards Ian Ian Goodfellow I.G.Goodfellow@reading.ac.uk From owner-proteins@net.bio.net Tue Nov 05 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!news.sgi.com!www.nntp.primenet.com!nntp.primenet.com!dispatch.news.demon.net!demon!netcom.net.uk!news.u-net.com!yama.mcc.ac.uk!liv!rgu.ac.uk!news From: 933467u@app_science.rgu.ac.uk Newsgroups: bionet.molbio.proteins Subject: Cost of Epogen Production / Treatment. Date: 1 Nov 1996 21:01:02 GMT Organization: Computer Sevices Unit, The Robet Gordon University, Aberdeen, Scotland Lines: 14 Message-ID: <55doee$smj@news.rgu.ac.uk> NNTP-Posting-Host: novixint.rgu.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Windows; I; 16bit) To anyone who can help me, I'm a forth year student at the Robert Gordon Universtiy in Aberdeen, Scotland. I'm desperately seeking information on some of the economic implications of synthetically produced Erythropoietin (AKA EPO, Epogen, etc.). Can anyone tell me how much it would cost to produce say, 100ml of the stuff? How much would it cost to treat a patient with EPO for a month? If no-one can answer my questions directly, I'd appreciate it if someone could suggest where I might find the information I need. Thanks for your time folks, Alan Mercer. From owner-proteins@net.bio.net Tue Nov 05 22:00:00 1996 Path: biosci!news.alaska.edu!newsfeed.acns.nwu.edu!news.cc.uic.edu!usenet From: "Edward B. Arias" Newsgroups: bionet.molbio.proteins Subject: Need help with GST fusion protein purification Date: Sun, 03 Nov 1996 19:15:26 -0500 Organization: University of Illinois at Chicago Lines: 19 Message-ID: <327D359E.3132@uic.edu> Reply-To: Dept., of, Physiology, &, Biophysics NNTP-Posting-Host: k034.phyb.uic.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0 (Win95; U) I'm having a problem purifying a 104 kDa GST fusion protein with the Pharmacia kit. I've tried most of the trouble shooting tips to no avail. One ref in the manual takes about losing GST fusion protein:glutathione sepharose beads affinity if your fusion protein is 80+ kDa. I'm a grad student and this is the last thing I must do to graduate...that is purify out some of my protein which is 75 kDa (+ the 29 kDa GST = 104 kDa fusion protein which gets bound to the beads). Any help or comments with those using the GST fusion protein kit from Pharmacia with similar problems is greatly appreciated. -- Edward B. Arias - "Just Say Know" UIC Dept of Physiology & Biophysics 835 S. Wolcott Ave. Chicago, IL 60612-7342 Tel: 312.996.4161 FAX: 312.996.1414 E-mail: earias1@uic.edu WWW: http://www.uic.edu/~earias1 From owner-proteins@net.bio.net Tue Nov 05 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!www.nntp.primenet.com!nntp.primenet.com!nntp.uio.no!news.nacamar.de!fu-berlin.de!zrz.TU-Berlin.DE!news-ber1.dfn.de!news-ham1.dfn.de!news-han1.dfn.de!news-koe1.dfn.de!news-kar1.dfn.de!news-stu1.dfn.de!news-mue1.dfn.de!news-nue1.dfn.de!uni-erlangen.de!winx03!wpxx02!not-for-mail From: krasel@wpxx02.toxi.uni-wuerzburg.de (Cornelius Krasel) Newsgroups: bionet.molbio.proteins Subject: Sensitive peptide assay? Date: 6 Nov 1996 17:55:34 GMT Organization: University of Wuerzburg, Germany Lines: 14 Message-ID: <55qjem$kt0@winx03.informatik.uni-wuerzburg.de> NNTP-Posting-Host: wpxx02.toxi.uni-wuerzburg.de X-Newsreader: TIN [UNIX 1.3 950824BETA PL0] I wonder whether there is a sensitive peptide assay being able to detect between 10 pmol and 1 nmol of a peptide. I was thinking about UV detection at 214 nm but I think that the extinction coefficient is not high enough (when I assume that it's 1000 per peptide bond -- which is just a wild guess -- I would be able to detect 1 nmol / 100 ul). Any ideas? --Cornelius. -- /* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */ /* D-97078 Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de SP3 */ /* "Science is the game we play with God to find out what His rules are." */ From owner-proteins@net.bio.net Tue Nov 05 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.erols.net!netnews.com!ix.netcom.com!news From: bellew@ix.netcom.com (Leo Bellew) Newsgroups: bionet.biology.cardiovascular,bionet.metabolic-reg,bionet.molbio.ageing,bionet.molbio.evolution,bionet.molbio.proteins,bionet.molecules.peptides,bionet.neuroscience Subject: Re: Physiologic Application of Molecular Biology Date: Thu, 07 Nov 1996 02:52:47 GMT Organization: Netcom Lines: 43 Message-ID: <32824be3.49540134@nntp.ix.netcom.com> References: Reply-To: bellew@ix.netcom.com NNTP-Posting-Host: phi-pa11-56.ix.netcom.com Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-NETCOM-Date: Wed Nov 06 6:56:03 PM PST 1996 X-Newsreader: Forte Agent .99f/32.299 Xref: biosci bionet.biology.cardiovascular:1267 bionet.metabolic-reg:870 bionet.molbio.ageing:3037 bionet.molbio.evolution:5290 bionet.molbio.proteins:9210 bionet.molecules.peptides:497 bionet.neuroscience:16596 I have been the architect or chief engineer on many high-tech computer software projects over the last 30 years where we have developed computer models of reality in many different situations utilizing engineering devices borrowed from areas such as artificial language and automata theory. I am very interested in the potential for utilizing these technologies in mapping the human genome to physiological function. In your note, you seem to imply that the relationships are known, at least in respect to the physiology techniques the course would be devoted to. Can you tell me if the problem of relating the human genome to physiological function is largely solved, and what formal mental, mathematical or simulative models provide the loci for that solution. Leo Bellew On 5 Nov 1996 16:38:08 -0800, David Stepp wrote: > >Many of us spend our days (and nights!) cloning, expressing and/or >knocking out genes that interest us. Often times, however, we are too >deep in the trees and lose sight of what our gene's function is in the >context of a whole animal or even at the organ level. I would be >interested to know what level of enthusiasm there is out there for >studying the physiology of your favorite gene. If you are overexpressing >a transcription factor that regulates cardiac gene expression, for >example, what effect does this have on cardiac performance, blood >pressure etc.? > >Granting agencies are asking for more than molecular biology these days; >they want relevance to a physiological or pathophysiological process. >Within this context, would you be interested in learning physiology >techniques (blood pressure measurements, cardiac and smooth contractility, >shear stress, electrophysiology etc) in a 2-3 week course as a means of >broadening your knowledge base and enhancing your experimental research?? > >Please forward your thoughts and comments to jmiano@post.its.mcw.edu. > >Thanks in advance! > > >Joe Miano & David W. Stepp From owner-proteins@net.bio.net Wed Nov 06 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!usenet.eel.ufl.edu!warwick!leicester!usenet From: "Dr E. Buxbaum" Newsgroups: bionet.molbio.proteins Subject: Re: basic molarity/curie question Date: 6 Nov 1996 19:24:46 GMT Organization: University of Leicester, UK (PCFS User) Lines: 21 Message-ID: <55qolu$q8o@falcon.le.ac.uk> References: NNTP-Posting-Host: pc47.msb.le.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) otterpop@pilot.msu.edu (Otter) wrote: >I am trying to figure out the molarity of my reaction. I am using >tritiated s-adenosyl-methionine which has a specific activity of 55.1 >Ci/mmol. I am using 2 micro Ci in my reactions. How many micro moles is >that? Since I am trying to learn how to perform this calculation, can you >please show me how you come up with the molarity. > >Thanks for your time and help, > >Otter if 1 mmol has 55.1 Ci, then 2 uCi correspond to 0.000002 Ci * 0.001 mol / 55.1 Ci = 36.3 pmol. Divide this by the volume of your assay (in liter) and you get your concentration in pmol/l or pM. The trick is always to work in basal units. Write down all walues with their units and check, that the result will have the correct unit. Then do the calculation itself. By the way, strictly speaking you should be working with Bq, not Ci. From owner-proteins@net.bio.net Wed Nov 06 22:00:00 1996 Path: biosci!daresbury!nntp-trd.UNINETT.no!sn.no!nntp.uio.no!nntp.zit.th-darmstadt.de!fu-berlin.de!news.belwue.de!news.uni-stuttgart.de!news From: Joerg Freigang Newsgroups: bionet.molbio.proteins Subject: Refolding problem Date: Thu, 07 Nov 1996 16:26:17 +0100 Organization: Universitaet Konstanz Lines: 14 Message-ID: <3281FF98.41C6@uni-konstanz.de> NNTP-Posting-Host: loop3.biologie.uni-konstanz.de Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.02 (X11; I; IRIX 5.3 IP9) I am trying to refold a protein belonging to the IGSF from inclusion bodies. The protein contains two disulfide bonds and a his-tag. I tried the refolding procedure developed by R. Rudolph for Fab-fragments as well as some other standart refolding procedures, but I couldn't get any detectable amounts of monodispers protein. Any suggestions what the problem could be and what else should be tried? Even 1 % of correctly folded protein would be enough. Joerg --- Joerg Freigang email: Joerg.Freigang@uni-konstanz.de Tel: +49 7531 88 2211 Fakultaet fuer Biologie, Universitaet Konstanz Postfach 5560, M 656, D-78434 Konstanz, Germany From owner-proteins@net.bio.net Wed Nov 06 22:00:00 1996 Path: biosci!rutgers!news.sgi.com!howland.erols.net!newsfeed.internetmci.com!in2.uu.net!rcogate.rco.qc.ca!altitude!usenet From: "Achim Recktenwald, PhD" Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: Best way to clean cuvettes? Date: Thu, 07 Nov 1996 07:36:22 -0500 Organization: IBEX Technologies, Inc., Biochemistry, 5485 Pare, Montreal, PQ, H4P 1P7, Canada Lines: 12 Message-ID: <3281D7C5.1699@ibex.ca> References: <3263EF78.3D54@topaz.microbio.uab.edu> <542agq$p33@winx03.informatik.uni-wuerzburg.de> <54kr3l$82f@info-server.surrey.ac.uk> <54n9td$1n3@sun0.urz.uni-heidelberg.de> <55d4lk$aed@info-server.surrey.ac.uk> <55mhd0$gpk@mtinsc01-mgt.ops.worldnet.att.net> NNTP-Posting-Host: ibex.hip.cam.org Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0 (Macintosh; I; PPC) To: clkstanley@postoffice.worldnet.att.net Xref: biosci bionet.molbio.methds-reagnts:51273 bionet.molbio.proteins:9215 clkstanley@postoffice.worldnet.att.net wrote: > > I have used both quartz and disposable, and quatrz bears the bell away as > far as reproducibility. Call me old-fashioned, but I would trust a result > from a quartz cuvette ove a result from a plastic one anyday. I agree, if you want to do serious spectroscopy, esp. in the lower wavelengths, you should use quartz cuvettes. For the range >400nm it might not be that necessary. Achim From owner-proteins@net.bio.net Wed Nov 06 22:00:00 1996 Path: biosci!CS.SANDIA.GOV!scistra From: scistra@CS.SANDIA.GOV (Sorin C. Istrail) Newsgroups: bionet.molbio.proteins Subject: RECOMB97: Hotel Reservation and Registration Information Date: 7 Nov 1996 15:39:59 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 114 Sender: daemon@net.bio.net Distribution: world Message-ID: <199611072333.QAA14895@frodo2.cs.sandia.gov> NNTP-Posting-Host: net.bio.net Please call the ElDorado hotel and make your reservation AS SOON AS POSSIBLE. This is a four-star and four-diamond hotel and we have a limited number of rooms avaiblable. ElDorado will direct the overflow to another hotel. FIRST ANNUAL INTERNATIONAL CONFERENCE ON COMPUTATIONAL MOLECULAR BIOLOGY RECOMB 97 http://www.cs.sandia.gov/recomb97 January 20-23, 1997 Eldorado Hotel Santa Fe, New Mexico Sponsored by SIGACT with support from SLOAN Foundation US Department of Energy ELDORADO HOTEL RESERVATIONS INFORMATION **** A four-star, four diamond-hotel **** Phones: 1-800 955-4455 505-988-4455 Fax: 505-995-4544 (reservations only) Webpage: http://www.eldoradohotel.com Email: rez@eldoradohotel.com Room Rates: Single/double $85 Sunday(1/19), Monday(1/20), Tuesday(1/21), Wednesday(1/22), Thursday(1/23), Friday(1/24) Saturday (1/25) Single/Double $135 Saturday(1/18) (For comparison, if reservations are not made at the RECOMB97 group rate, the average single/double rate would be $144 for these types of rooms.) RECOMB97 REGISTRATION INFORMATION A registration form can be obtained from the conference web page http://www.cs.sandia.gov/recomb97 Advanced Registration ACM Member Non-ACM Member $285 $360 Late/On-site Registration (After December 12) ACM Member Non-ACM Member $360 $435 Full time student Advanced Registration $160 Late/On-site Registration (After Decemebr 12) $210 Registration fee includes: one copy of the proceedings, four lunches, eight coffee breaks, and a banquet ticket. Student registration fee includes all of the above except the banquet ticket. For more information check out the conference web page http://www.cs.sandia.gov/recomb97 or contact Sorin Istrail RECOMB97 Conference Chair Sandia National Laboratories Massively Parallel Computing Research Laboratory MS 1110 Albuquerque, NM 87185-1110 Phone: (505) 845-7612 Fax : (505) 845-7442 Secretary: (505) 845-7432 Email: scistra@cs.sandia.gov www : http://www.cs.sandia.gov/~scistra From owner-proteins@net.bio.net Thu Nov 07 22:00:00 1996 Path: biosci!iata.csic.es!poseva From: poseva@iata.csic.es (Eva Dupille) Newsgroups: bionet.molbio.proteins Subject: TCA precipitation Date: 8 Nov 1996 01:11:23 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 11 Sender: daemon@net.bio.net Distribution: world Message-ID: <01BBCD5C.B2A71400@alzacarias.iata.csic.es> NNTP-Posting-Host: net.bio.net Hello, I have a protein problem precipitation. I did a TCA precipitation, 15 % = for 1 h at 4 C, and I got, nothing at all. Sure that I have proteins, I get enzyme activity and with biorad, before = precipitation, I have around 10 micrograms/ml. Is it a problem of temperature or incubation time ?? Please, if you have some comments, let me know. That s boring to lose so = many time in protein purification, to lose all the samples for a protein = precipitation problem .. Thanks in advance, sorry for the approximative english. Eve From owner-proteins@net.bio.net Thu Nov 07 22:00:00 1996 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed1.bbnplanet.com!news.bbnplanet.com!cpk-news-hub1.bbnplanet.com!www.nntp.primenet.com!nntp.primenet.com!usenet.eel.ufl.edu!warwick!bignews.shef.ac.uk!usenet From: PMA95SSK@shef.ac.uk (Fade To Black) Newsgroups: bionet.molbio.proteins Subject: REQ: Amonia disease ( sad but true ) Date: 8 Nov 1996 23:35:46 GMT Organization: Nothing man Lines: 64 Message-ID: <560g4i$ijg@bignews.shef.ac.uk> Reply-To: PMA95SSK@shef.ac.uk ( Fade To Black) NNTP-Posting-Host: pc021049.shef.ac.uk Mime-Version: 1.0 Content-Type: Text/Plain; charset=US-ASCII X-Newsreader: WinVN 0.99.7 Hello, I would like to know about 'Amonia disease' known as 'body odor. Since I have a serious problem with that disease, I have to know about this as possible as I can. What an annoying disease I have! My life is getting worse day by day. I just can't stand this feeling anymore. I need to define things stighten out my brians otherwise I'll go insane. Of course,Sure I got a pride. I 've been hurt by many people . They just can't stand myself. If they are jealous of me in many ways, it's so easy for them to make fun of me in public. I was forced to sacrifice lots of things due to that disease. I'm not fat. I do exercise everyday . ( Running , playing sports , going to gym ) I just always had to face the fact. Time is the time, I have a right to live like others. Obviously, I went to see the doctor about my rare but awful disease. Initially, I was branded as ' paranoid ' by him. Believe me, I had to persuade him in 10 minteus. It's weird, cos doctor's are used to patient's various odor. So basically, they don't have a sensitive nose as others do. The truth is not to be denied. The truth surely pays. I fully understand my GP's situation. My case is very rare. I'm not intelligent or talented on particular thing. But I 'm very careful & patient man. In fact, it's a sad thing to know that my life is going to the edge. I have always believed that the search for the meaning of life has been a futile one. It's a cliche, but the meaning of life is simply to enjoy every minute, every second, that life throws at me. I became 21 recently. Life is really crap on the work front. But I 'm thankful to the people who were always cheering me up. Yeap , people have a tendency to make fun of other's weakness , sadly I am one of those. The guilty surely pays. I did my best. I just can't help it. It's natural. Well, I do take a shower or bath as many times as I can. Doesn't work. I invested lots of money for perfume. It wasn't helpful neither. Do I need a diet? Well. Thanks a lot. Yours sincerely. P.S This is serious. Believe or not, my account ( pc novell ) was disabled by the supervisor , cos some people had compalined about my article last june. -- __ _ _ __| | | / \ | |[] - - - - - - - - - - - - - - - - [] \__ | | / | \ [] -*REMEMBER:Death is nature's way[] \ _ |\ [] -* to tell you to slow down. [] _ ___/ |_ / \ _ _|[] -* [] Stan 's web page : http://www.shef.ac.uk/students/pm/pma95ssk ======= E-Mail :PMA95SSK@shef.ac.uk ====== __~@ __~@ __~@ Run ----- __~@ _-\__<,_-\__<, _-\__<, for your life... ----- _-\__<, (*)/' ((*)/' (*) (*)/' (*) ----- (*)/' (*) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ * This sadness must go on - From the late Van Gough's last word ** From owner-proteins@net.bio.net Thu Nov 07 22:00:00 1996 Path: biosci!MANI.CBS.UMN.EDU!npv From: npv@MANI.CBS.UMN.EDU (Nora Plesofsky-Vig) Newsgroups: bionet.molbio.proteins Subject: re: TCA ppt Date: 8 Nov 1996 12:16:54 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 7 Sender: daemon@net.bio.net Distribution: world Message-ID: <9611082017.AA02114@mani.cbs.umn.edu> Reply-To: nora@biosci.cbs.umn.edu NNTP-Posting-Host: net.bio.net I have had trouble in the past with TCA precipitation, due to not removing enough of the TCA after the precipitation. I have found that washing three times with cold acetone after TCA precipitation gave fine yield of protein. Nora Plesofsky-Vig From owner-proteins@net.bio.net Thu Nov 07 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.erols.net!feed1.news.erols.com!hunter.premier.net!hammer.uoregon.edu!csulb.edu!not-for-mail From: cohlberg@csulb.edu (Jeff Cohlberg) Newsgroups: bionet.molbio.proteins Subject: Re: TCA precipitation Date: 8 Nov 1996 17:35:52 GMT Organization: Cal State Long Beach Lines: 14 Distribution: world Message-ID: <55vr1o$ku0@hatathli.csulb.edu> References: <01BBCD5C.B2A71400@alzacarias.iata.csic.es> NNTP-Posting-Host: skimmer.csulb.edu X-Newsreader: TIN [UNIX 1.3 950824BETA PL0] Eva Dupille (poseva@iata.csic.es) wrote: : Hello, : I have a protein problem precipitation. I did a TCA precipitation, 15 % = : for 1 h at 4 C, and I got, nothing at all. The efficiency of TCA precipitation often depends on salt concentration. If you're trying to precipitate from a dilute buffer, try adding 0.2 M NaCl before adding the TCA. -- Jeffrey A. Cohlberg, Professor Department of Chemistry and Biochemistry California State University, Long Beach 1250 Bellflower Boulevard, Long Beach, CA 90840 Phone: (310) 985-4944 FAX: (310) 985-8557 E-mail: cohlberg@csulb.edu From owner-proteins@net.bio.net Thu Nov 07 22:00:00 1996 Path: biosci!daresbury!nntp-trd.UNINETT.no!nntp.uio.no!www.nntp.primenet.com!nntp.primenet.com!howland.erols.net!math.ohio-state.edu!jussieu.fr!oleane!plug.news.pipex.net!pipex!hole.news.pipex.net!pipex!bowl.news.pipex.net!pipex!news00.sunet.se!sunic!news99.sunet.se!umdac!news From: "Pär Wästerby" Newsgroups: bionet.cellbiol,bionet.general,bionet.info-theory,bionet.metabolic-reg,bionet.molbio.proteins,bionet.molecules.peptides,bionet.structural-nmr,sci.med.pharmacy Subject: A novel Progesterone Researsh Project Date: 8 Nov 1996 13:40:17 GMT Organization: Fysikalisk kemi Lines: 7 Message-ID: <01bbcd58$e8fc8520$3024ef82@pepsi> NNTP-Posting-Host: pepsi.chem.umu.se X-Newsreader: Microsoft Internet News 4.70.1155 Xref: biosci bionet.cellbiol:5894 bionet.general:23924 bionet.info-theory:4368 bionet.metabolic-reg:871 bionet.molbio.proteins:9221 bionet.molecules.peptides:501 bionet.structural-nmr:1598 sci.med.pharmacy:36113 Dear all, we would really apprecieate if there is someone that could give us information about Progesterone and its metabolites. Since we are going to do chromotographic separation, we are interested in physical data, structure data and acid - base properties (pKa values). It would also be very helpful to find a review aritcle in the area. From owner-proteins@net.bio.net Thu Nov 07 22:00:00 1996 Path: biosci!bloom-beacon.mit.edu!howland.erols.net!EU.net!usenet2.news.uk.psi.net!uknet!usenet1.news.uk.psi.net!uknet!uknet!bhamcs!news.ox.ac.uk!is.bbsrc.ac.uk!news From: Mick Partis Newsgroups: bionet.molbio.proteins Subject: Re: TCA precipitation Date: 8 Nov 1996 10:53:07 GMT Organization: Horticulture Research International Lines: 27 Message-ID: <55v3ej$ius@is.bbsrc.ac.uk> References: <01BBCD5C.B2A71400@alzacarias.iata.csic.es> NNTP-Posting-Host: pc0233.hriw.bbsrc.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Windows; I; 16bit) poseva@iata.csic.es (Eva Dupille) wrote: >Hello, >I have a protein problem precipitation. I did a TCA precipitation, 15 % >for 1 h at 4 C, and I got, nothing at all. >Sure that I have proteins, I get enzyme activity and with biorad, before >precipitation, I have around 10 micrograms/ml. >Is it a problem of temperature or incubation time ?? >Please, if you have some comments, let me know. That s boring to lose so >many time in protein purification, to lose all the samples for a protein >precipitation problem .. >Thanks in advance, sorry for the approximative english. >Eve You don't say why you want to precipitate your protein: if you are running assays and just want to get rid of the protein then 15% TCA should inactivate. If you need a precipitate then why not add a carrier protein? Adding some serum albumin (or any cheap, pure protein) at about 1 mg/ml before adding your TCA and your protein should co-precipitate. If you are precpitating prior to running SDS-PAGE, just make sure the MW of the carrier protein is different from that of your protein. Mick -- mick.partis@hri.ac.uk Horticulture Research International http://www.geocities.com/CapeCanaveral/1957/ From owner-proteins@net.bio.net Thu Nov 07 22:00:00 1996 Path: biosci!daresbury!nntp-trd.UNINETT.no!news-stkh.gsl.net!news.gsl.net!eru.mt.luth.se!www.nntp.primenet.com!nntp.primenet.com!news.mathworks.com!enews.sgi.com!news-feed.inet.tele.dk!cph-1.news.DK.net!dkuug!dknet!funny.bahnhof.se!seunet!news2.swip.net!mn6.swip.net!news00.sunet.se!sunic!news99.sunet.se!umdac!news From: "Pär Wästerby" Newsgroups: bionet.general,bionet.info-theory,bionet.metabolic-reg,bionet.molbio.proteins,bionet.molecules.peptides,sci.med.pharmacy Subject: A novel Progesterone Researsh Project Date: 8 Nov 1996 13:52:12 GMT Organization: Fysikalisk kemi Lines: 7 Message-ID: <01bbcd7b$e9d3bd60$3024ef82@pepsi> NNTP-Posting-Host: pepsi.chem.umu.se X-Newsreader: Microsoft Internet News 4.70.1155 Xref: biosci bionet.general:23937 bionet.info-theory:4372 bionet.metabolic-reg:873 bionet.molbio.proteins:9226 bionet.molecules.peptides:504 sci.med.pharmacy:36130 Dear all, we would really apprecieate if there is someone that could give us information about Progesterone and its metabolites. Since we are going to do chromotographic separation, we are interested in physical data, structure data and acid - base properties (pKa values). It would also be very helpful to find a review aritcle in the area. From owner-proteins@net.bio.net Fri Nov 08 22:00:00 1996 Path: biosci!daresbury!lyra.csx.cam.ac.uk!uknet!usenet2.news.uk.psi.net!uknet!usenet1.news.uk.psi.net!uknet!dispatch.news.demon.net!demon!www.nntp.primenet.com!nntp.primenet.com!feed1.news.erols.com!howland.erols.net!agate!ihnp4.ucsd.edu!news1.ucsd.edu!usenet From: Dmitry Beransky Newsgroups: bionet.molbio.proteins Subject: PDB parsing [Q] Date: Sat, 09 Nov 1996 14:21:44 -0800 Organization: UCSD Lines: 25 Message-ID: <328503F4.BE0@ucsd.edu> Reply-To: dberansky@ucsd.edu NNTP-Posting-Host: bio-york120.ucsd.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0 (Macintosh; I; PPC) Hello all, I'm not sure if this is the right newsgroup to post such a message, but it is the closest I could find. I need to write a program that among other things parses PDB files. I'm a programmer and I've never before dealt with PDB files. I know that there are currently two formats in which , PDB files can be found: v.1 and v.2. My question is this: is it possible to tell the difference between the two formats programmatically? If there is another place more suited to the nature of my question, do let me know. Thanks in advance ==================================================== Dmitry Beransky University of California, San Diego Department of Computer Science and Engineering Multimedia Interactive Learning Laboratory (MILL)|Tel. 619/534-8560 Department of Biology, UC San Diego |Fax 619/534-1835 Web: http://millftp.ucsd.edu/ From owner-proteins@net.bio.net Fri Nov 08 22:00:00 1996 Path: biosci!daresbury!nntp-trd.UNINETT.no!news-stkh.gsl.net!news.gsl.net!news-peer.gsl.net!news.gsl.net!www.nntp.primenet.com!nntp.primenet.com!uwm.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!daemon From: klenchin@facstaff.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins Subject: Re: Big Problems with Novagen´s HisTag Protein Purification Date: Sat, 09 Nov 96 20:44:48 GMT Organization: UW-Madison Lines: 25 Message-ID: <562n1r$1s10@news.doit.wisc.edu> References: <01bbce57$8fc130a0$09019386@Schmidt.rz.ruhr-uni-bochum.de> NNTP-Posting-Host: f182-120.net.wisc.edu Mime-Version: 1.0 Content-Type: text/plain; charset=KOI8-R Content-Transfer-Encoding: 8bit X-No-Archive: Yes In article <01bbce57$8fc130a0$09019386@Schmidt.rz.ruhr-uni-bochum.de>, "Thorsten Schmidt" wrote: #Dear Sir or Madam! # #I have big Problems with Novagen`s HisTag Protein Purification-Kit. #One should get 50-100 mg total protein after cell lysis and -after #purification- 20 mg target protein from one 100 ml culture. #I followed strikly Novagen´s purification protocol but the problems begin #with the #total protein. Instead of the predicted 50-100 mg, I get only 2 (!). Did you have 0.5-1 mg/ml to start with? Not all proteins are expressed in soluble form to this level. #The second problem is the colomn chromatographie: .. #How can I increase the column´s flow rate? 1) use "real" chromatography with peristaltic pump 2) connect tubing to the outlet of you gravity column; the lenth of tubing will control flow rate 3) [maybe] dilute you lysate - Dima From owner-proteins@net.bio.net Fri Nov 08 22:00:00 1996 Path: biosci!bloom-beacon.mit.edu!panix!feed1.news.erols.com!uunet!in1.uu.net!fu-berlin.de!news-ber1.dfn.de!news-ham1.dfn.de!news-han1.dfn.de!news-koe1.dfn.de!news.ruhr-uni-bochum.de!usenet From: "Thorsten Schmidt" Newsgroups: bionet.molbio.proteins Subject: Big Problems with Novagen´s HisTag Protein Purification Date: 9 Nov 1996 16:03:40 GMT Organization: Ruhr-Universitaet Bochum, Rechenzentrum Lines: 48 Message-ID: <01bbce57$8fc130a0$09019386@Schmidt.rz.ruhr-uni-bochum.de> NNTP-Posting-Host: dialppp-9.rz.ruhr-uni-bochum.de X-Newsreader: Microsoft Internet News 4.70.1155 Dear Sir or Madam! I have big Problems with Novagen`s HisTag Protein Purification-Kit. I use the pET30-Vector which allows translation of my target protein containing 6 His-residues. The target protein expression is inhibited during cell growth and induced by IPTG addition. The 6 Histidines are used for protein-purification: The Histidines bind to a "His bind resin" carrying Ni ions. One should get 50-100 mg total protein after cell lysis and -after purification- 20 mg target protein from one 100 ml culture. But which differences between theory and my experience!!! I followed strikly Novagen´s purification protocol but the problems begin with the total protein. Instead of the predicted 50-100 mg, I get only 2 (!). But there might be no fault possible: One just harvests the cells by centrifugation, resuspends the cells in a buffer (containing 5 mM imidazol, 0,5 M NaCl and 20 mM Tris-HCl pH 7,9), sonicates and then removes debris by ultra centrifugation (39.000 g). Changes in the protocol (addition of Lysozyme, 0,1 % Triton) increases the total protein amount only to 30 mg. This leads to my first question: What can I also do to increase the total protein yield? May Lysozyme or Triton lead to problems with the HisTag purification? The second problem is the colomn chromatographie: According to the protocol I expect a flow rate of 25 ml/h. But the real flow rate is just 10 ml/h. Filtration of the cell lysate or new packing of the column did not help. Although SDS-PAGE analysis showed that the target protein is expressed, I got no target protein (0,04 mg instead of 20 mg!). Have you made experiences with this purification kit? Why is the total protein concentration so low? How can I increase the column´s flow rate? Why is it impossible to purify my target protein? Can you help me? Thank you for your answer! yours sincerely Thorsten Schmidt From owner-proteins@net.bio.net Fri Nov 08 22:00:00 1996 Path: biosci!daresbury!yama.mcc.ac.uk!viking.ucsalf.ac.uk!news.salford.ac.uk!aber!bath.ac.uk!dcl-cs!strath-cs!queens-belfast.ac.uk!queens-belfast.ac.uk!nntp Newsgroups: bionet.molbio.proteins Subject: Visiting Studentships at QUB Message-ID: <327F090C.5C20@qub.ac.uk> From: Andrew Wallace Date: Tue, 05 Nov 1996 09:29:48 +0000 Reply-To: a.wallace@queens-belfast.ac.uk Organization: Queens University Belfast Nntp-Posting-Host: awall.bc.qub.ac.uk X-Mailer: Mozilla 2.0 (Macintosh; I; PPC) MIME-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit Lines: 26 Three Visiting Studentships, tenable for two years initially with the possibility of renewal for a third year, are available for postgraduate research in the Colleges of Engineering, Health Sciences and Science & Agriculture. Their value is currently 6060 pounds Sterling per annum, plus exemption from enrolment and tuition fees of the University. Applicants must have at least a 2.1 honours degree (or its equivalent) from a University other than Queens, and must have shown aptitude for research or other original work. Students who have not yet graduated and postgraduate students already registered for a doctorate at any University are ineligible to apply. Application forms are available from the Academic Council Office, The Queen's University of Belfast, Belfast BT7 1NN, Northern Ireland (UK). The closing date is 1st December 1996. Email: joyce.thompson@qub.ac.uk Fax: +44-1232-313537 Tel: +44-1232-245133 ext 3004/5/6 -- ================================================================== Andrew Wallace,Ph.D., Queen's University Belfast, N. Ireland (UK) a.wallace@qub.ac.uk http://web.qub.ac.uk/bb/awpage/wallace.html ================================================================== From owner-proteins@net.bio.net Fri Nov 08 22:00:00 1996 Path: biosci!agate!howland.erols.net!www.nntp.primenet.com!nntp.primenet.com!dispatch.news.demon.net!demon!btnet!netcom.net.uk!news.u-net.com!yama.mcc.ac.uk!viking.ucsalf.ac.uk!news.salford.ac.uk!aber!bath.ac.uk!dcl-cs!strath-cs!str-ccsun!intsvr1.bell.ac.uk!root From: helen Newsgroups: bionet.molbio.proteins Subject: Troponon I - query Date: Tue, 05 Nov 1996 21:09:26 +0000 Organization: a Digital Internet AlphaServer Site Lines: 2 Message-ID: <327FAD06.509E@hamilton.bell.ac.uk> Reply-To: student5@hamilton.bell.ac.uk NNTP-Posting-Host: cscpc35.bell.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (Win95; I) Does anyone know of a homology between animal and human structure for Troponin I ? From owner-proteins@net.bio.net Fri Nov 08 22:00:00 1996 Path: biosci!rutgers!gatech!csulb.edu!hammer.uoregon.edu!arclight.uoregon.edu!news.sprintlink.net!news-peer.sprintlink.net!howland.erols.net!agate!nature.berkeley.edu!lhom From: lhom@nature.berkeley.edu (Louis Hom) Newsgroups: bionet.molbio.proteins Subject: Disulfides & organelles Date: 10 Nov 1996 00:18:09 GMT Organization: University of California, Berkeley Lines: 13 Message-ID: <563701$3fq@agate.berkeley.edu> NNTP-Posting-Host: nature.berkeley.edu So, as far as I know, the cytoplasm is a reducing environment, so that the tendency is for cysteins to be found in the reduced state. In contrast, the extracellular environment favors disulfide formation (I hope I'm right so far). So my question is, is it generally true that you'd expect to see disulfide formation in structures/organelles that are topologically equivalent to the outside of the cell? -- _______________________________________________________________________________ Lou Hom >K '93 "Into each life a little rain lhom@nature.berkeley.edu must fall, even in San Diego." http://www.ocf.berkeley.edu/~lhom -- from "My Blue Heaven" From owner-proteins@net.bio.net Sat Nov 09 22:00:00 1996 Path: biosci!rutgers!uwm.edu!fnnews.fnal.gov!lakesis.fapesp.br!jazz.cr-df.rnp.br!news.dcc.ufmg.br!mono.icb.ufmg.br!waisberg From: Michael Waisberg Newsgroups: bionet.molbio.proteins Subject: Ab against a receptor??? Date: Thu, 7 Nov 1996 13:43:06 -0300 Organization: Universidade Federal de Minas Gerais Lines: 18 Message-ID: NNTP-Posting-Host: mono.icb.ufmg.br Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII I would like to produce ab's against one receptor in order to analize it's properties but I don't have enough receptors to make the immunization. I would like to know if there is a simple way to extract one amount large enough to make this immunization or even if it is possible to produce these ab' without extracting the receptor.. Thanks. _________________________________________________________________________ Michael Waisberg Adress: R. Desemb. Alarico Barroso n 630 Bairro: Ouro Preto BH/ MG Brazil Cep: 31310-380 Tel: (031)4981773 waisberg@mono.icb.ufmg.br From owner-proteins@net.bio.net Sat Nov 09 22:00:00 1996 Path: biosci!agate!howland.erols.net!www.nntp.primenet.com!nntp.primenet.com!dispatch.news.demon.net!demon!netcom.net.uk!news.u-net.com!yama.mcc.ac.uk!liv!rgu.ac.uk!news From: 933467u@app_science.rgu.ac.uk Newsgroups: bionet.molbio.proteins Subject: EPOGEN and PROCRIT Date: 5 Nov 1996 15:03:24 GMT Organization: Computer Sevices Unit, The Robet Gordon University, Aberdeen, Scotland Lines: 18 Message-ID: <55nkvs$cgm@news.rgu.ac.uk> NNTP-Posting-Host: grays.rgu.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Windows; I; 16bit) To anyone who can help me, I understand that EPOGEN is given to dialysis patents and PROCRIT is given to people suffering from cancer and HIV / AIDS. Can anyone tell me if there's any difference between these drugs at the molecular level? As they're given to different classes of patient, I assume that they're different drugs? On the other hand it MIGHT be that due to some legal ruling, the manufacturers of the drugs (Amgen and Ortho Biotech) have been told to stick to seperate areas of the market. If this is the case, perhaps their products have different names simply to help people differentiate? In effect, one drug has been given two names??? If no-one can answer these questions directly, I'd appreciate it if someone can advise me as to where I might find the information. Thanks for your time folks, Alan Mercer. From owner-proteins@net.bio.net Sat Nov 09 22:00:00 1996 Path: biosci!bloom-beacon.mit.edu!eru.mt.luth.se!www.nntp.primenet.com!nntp.primenet.com!usenet.eel.ufl.edu!news-feed-1.peachnet.edu!newsgate.duke.edu!news.duq.edu!newsfeed.pitt.edu!portc02.blue.aol.com!portc01.blue.aol.com!audrey01.news.aol.com!not-for-mail From: researchd@aol.com Newsgroups: bionet.molbio.proteins Subject: Re: Troponon I - query Date: 10 Nov 1996 21:39:01 GMT Organization: America Online, Inc. (1-800-827-6364) (1.10) Lines: 4 Sender: news@aol.com Message-ID: <19961110214100.QAA13212@ladder01.news.aol.com> References: <327FAD06.509E@hamilton.bell.ac.uk> NNTP-Posting-Host: ladder01.news.aol.com X-Newsreader: AOL Offline Reader See our antibodies to troponin and pure troponin standrad at http://www.researchd.com direct page access: http://www.researchd.com/troponin/troponin.htm From owner-proteins@net.bio.net Sun Nov 10 22:00:00 1996 Path: biosci!JASON.UTHCT.EDU!SHAUN From: SHAUN@JASON.UTHCT.EDU ("Shaun D. Black") Newsgroups: bionet.molbio.proteins Subject: Re: Specificity of Endoproteinase Asp-N Date: 11 Nov 1996 16:18:12 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 24 Sender: daemon@net.bio.net Distribution: world Message-ID: <961111181714.725d@jason.uthct.edu> NNTP-Posting-Host: net.bio.net Howdy all, There are two ways to deal with cleavage at Cys residues, as best I know: 1. Modify with ethyleneimine. This converts Cys into a derivative that is isosteric with Lys, so trypsin will cleave such modified AE-Cys residues. 2. Cyanylate Cys with Cyano-Thionitrobenzoate (CTNB). Chemical peptide cleavage at the Cyano-Cys residues can be effected under alkaline conditions. Methods in Enzymology has protocols for both of these procedures. I hope this helps a bit. Best regards, Shaun =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= = Shaun D. Black, PhD | Internet address: shaun@jason.uthct.edu = = Dept. of Biochemistry | University of Texas Health Center, at Tyler = = World Wide Web: http://pegasus.uthct.edu/UTHCT-Home/Welcome.html = = Network of Emerging Scientists: http://pegasus.uthct.edu/nes/nes.html = =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= From owner-proteins@net.bio.net Sun Nov 10 22:00:00 1996 Path: biosci!botany.uq.edu.au!J.Marcus From: J.Marcus@botany.uq.edu.au ("Marcus, Dr J.") Newsgroups: bionet.molbio.proteins Subject: Re: Specificity of Endoproteinase Asp-N Date: 11 Nov 1996 14:34:49 -0800 Organization: Dept of Botany, Univ of Qld Lines: 36 Sender: daemon@net.bio.net Distribution: world Message-ID: Reply-To: Marcus@tpp.uq.edu.au NNTP-Posting-Host: net.bio.net Dear Kali, You wrote: > I am looking for something to specifically cleave at Cys residues. I > am aware of Endoproteinase Asp-N which cleaves at Aspartic acid also. > Does anyone know of anything I could use? I am looking forward to your > great ideas. > Thanks for the help, Kali > If you are really desparate you might try modifying Cys residues with iodoacetamide. Once modified, I think it allows trypsin to cleave at that position (maybe not efficiently but you should get some cleavage). You would have to modify both arginines and lysines (see Pierce catalog) so that they are not susceptible to trypsin cleavage. A bit of work, but if you are desparate, it might work. Hope that helps. John _________________________________________________________ John Marcus Marcus@tpp.uq.edu.au (Dr J.Marcus) Cooperative Research Centre for Tropical Plant Pathology 5th Level John Hines Building University of Queensland St. Lucia, QLD 4072 AUSTRALIA Fax: 61-7-3365-4771 Phone: 61-7-3365-4764 From owner-proteins@net.bio.net Sun Nov 10 22:00:00 1996 Path: biosci!vt.edu!kkniel From: kkniel@vt.edu (Kali Phelps) Newsgroups: bionet.molbio.proteins Subject: Specificity of Endoproteinase Asp-N Date: 11 Nov 1996 13:06:57 -0800 Organization: student Lines: 5 Sender: daemon@net.bio.net Distribution: world Message-ID: <32879636.652B@vt.edu> Reply-To: kkniel@vt.edu NNTP-Posting-Host: net.bio.net I am looking for something to specifically cleave at Cys residues. I am aware of Endoproteinase Asp-N which cleaves at Aspartic acid also. Does anyone know of anything I could use? I am looking forward to your great ideas. Thanks for the help, Kali From owner-proteins@net.bio.net Sun Nov 10 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.erols.net!vixen.cso.uiuc.edu!uwm.edu!news.he.net!stc06.ctd.ornl.gov!fnnews.fnal.gov!cbgw1.lucent.com!cbgw3.lucent.com!news.pbi.net!samba.rahul.net!rahul.net!a2i!bug.rahul.net!rahul.net!a2i!in-news.erinet.com!news.izzy.net!aanews.merit.net!news.gmi.edu!msunews!NewsWatcher!user From: 23144jda@msu.edu (Jenny Davila-Aponte) Newsgroups: bionet.molbio.proteins Subject: Affinity chromatography Followup-To: bionet.molbio.proteins Date: Mon, 11 Nov 1996 17:01:42 -0400 Organization: Michigan State University Lines: 15 Message-ID: <23144jda-111196170142@35.8.197.48> NNTP-Posting-Host: 35.8.197.48 I am trying to purify a fucosyltransferase that is involved in cell wall synthesis in higher plants, and one of the steps of my purification strategy is a GDP affinity column step. This step gives me good enrichment, but the recovery of activity is mediocre (15 - 20%). Obtaining tissue for protein extraction is laborious, so I would like to improve my recovery of activity. I have tried changing several ingredients of my column buffer: buffer, pH, detergent, divalent cations, ionic strength, and stabilizing agents; but have not been able to improve my recovery. The column buffer that I am currently using contains 20% glycerol, 50 mM pipes KOH pH 6.2, 100 mM KCl, 0.1% decylmaltoside, aprotinin, caproic acid, leupeptin, and pepstatin. I would appreciate any advice or useful suggestions Amy DeRocher deroche1@pilot.msu.edu From owner-proteins@net.bio.net Sun Nov 10 22:00:00 1996 Path: biosci!rutgers!news.sgi.com!su-news-hub1.bbnplanet.com!news.bbnplanet.com!cpk-news-hub1.bbnplanet.com!www.nntp.primenet.com!nntp.primenet.com!feed1.news.erols.com!howland.erols.net!news.sprintlink.net!news-peer.sprintlink.net!newsfeed.internetmci.com!in1.uu.net!amgen!usenet From: John Philo Newsgroups: bionet.molbio.proteins Subject: Re: Big Problems with =?iso-8859-1?Q?Novagen=B4s?= HisTag Protein Purification Date: Mon, 11 Nov 1996 09:09:47 -0800 Organization: Amgen Inc. Lines: 68 Message-ID: <32875DDB.3D50@amgen.com> References: <01bbce57$8fc130a0$09019386@Schmidt.rz.ruhr-uni-bochum.de> Reply-To: jphilo@amgen.com Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: quoted-printable X-Mailer: Mozilla 3.0Gold (Win95; I) To: Thorsten Schmidt Thorsten Schmidt wrote: > = > Dear Sir or Madam! > = > I have big Problems with Novagen`s HisTag Protein Purification-Kit. > = > (snip) > = > I followed strikly Novagen=B4s purification protocol but the problems b= egin > with the > total protein. Instead of the predicted 50-100 mg, I get only 2 (!). > (snip) > Changes in the protocol (addition of Lysozyme, 0,1 % Triton) increases = the > total protein amount only to 30 mg. > This leads to my first question: > What can I also do to increase the total protein yield? Expression levels of recombinant proteins can vary widely even when using the same expression vector and system. For example, proteins whose N-terminal sequences contain several grouped proline residues often express poorly. Even for identical polypeptide sequences, expression can vary depending on which of the redundant nucleotide codons are used (many mammalian proteins do not express optimally in E. coli because E. coli has different codon preferences). = > May Lysozyme or Triton lead to problems with the HisTag purification? Lysozyme shouldn't, and I doubt Triton will either, but I have no personal experience with either. > (snip) > Although SDS-PAGE analysis showed that the target protein is expressed,= I > got no target protein (0,04 mg instead of 20 mg!). > (snip) > Why is it impossible to purify my target protein? While I have not done this myself, I know the experience of my Amgen colleagues is that purification of His-tagged proteins on the Ni resin fails fairly often, either because the protein hardly binds at all, or because many other proteins are eluted along with the one of interest. The view here is that His-tag purification of FOLDED proteins seldom works, probably because the tag is sterically restrained from binding well to the resin. You may have better success if you explicitly run your Ni column under conditions where the protein will be denatured (perhaps adding urea or guanidine-HCl), but then you will have to refold the protein in a separate step. If you are hoping to purify a folded protein using the Ni resin, you might want to try adding a spacer sequence between your protein and the His tag so it is more exposed. The view here is also that protein binding to the Ni resin is less specific than one might wish. It seems that often other proteins are bound and elute with the tagged protein. Presumably these other proteins accidentally contain clusters of histidines. 'Hope this helps. Good luck. John Philo, Protein Chemistry Amgen Inc., Thousand Oaks, CA jphilo@amgen.com *** Disclaimer: These are the opinions of the poster not Amgen Inc.*** From owner-proteins@net.bio.net Sun Nov 10 22:00:00 1996 Path: biosci!rutgers!gatech!csulb.edu!not-for-mail From: cohlberg@csulb.edu (Jeff Cohlberg) Newsgroups: bionet.molbio.proteins Subject: Re: Disulfides & organelles Date: 11 Nov 1996 17:14:25 GMT Organization: Cal State Long Beach Lines: 17 Message-ID: <567mth$m4e@hatathli.csulb.edu> References: <563701$3fq@agate.berkeley.edu> NNTP-Posting-Host: gull.csulb.edu X-Newsreader: TIN [UNIX 1.3 950824BETA PL0] Louis Hom (lhom@nature.berkeley.edu) wrote: : So my question is, is it generally true that you'd expect to see : disulfide formation in structures/organelles that are topologically : equivalent to the outside of the cell? Yes. For example, my understanding is that disulfide bonds are introduced into secreted proteins by enzymes localized in the ER lumen, and the oxidizing potential of the ER lumen and the various Golgi compartments preserves the SS bond until the protein is secreted. My uninformed guess would be that lysosomal enzymes contain SS bonds to help preserve their conformation in the extreme lysosomal environment. Jeffrey A. Cohlberg, Professor Department of Chemistry and Biochemistry California State University, Long Beach 1250 Bellflower Boulevard, Long Beach, CA 90840 Phone: (310) 985-4944 FAX: (310) 985-8557 E-mail: cohlberg@csulb.edu From owner-proteins@net.bio.net Sun Nov 10 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!www.nntp.primenet.com!nntp.primenet.com!feed1.news.erols.com!uunet!in1.uu.net!amgen!usenet From: John Philo Newsgroups: bionet.molbio.proteins Subject: Re: EPOGEN and PROCRIT Date: Mon, 11 Nov 1996 08:46:51 -0800 Organization: Amgen Inc. Lines: 26 Message-ID: <3287587B.764A@amgen.com> References: <55nkvs$cgm@news.rgu.ac.uk> Reply-To: jphilo@amgen.com Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (Win95; I) To: 933467u@app_science.rgu.ac.uk 933467u@app_science.rgu.ac.uk wrote: > > I understand that EPOGEN is given to dialysis patents and PROCRIT is > given to people suffering from cancer and HIV / AIDS. Can anyone tell me > if there's any difference between these drugs at the molecular level? > > As they're given to different classes of patient, I assume that they're > different drugs? On the other hand it MIGHT be that due to some legal > ruling, the manufacturers of the drugs (Amgen and Ortho Biotech) have > been told to stick to seperate areas of the market. If this is the case, > perhaps their products have different names simply to help people > differentiate? In effect, one drug has been given two names??? EPOGEN and PROCRIT are identical at the molecular level. Ortho provided financial support to Amgen for the development of erythropoietin as a therapeutic. In return, they obtained licensing rights to market the drug for all indications in the U.S. other than kidney dialysis. In fact, fairly often hospital pharmacies only carry one of these two products, so PROCRIT can end up being used in dialysis patients or EPOGEN in AIDS patients. The two companies have a agreement on how to compensate each other for such "out of market" sales. John Philo, Protein Chemistry Amgen Inc., Thousand Oaks, CA jphilo@amgen.com *** Disclaimer: These are the opinions of the poster not Amgen Inc.*** From owner-proteins@net.bio.net Sun Nov 10 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!www.nntp.primenet.com!nntp.primenet.com!news.bbnplanet.com!cpk-news-hub1.bbnplanet.com!newsfeed.internetmci.com!feed1.news.erols.com!uunet!in1.uu.net!amgen!usenet From: John Philo Newsgroups: bionet.molbio.proteins Subject: Re: Cost of Epogen Production / Treatment. Date: Mon, 11 Nov 1996 08:38:20 -0800 Organization: Amgen Inc. Lines: 40 Message-ID: <3287567C.36DF@amgen.com> References: <55doee$smj@news.rgu.ac.uk> Reply-To: jphilo@amgen.com Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (Win95; I) To: 933467u@app_science.rgu.ac.uk 933467u@app_science.rgu.ac.uk wrote: > > To anyone who can help me, > > I'm a forth year student at the Robert Gordon Universtiy in Aberdeen, > Scotland. I'm desperately seeking information on some of the economic > implications of synthetically produced Erythropoietin (AKA EPO, Epogen, > etc.). Can anyone tell me how much it would cost to produce say, 100ml > of the stuff? How much would it cost to treat a patient with EPO for a > month? > > If no-one can answer my questions directly, I'd appreciate it if someone > could suggest where I might find the information I need. > > Thanks for your time folks, Alan Mercer. You should be able to get information about the costs of EPO treatment from the Amgen sales and marketing people. We have an office in Cambridge, UK, tel. 011 44 1223 420305. You would probably also be able to get such information from organizations providing support for kidney disease patients. You should probably look at the info available about EPO on the Amgen web site, www.amgen.com. The specific link to a page about EPO is http://www.Amgen.com/cgi-bin/genobject/productEpogen/tigxbJPgVC9 This page leads to background info about EPO and its actions, physician prescription info, and information about our Patient Assistance Program for those who cannot afford to pay for EPO treatment, and pointers to various kidney disease organizations. EPO, a protein hormone, is not measured in milliliters. Internally we measure it in micrograms, but for prescription purposes it is measured in activity units, based on an international standard. I believe those international units are based on formation of red blood cell precursors in an in vitro bone marrow assay, but I am not sure about that. John Philo, Protein Chemistry Amgen Inc., Thousand Oaks, CA jphilo@amgen.com *** Disclaimer: These are the opinions of the poster not Amgen Inc.*** From owner-proteins@net.bio.net Sun Nov 10 22:00:00 1996 Path: biosci!CENTRAL.MURDOCH.EDU.AU!ppanutat From: ppanutat@CENTRAL.MURDOCH.EDU.AU (Panomporn Panutat) Newsgroups: bionet.molbio.proteins Subject: Ni column problem Date: 11 Nov 1996 02:25:18 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 7 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net I have a problem with eluting protein from Ni-column (QIAGEN) . I followed spin column protocol described by the manufacturers but the protein remains bound to the column. Does anyone have any suggestions as to how I can elute my protein without denaturing it. Any suggestions greatly appreciated. Thanks Panomporn From owner-proteins@net.bio.net Sun Nov 10 22:00:00 1996 Path: biosci!rutgers!uwm.edu!news.he.net!www.nntp.primenet.com!nntp.primenet.com!mr.net!news.mid.net!nntp.ksu.edu!news.physics.uiowa.edu!news.uiowa.edu!phloem.biology.uiowa.edu!user From: r-sjolund@uiowa.edu (Richard Sjolund) Newsgroups: bionet.molbio.proteins Subject: Postdoc in Plant Cell and Mol. Biology Date: Mon, 11 Nov 1996 12:38:41 -0500 Organization: Univ. of Iowa Lines: 21 Message-ID: NNTP-Posting-Host: phloem.biology.uiowa.edu A Postdoctoral position is available immediately to conduct research on phloem sieve elements in plant tissue cultures using biochemical, cellular, and molecular techniques. (See Wang, Monroe and Sjolund, 1995, Plant Physiology, 109:743-750.) Applicants must have a Ph.D. in a plant science, and direct experience in plant molecular biology and plant biochemistry. Experience in SDS-polyacrylamide gel electrophoresis, western blotting techniques, and protein purification is desirable. Please send a letter describing your research and training, a C.V., and three letters of recommendation to: Dr. Richard Sjolund Department of Biological Sciences Room 222 Chemistry Bld. University of Iowa Iowa City, Iowa USA 52242. Fax: 319: 335 3620. Email: r-sjolund@uiowa.edu The University of Iowa is an equal opportunity employer. Women and minority candidates are encouraged to apply. From owner-proteins@net.bio.net Sun Nov 10 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!www.nntp.primenet.com!nntp.primenet.com!dispatch.news.demon.net!demon!news.good.net!news.good.net!news.he.net!night.primate.wisc.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!martinlab From: klenchin@facstaff.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins Subject: Re: Affinity chromatography Date: 11 Nov 1996 23:51:06 GMT Organization: UW-Madison Lines: 39 Message-ID: <568e5a$13mk@news.doit.wisc.edu> References: <23144jda-111196170142@35.8.197.48> NNTP-Posting-Host: martinlab.biochem.wisc.edu X-Newsreader: News Xpress Version 1.0 Beta #4 X-No-Archive: Yes In article <23144jda-111196170142@35.8.197.48>, 23144jda@msu.edu (Jenny Davila-Aponte) wrote: ->I am trying to purify a fucosyltransferase that is involved in cell wall ->synthesis in higher plants, and one of the steps of my purification ->strategy is a GDP affinity column step. This step gives me good ->enrichment, but the recovery of activity is mediocre (15 - 20%). Obtaining ->tissue for protein extraction is laborious, so I would like to improve my ->recovery of activity. I have tried changing several ingredients of my ->column buffer: buffer, pH, detergent, divalent cations, ionic strength, and ->stabilizing agents; but have not been able to improve my recovery. The ->column buffer that I am currently using contains 20% glycerol, 50 mM pipes ->KOH pH 6.2, 100 mM KCl, 0.1% decylmaltoside, aprotinin, caproic acid, ->leupeptin, and pepstatin. I would appreciate any advice or useful ->suggestions Many possibilities: 1. Another factor stimulates activity. Try adding flow-through back to eluted fractions and see if activity is any different. 2. What is eluent? Affinity (GDP*Mg)? Try higher concentrations and/or longer elution times - when the affinity for GDP is high, k(off) is frequently rate-limiting step. In this case filling the column with eluent, stopping the flow for 30-60 min, then eluting helps. 3. You have an inherently labile protein. Not much can be done here. Live with it and hope that one day you hit some magic trick that will stabilize activity. As a matter of fact, I envy you. I've not been able to push recovery of "my" protein above 5%. Good luck, - Dima From owner-proteins@net.bio.net Sun Nov 10 22:00:00 1996 Path: biosci!rutgers!gatech!csulb.edu!newshub.csu.net!www.nntp.primenet.com!nntp.primenet.com!news.bbnplanet.com!cpk-news-hub1.bbnplanet.com!cpk-news-feed4.bbnplanet.com!news.fsu.edu!fn3.freenet.tlh.fl.us!usenet From: "mehboob b.sheikh" Newsgroups: bionet.jobs,bionet.molbio.proteins,bionet.molbio.rapd,bionet.plants Subject: Post-doc/visiting scientist position available Date: Mon, 11 Nov 1996 17:45:29 -0500 Organization: FAMU Lines: 25 Message-ID: <3287AC88.5231@freenet3.scri.fsu.edu> NNTP-Posting-Host: fts4p8-bfs.scri.fsu.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (Win95; I) Xref: biosci bionet.molbio.proteins:9246 bionet.molbio.rapd:1710 bionet.plants:13225 POSITION ANNOUNCEMENT Research Associate - Molecular Biology. Two postdoctoral positions are available to work on peanut molecular biology. The successful candidates will conduct research on the molecular biology of drought and disease resistance, and improving protein quality of peanut. Major goals will include identifying molecular markers for disease and drought resistance, cloning methionine-rich protein gene, and transformation of peanut. The appointee will be expected to have demonstrated experience in modern molecular biology techniques, which may include mRNA isolation and in vitro translation, southern and northern hybridization, RT-PCR, gene cloning/vector construction, screening cDNA library using polyclonal antibodies, DNA sequencing and related techniques, to detect, describe and manage the behavior of the genes. The laboratory is fully equipped with necessary instrumentation and supplies for the above research. A Ph.D. in plant science with extensive knowledge and experience in plant molecular biology is required. Applicants must submit a letter of application describing research and career interests and experience, resume, transcripts and three references to Dr. Mehboob B. Sheikh, Plant Biotechnology Program, Division of Agricultural Sciences, Florida A&M University, Tallahassee, Florida 32307-4100. Phone: (904) 561-2218; E-Mail: Sheikh1@juno.com. Florida A&M University is an Equal Opportunity/Affirmative Action Employer. Minority candidates and women are encouraged to apply. From owner-proteins@net.bio.net Sun Nov 10 22:00:00 1996 Path: biosci!botany.uq.edu.au!J.Marcus From: J.Marcus@botany.uq.edu.au ("Marcus, Dr J.") Newsgroups: bionet.molbio.proteins Subject: Re: Specificity of Endoprot Date: 11 Nov 1996 20:30:41 -0800 Organization: Dept of Botany, Univ of Qld Lines: 54 Sender: daemon@net.bio.net Distribution: world Message-ID: Reply-To: Marcus@tpp.uq.edu.au NNTP-Posting-Host: net.bio.net Dear netters: In an earlier post I suggested using Iodoacetamide to modify Cys residues to make them susceptible to Trypsin cleavage. I was quite mistaken, as Daniel Milligan kindly pointed out to me. Shaun Black's suggestion to use the ethyleneimine is the suggestion that I should have made. Sorry if I caused any confusion. Regards, John > > -------------------------------------- > Date: 11/11/96 4:13 PM > To: protein-analysis@net.bio.net > From: Marcus@tpp.uq.edu.au > Dear Kali, > > You wrote: > > > I am looking for something to specifically cleave at Cys residues. I > > am aware of Endoproteinase Asp-N which cleaves at Aspartic acid also. > > Does anyone know of anything I could use? I am looking forward to your > > great ideas. > > Thanks for the help, Kali > > > If you are really desparate you might try modifying Cys > residues with iodoacetamide. Once modified, I think it > allows trypsin to cleave at that position (maybe not > efficiently but you should get some cleavage). You would > have to modify both arginines and lysines (see Pierce > catalog) so that they are not susceptible to trypsin > cleavage. A bit of work, but if you are desparate, it > might work. > > Hope that helps. > > John > _________________________________________________________ John Marcus Marcus@tpp.uq.edu.au (Dr J.Marcus) Cooperative Research Centre for Tropical Plant Pathology 5th Level John Hines Building University of Queensland St. Lucia, QLD 4072 AUSTRALIA Fax: 61-7-3365-4771 Phone: 61-7-3365-4764 From owner-proteins@net.bio.net Mon Nov 11 22:00:00 1996 Path: biosci!NMSU.EDU!hroychow From: hroychow@NMSU.EDU (Hiranya Roychowdhury) Newsgroups: bionet.molbio.proteins Subject: Re: small volume dialysis Date: 12 Nov 1996 14:41:50 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 21 Sender: daemon@net.bio.net Distribution: world Message-ID: <199611122241.PAA10831@NMSU.Edu> NNTP-Posting-Host: net.bio.net At 09:22 AM 11/12/96 -0800, kewugator@UFCC.UFL.EDU wrote: >Hi, there: >I am going to dialysis some proteins (30 KDa) in very small volume (20 ul). >Any body has any idea that how can I have a good recovery of the proteins and >what kind of dialysis apparatus can be used to get the maximum recovery of the >proteins. >Thanks. > > Ke Wu > > Try microdialysis using VSWP membrane filters from Milipore. Dr. Hiranya Sankar Roychowdhury Plant Genetic Engineering Lab. New Mexico State University Las Cruces, NM 88003 Ph. (505) 646-5785 hroychow@nmsu.edu From owner-proteins@net.bio.net Mon Nov 11 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.erols.net!www.nntp.primenet.com!nntp.primenet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!su-news-hub1.bbnplanet.com!arclight.uoregon.edu!news.bc.net!torn!resunix.sickkids.on.ca!NewsWatcher!user From: michael.didonato@utoronto.ca (Michael DiDonato) Newsgroups: bionet.molbio.proteins Subject: Re: small volume dialysis Date: Tue, 12 Nov 1996 15:19:06 -0500 Organization: The Hospital for Sick Children Lines: 19 Distribution: world Message-ID: References: <009AB424.1AC6CEA0.10@UFCC.UFL.EDU> NNTP-Posting-Host: 142.20.36.38 In article <009AB424.1AC6CEA0.10@UFCC.UFL.EDU>, kewugator@UFCC.UFL.EDU wrote: > Hi, there: > I am going to dialysis some proteins (30 KDa) in very small volume (20 ul). > Any body has any idea that how can I have a good recovery of the proteins and > what kind of dialysis apparatus can be used to get the maximum recovery of the > proteins. > Thanks. > > Ke Wu Dialysis buttons would be the best in this case I think. You can get them in a range of sizes from 5 micro liters to ~500 micro liters I think. Unfortunately I don't know who supplies them. Good Luck.. Mike From owner-proteins@net.bio.net Mon Nov 11 22:00:00 1996 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed1.bbnplanet.com!news.bbnplanet.com!cpk-news-hub1.bbnplanet.com!www.nntp.primenet.com!nntp.primenet.com!mr.net!news.mid.net!nntp.ksu.edu!news.cis.okstate.edu!usenet From: Ron Tate Newsgroups: bionet.molbio.proteins Subject: Re: small volume dialysis Date: Tue, 12 Nov 1996 14:33:15 -0600 Organization: Oklahoma State University, Biochemistry and Molecular Biology Lines: 31 Message-ID: <3288DF0A.524C@bmb-fs1.biochem.okstate.edu> References: <009AB424.1AC6CEA0.10@UFCC.UFL.EDU> NNTP-Posting-Host: firefly3.biochem.okstate.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0 (Win95; I) kewugator@UFCC.UFL.EDU wrote: > > Hi, there: > I am going to dialysis some proteins (30 KDa) in very small volume (20 ul). > Any body has any idea that how can I have a good recovery of the proteins and > what kind of dialysis apparatus can be used to get the maximum recovery of the > proteins. > Thanks. > > Ke Wu I posted this suggestion a month or so ago also, BioTechniques published a way to do this last year, Vol. 19, No.2 (1995) (I don't have the page number) the article was titled "Waterbug Dialysis". It involved cutting the top portion off of a microfuge tube and closing a piece of dialysis membrane in the lid with your sample inside the lid and then floating the thing in a beaker of buffer. Its one of those "Why didn't I think of that" kind of ideas. If you don't have access to back issues of BioTechniques they are online with their back issues and a searchable d-base. Happy Hunting -- >>>>>>---------------------------> Ron Tate Lab of Franklin Leach Dept. of Biochem. & Molecular Biology Oklahoma State University rtate@bmb-fs1.biochem.okstate.edu (405) 744-9326 --------------------------------------------- From owner-proteins@net.bio.net Mon Nov 11 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!www.nntp.primenet.com!nntp.primenet.com!news.enteract.com!news.inetnebr.com!netserv.unmc.edu!netserv!gspahwa From: gspahwa@netserv.unmc.edu (Gurcharan Pahwa) Newsgroups: bionet.molbio.proteins Subject: Secretory protein Date: 12 Nov 1996 21:16:24 GMT Organization: University of Nebraska Medical Center Lines: 1 Message-ID: <56apf8$s0d1@netserv.unmc.edu> NNTP-Posting-Host: 137.197.98.10 X-Newsreader: TIN [version 1.2 PL2] From owner-proteins@net.bio.net Mon Nov 11 22:00:00 1996 Path: biosci!UFCC.UFL.EDU!kewugator From: kewugator@UFCC.UFL.EDU Newsgroups: bionet.molbio.proteins Subject: small volume dialysis Date: 12 Nov 1996 09:22:05 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 8 Sender: daemon@net.bio.net Distribution: world Message-ID: <009AB424.1AC6CEA0.10@UFCC.UFL.EDU> NNTP-Posting-Host: net.bio.net Hi, there: I am going to dialysis some proteins (30 KDa) in very small volume (20 ul). Any body has any idea that how can I have a good recovery of the proteins and what kind of dialysis apparatus can be used to get the maximum recovery of the proteins. Thanks. Ke Wu From owner-proteins@net.bio.net Mon Nov 11 22:00:00 1996 Path: biosci!daresbury!bioftp.unibas.ch!infobiogen.fr!pasteur.fr!oleane!in2p3.fr!swidir.switch.ch!swsbe6.switch.ch!scsing.switch.ch!elna.ethz.ch!bclutz2.ethz.ch!user From: hornig@bc.biol.ethz.ch (Ruediger Hornig) Newsgroups: bionet.molbio.proteins Subject: Re: Specificity of Endoproteinase Asp-N Date: Tue, 12 Nov 1996 16:54:04 +0100 Organization: ETH Zurich Lines: 29 Distribution: world Message-ID: References: <32879636.652B@vt.edu> NNTP-Posting-Host: bclutz2.ethz.ch In article <32879636.652B@vt.edu>, kkniel@vt.edu wrote: > I am looking for something to specifically cleave at Cys residues. I > am aware of Endoproteinase Asp-N which cleaves at Aspartic acid also. > Does anyone know of anything I could use? I am looking forward to your > great ideas. > Thanks for the help, Kali Dear Kali A chemical way of cleaving a protein before C is cutting with NTCB. The procedure requires complete denaturation in SDS and reduction of all sulfhydryl side chains with DTT. Contrary to Asp-N and the proposed Iodoacetamide extension of C, it's pretty easy and specific. Hope this helps Ruedi ---------------------------------------------------------------------- _/_/_/_/_/_/_/_/_/ _/ ETH Zurich _/ _/ _/ _/ Biochemie II _/_/_/ _/ _/_/_/_/ Rüdiger Hornig (hornig@bc.biol.ethz.ch) _/ _/ _/ _/ Universitaetstr. 16, ETH Zentrum _/_/_/_/ _/ _/ _/ CH-8092 Zurich (Switzerland) Phone: +41 1 632 3007 FAX: +41 1 632 12 69 http://www.bc.biol.ethz.ch/BiochemistryII/BioII.html ---------------------------------------------------------------------- From owner-proteins@net.bio.net Mon Nov 11 22:00:00 1996 Path: biosci!EMPIRE.NET!wepayne From: wepayne@EMPIRE.NET (William E. Payne) Newsgroups: bionet.molbio.proteins Subject: subscribe Date: 12 Nov 1996 06:17:35 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 12 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net subscribe protein-analysis William E. Payne - - - - - - - - - - - - - - - William E. Payne 40A Wheeler Road Pepperell, MA 01463 (508) 433-8940 Phone (508) 433-2971 Fax wepayne@empire.net - - - - - - - - - - - - - - - From owner-proteins@net.bio.net Mon Nov 11 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.erols.net!math.ohio-state.edu!news.cis.ohio-state.edu!nntp.sei.cmu.edu!fs7.ece.cmu.edu!casaba.srv.cs.cmu.edu!das-news2.harvard.edu!fas-news.harvard.edu!husc7.harvard.edu!mlevin From: mlevin@husc7.harvard.edu (Michael Levin) Newsgroups: bionet.cellbiol,bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: need a protocol to do a Western blot for membrane protein from Xenopus Date: 12 Nov 1996 13:12:12 GMT Organization: Harvard University, Cambridge, MA Lines: 14 Distribution: usa Message-ID: <569t3c$10r@decaxp.harvard.edu> NNTP-Posting-Host: husc7.harvard.edu Xref: biosci bionet.cellbiol:5930 bionet.molbio.methds-reagnts:51411 bionet.molbio.proteins:9251 This is probably a pretty easy question, but I need a protocol for isolating protein (making sure I get membrane proteins) from Xenopus and probing it with an antibody (whether on a gel or just on some kind of "spot blot"). While I know the overall idea of how to do it, I haven't worked with proteins before, so I really need a detailed protocol, which spells out exactly what to do - which solutions (and how to make 'em), for how long, etc. If you have such a protocol (in your lab's computer, for example), or know where I can get one, please email me at mlevin@husc.harvard.edu - I would greatly appreciate it. Thanks in advance, Mike Levin From owner-proteins@net.bio.net Mon Nov 11 22:00:00 1996 Path: biosci!agate!usenet.kornet.nm.kr!newsfeed.dacom.co.kr!arclight.uoregon.edu!nntp.primenet.com!nntp.uio.no!nntp.zit.th-darmstadt.de!fu-berlin.de!informatik.tu-muenchen.de!lrz-muenchen.de!news From: Boris Steipe Newsgroups: bionet.molbio.proteins Subject: Re: Ni column problem Date: Tue, 12 Nov 1996 11:34:09 +0000 Organization: Genzentrum Lines: 28 Distribution: world Message-ID: <32886033.4F59@LMB.uni-muenchen.de> References: Reply-To: steipe@LMB.uni-muenchen.de NNTP-Posting-Host: bs_boris.lmb.uni-muenchen.de Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (Macintosh; I; PPC) Panomporn Panutat wrote: > > I have a problem with eluting protein from Ni-column (QIAGEN) . I > followed spin column protocol described by the manufacturers but the > protein remains bound to the column. Does anyone have any suggestions as > to how I can elute my protein without denaturing it. Any of the following should elute protein, that is specifically bound: Imidazole: washing with 300 mM should elute everything pH: protonated Histidines will not bind, pH below 6.0 will elute. EDTA: will wash down complexed Ni - try 5mM. BUT: if you have followed the instructions, most likely your protein is denatured and/or nonspecifically bound to the column. Then none of the above is likely to work. Try 8M Urea (7M if you work in the coldroom), 1 M NaCl, 10 mM EDTA. This also will clean your column between runs. Good luck, Boris -- +---Dr. Boris Steipe---------------+ | Genzentrum | | Feodor-Lynen Str. 25 Tel +49 (0)89 74017-417 | | 81377 Muenchen, Germany Fax - 448 | +------+ From owner-proteins@net.bio.net Mon Nov 11 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!news.sprintlink.net!news-peer.sprintlink.net!uunet!in2.uu.net!newsfeed.pitt.edu!mv29.pathology.pitt.edu!user From: pxpst2@vms.cis.pitt.edu (THE GREEK) Newsgroups: bionet.molbio.proteins Subject: Coagulation proteins Date: 12 Nov 1996 20:18:03 GMT Organization: USA Lines: 3 Message-ID: NNTP-Posting-Host: mv29.pathology.pitt.edu Any body out there with experience with the coagulation proteins? I am most interested in factor Xa and its reaction requirements. Peter Pediaditakis pxpst2@vms.cis.pitt.edu From owner-proteins@net.bio.net Mon Nov 11 22:00:00 1996 Path: biosci!CSHL.ORG!leemor From: leemor@CSHL.ORG (Leemor Joshua-Tor) Newsgroups: bionet.molbio.proteins Subject: Re: small volume dialysis Date: 12 Nov 1996 15:09:17 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 60 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net >In article <009AB424.1AC6CEA0.10@UFCC.UFL.EDU>, kewugator@UFCC.UFL.EDU wrote: > >> Hi, there: >> I am going to dialysis some proteins (30 KDa) in very small volume (20 ul). >> Any body has any idea that how can I have a good recovery of the >>proteins and >> what kind of dialysis apparatus can be used to get the maximum recovery >of the >> proteins. >> Thanks. >> >> Ke Wu > >Dialysis buttons would be the best in this case I think. You can get them >in a range of sizes from >5 micro liters to ~500 micro liters I think. Unfortunately I don't know >who supplies them. > >Good Luck.. >Mike You can find the buttons through Hampton Research. They normally sell stuff for crystallization but they sell these dialysis buttons. Check out their home page: http://www.hamptonresearch.com However, Ron Tate wrote: >I posted this suggestion a month or so ago also, BioTechniques published >a way to do this last year, Vol. 19, No.2 (1995) (I don't have the page >number) the article was titled "Waterbug Dialysis". It involved cutting >the top portion off of a microfuge tube and closing a piece of dialysis >membrane in the lid with your sample inside the lid and then floating >the thing in a beaker of buffer. Its one of those "Why didn't I think >of that" kind of ideas. >If you don't have access to back issues of BioTechniques they are online >with their back issues and a searchable d-base. >Happy Hunting >-- >>>>>>---------------------------> >Ron >Tate We use this method often for very small volumes and it works well. Another way of making the hole in the tube cap is by using a hot glass rod, the hole is less sharp and less likely to puncture your dialysis membrane. good luck, Leemor ******************************************************************* Leemor Joshua-Tor, Ph.D. Assistant Investigator Cold Spring Harbor Laboratory Tel. (516) 367 8821 1 Bungtown Road Fax (516) 367 8873 Cold Spring Harbor, NY 11724 e-mail: leemor@cshl.org ******************************************************************* From owner-proteins@net.bio.net Mon Nov 11 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.erols.net!www.nntp.primenet.com!nntp.primenet.com!news.enteract.com!news.inetnebr.com!netserv.unmc.edu!netserv!gspahwa From: gspahwa@netserv.unmc.edu (Gurcharan Pahwa) Newsgroups: bionet.molbio.proteins Subject: Secretory protein Date: 12 Nov 1996 22:49:52 GMT Organization: University of Nebraska Medical Center Lines: 14 Message-ID: <56auug$s0d3@netserv.unmc.edu> NNTP-Posting-Host: 137.197.98.10 Keywords: proteins,o-glycosylation X-Newsreader: TIN [version 1.2 PL2] I intend to study the O-linked glycosylation of a polypeptide in cells. For that matter I intend to use a custom designed oligonucleotide for its stable expression in mammalian cell cultures. I need an eukaryotic expression vector with features such as a strong promotor (SV40 etc), a selection marker, signal peptide for secretary proteins, polypeptide sequence for processing in the golgi.I will need complete restriction map to design my oilgo. I shall greatly admire to have such an expression vector so that we are able to insert our oligonucleotide for expression of the corresponding protein and further secretion into the medium. I look forward to your valuable advice. Thanks Jaswant. From owner-proteins@net.bio.net Mon Nov 11 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.erols.net!www.nntp.primenet.com!nntp.primenet.com!news.enteract.com!news.inetnebr.com!netserv.unmc.edu!netserv!gspahwa From: gspahwa@netserv.unmc.edu (Gurcharan Pahwa) Newsgroups: bionet.molbio.proteins Subject: Secretory protein Date: 12 Nov 1996 22:52:27 GMT Organization: University of Nebraska Medical Center Lines: 13 Message-ID: <56av3b$s0d4@netserv.unmc.edu> NNTP-Posting-Host: 137.197.98.10 X-Newsreader: TIN [version 1.2 PL2] I intend to study the O-linked glycosylation of a polypeptide in cells. For that matter I intend to use a custom designed oligonucleotide for its stable expression in mammalian cell cultures. I need an eukaryotic expression vector with features such as a strong promotor (SV40 etc), a selection marker, signal peptide for secretary proteins, polypeptide sequence for processing in the golgi.I will need complete restriction map to design my oilgo. I shall greatly admire to have such an expression vector so that we are able to insert our oligonucleotide for expression of the corresponding protein and further secretion into the medium. I look forward to your valuable advice. Thanks Jaswant. From owner-proteins@net.bio.net Mon Nov 11 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!swrinde!www.nntp.primenet.com!nntp.primenet.com!mr.net!news.mid.net!netserv.unmc.edu!netserv!jsingh From: jsingh@netserv.unmc.edu (Jaswant Singh) Newsgroups: bionet.molbio.proteins Subject: test Date: 12 Nov 1996 22:24:20 GMT Organization: University of Nebraska Medical Center Lines: 2 Message-ID: <56atek$s0d2@netserv.unmc.edu> NNTP-Posting-Host: 137.197.98.10 X-Newsreader: TIN [version 1.2 PL2] test,please ignore From owner-proteins@net.bio.net Mon Nov 11 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.erols.net!www.nntp.primenet.com!nntp.primenet.com!nntp.uio.no!nntp.zit.th-darmstadt.de!news.th-darmstadt.de!odb!photon.ON-Luebeck.DE!msms From: Manfred Schuermann Newsgroups: bionet.molbio.proteins Subject: orosomucoid genotyping Date: 13 Nov 1996 05:40:35 GMT Organization: Offenes Netz Luebeck e.V. Lines: 21 Message-ID: <56bn0j$3hf@photon.ON-Luebeck.DE> NNTP-Posting-Host: on.on-luebeck.de Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Newsreader: TIN [UNIX 1.3 unoff BETA release 961005] Orosomucoid (ORM) is a highly polymorphic protein. Genotyping is usually done by isoelectric focusing. I would like to know, if there is a *DNA / PCR based* method to demonstrate ORM alleles. (The Genome Data Base -GDB- does not list ORM variants) Thank you very much for helpful hints! Manfred Schuermann, M.D. Institute of Human Genetics University of Luebeck Medical School, Germany e-mail msms@on-luebeck.de phone +49 451 500 2620 fax +49 451 500 4187 From owner-proteins@net.bio.net Mon Nov 11 22:00:00 1996 Path: biosci!bloom-beacon.mit.edu!eru.mt.luth.se!www.nntp.primenet.com!nntp.primenet.com!nntp.uio.no!nntp.zit.th-darmstadt.de!news.th-darmstadt.de!odb!photon.ON-Luebeck.DE!msms From: Manfred Schuermann Newsgroups: bionet.molbio.proteins Subject: haptoglobin genotyping Date: 13 Nov 1996 05:39:41 GMT Organization: Offenes Netz Luebeck e.V. Lines: 24 Message-ID: <56bmut$3hf@photon.ON-Luebeck.DE> NNTP-Posting-Host: on.on-luebeck.de Mime-Version: 1.0 Content-Type: text/plain; charset=US-ASCII Content-Transfer-Encoding: 8bit X-Newsreader: TIN [UNIX 1.3 unoff BETA release 961005] Haptoglobin (HP) is a highly polymorphic protein. Genotyping is usually done by isoelectric focusing. I would like to know, if there is a * PCR based* method to demonstrate HP alleles. It should be possible as the underlying DNA variations are known. (The Genome Data Base -GDB- lists HP variants that are detected by Southern blotting and HP probe hybridization ) Thank you very much for helpful hints! Manfred Schuermann, M.D. Institute of Human Genetics University of Luebeck Medical School, Germany e-mail msms@on-luebeck.de phone +49 451 500 2620 fax +49 451 500 4187 From owner-proteins@net.bio.net Mon Nov 11 22:00:00 1996 Path: biosci!rutgers!news.sgi.com!www.nntp.primenet.com!nntp.primenet.com!feed1.news.erols.com!panix!news.panix.com!not-for-mail From: iayork@panix.com (Ian A. York) Newsgroups: bionet.molbio.proteins Subject: Re: small volume dialysis Date: 12 Nov 1996 19:07:11 -0500 Organization: Panix Lines: 18 Message-ID: <56b3ff$1jr@panix.com> References: <009AB424.1AC6CEA0.10@UFCC.UFL.EDU> NNTP-Posting-Host: panix.com In article <009AB424.1AC6CEA0.10@UFCC.UFL.EDU>, wrote: > >I am going to dialysis some proteins (30 KDa) in very small volume (20 ul). You might try floating dialysis. Float a small piece of filter paper on water (or whatever you're dialyzing into), and gently pipette the sample on top of the filter. After you're done, gently pipette it off again. If your hands aren't shaky it's very easy. I don't know what filter would be the best - check with MilliPore for the appropriate one. Ian -- Ian York (iayork@panix.com) "-but as he was a York, I am rather inclined to suppose him a very respectable Man." -Jane Austen, The History of England From owner-proteins@net.bio.net Mon Nov 11 22:00:00 1996 Path: biosci!rutgers!uwm.edu!news.he.net!www.nntp.primenet.com!nntp.primenet.com!EU.net!news.sprintlink.net!news-peer.sprintlink.net!uunet!in3.uu.net!news.u.washington.edu!saul1.u.washington.edu!egn From: "E. Kolker" Newsgroups: bionet.general,bionet.info-theory,bionet.molbio.proteins,sci.med.pharmacy Subject: Abstract Date: Tue, 12 Nov 1996 15:58:30 -0800 Organization: University of Washington Lines: 81 Message-ID: References: <01bbcd7b$e9d3bd60$3024ef82@pepsi> NNTP-Posting-Host: saul1.u.washington.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII NNTP-Posting-User: egn In-Reply-To: <01bbcd7b$e9d3bd60$3024ef82@pepsi> Xref: biosci bionet.general:23997 bionet.info-theory:4377 bionet.molbio.proteins:9265 sci.med.pharmacy:36407 ANNOUNCEMENT AND CALL FOR PAPERS Computational Biology Session: "Computing in the Genome Era" Eleventh International Conference on Mathematical and Computer Modelling and Scientific Computing March 31 - April 3, 1997 Georgetown University Conference Center Washington, DC, USA The Eleventh International Conference on Mathematical and Computer Modelling and Scientific Computing is scheduled to take place March 31 - April 3, 1997 at the Georgetown University Conference Center, Washington, DC, U.S.A. Plenary lectures by world-renowned scientists and sessions on many recent developments in engineering and sciences comprise a long-standing tradition at the ICMCM's. Mathematical and computer modelling and scientific computing have become powerful tools for solving complex problems and providing greater insights into the future. The objective of the conference is to bring together researchers from various disciplines including the traditional and emerging areas of engineering and sciences for cross-fertilization of ideas and exchange of information. The objective of the Computational Biology Session "Computing in the Genome Era" is to discuss the current state of computational biology, its approaches, methods, general problems, achievements, and future developments with emphasis on sequence research and analysis for the Genome Projects. Speakers of the session include: S. Altschul (NCBI, NIH, Bethesda), A. Bairoch (Geneva Univ., Switzerland), W. Gish (Wash. Univ., St. Louis), P. Green (Univ. of Wash., Seattle), S. Henikoff (FHCRC, HHMC, Seattle), L. Hood (Univ. of Wash., Seattle), E. Koonin (NCBI, NIH, Bethesda), D. Searls (SmithKline Beecham, King of Prussia), E. Trifonov (Weizmann Inst., Israel). Papers (Abstracts) are invited on all relevant aspects of computational biology for presentation at the session, to be selected on competitive basis by a steering committee. One-page abstracts (about 200 words) should clearly describe the work and its conclusions. Full length manuscripts (limited to six pages) of papers presented at the conference will be published in the Conference Proceedings, in a special issue of the journal MATHEMATICAL MODELLING AND SCIENTIFIC COMPUTING, Vol. 8, 1997 (ISSN 1067-0688). The manuscripts for the special issue are due June 15, 1997. The special issue of the journal will be published by September 1997. All participants shall pay the registration fee (pre-registration before February 15, 1997: IAMCM or IMACS members - $330, non-members - $375, graduate students - $210, undergraduate students - $175). Abstracts may be submitted in hard copy or via fax or by e-mail (preferred!) under subject "Abstract". The abstracts must be formatted to fit on 8-1/2 x 11 inch (approximately 21.5 cm x 28 cm or European Standard A-4 size) paper, typed in single space. The title must be capitalized and centered followed by the author's name(s), institution, and full address, including fax and e-mail. Send two copies (ONE copy if sent by fax or e-mail) of the abstract to the session organizer BEFORE November 30, 1996: Eugene Kolker Dept of Molecular Biotechnology and Genome Center Box 357730, University of Washington Seattle, WA 98195-7730, USA Fax: +1-206-685-7301 E-mail: egn@u.washington.edu Important dates: Closing date for receiving abstracts: November 30, 1996 Notification of acceptance: December 27, 1996 The Computational Biology Session is sponsored by SILICON GRAPHICS. Companies are welcome to participate in the session, present their tools and products, and/or co-sponsor the session. The Web site for the session is under contruction and will include information on the session, a list of related sites, links to the sponsors etc. From owner-proteins@net.bio.net Mon Nov 11 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.erols.net!vixen.cso.uiuc.edu!news.uoregon.edu!waikato!auckland.ac.nz!news From: mmm_klehnert@mednov1.auckland.ac.nz (Klaus Lehnert) Newsgroups: bionet.molbio.proteins Subject: Re: small volume dialysis Date: 12 Nov 1996 20:59:28 GMT Organization: University of Auckland Lines: 32 Distribution: world Message-ID: <56aofg$80k@scream.auckland.ac.nz> References: <009AB424.1AC6CEA0.10@UFCC.UFL.EDU> NNTP-Posting-Host: mmm-55-82.mmm.auckland.ac.nz Mime-Version: 1.0 X-Newsreader: WinVN 0.93.11 In article , says... > >Hi, there: >I am going to dialysis some proteins (30 KDa) in very small volume (20 ul). Hi Ke Wu, it doesn't get any cheaper and easier than that: Take an eppendorf tube (0.5-1.5ml) punch a hole in the lid fill your sample take a coin-sized piece of dialysis tubing (one layer) put on top of tube close lid, thereby squeezing the dialysis membrane (opt: seal the rim of lid/tube with small strip of parafilm; necessary only foe low-quality tubes). put tube into floater put upside-down into dialysis buffer make sure there is no air bubble trapped in the punched-out hole. invert from time to time to make sure all your protein comes into contact with the membrane. After dialysis, simply spin tube, this gets you all liquid to the bottom. Works always well for me, no significant loss, very little increase in volume Have fun KLAUS From owner-proteins@net.bio.net Tue Nov 12 22:00:00 1996 Path: biosci!internet!biosci!not-for-mail From: biohelp (BIOSCI Administrator) Newsgroups: bionet.molbio.proteins Subject: BIOSCI/bionet miniFAQ & Fundraiser Date: 13 Nov 1996 02:00:44 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 239 Sender: daemon@net.bio.net Distribution: world Message-ID: <199611131000.CAA22592@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/. 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