From owner-proteins@net.bio.net Sun Aug 01 09:16:00 1999 Path: biosci!fcs280s.ncifcrf.gov!fcrdcnews.NCIFCRF.GOV!washdc3-snf1!crtntx1-snh1.gtei.net!su-news-hub1.bbnplanet.com!news.gtei.net!news.good.net!news-dc.gip.net!news-penn!news.gsl.net!gip.net!iafrica.com!news.adamastor.ac.za!not-for-mail From: Dr Lester Davids Newsgroups: bionet.molbio.proteins Subject: Inclusion Bodies Date: Fri, 30 Jul 1999 15:13:28 +0200 Organization: University of Cape Town Lines: 8 Message-ID: <37A1A4F8.29B8@uctgsh1.uct.ac.za> Reply-To: lester@uctgsh1.uct.ac.za NNTP-Posting-Host: pc107.mdn.uct.ac.za Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Win95; I) I am currently trying to get proteins out of inclusion bodies in E.coli after an overnight culture growth and am really battling. Anyone out there with advice to get prots out of inclusion bodies without destroying/denaturing the proteins and rendering them inactive? Looking for help Dr L.Davids, Cape Town, South Africa From owner-proteins@net.bio.net Sun Aug 01 09:18:00 1999 Path: biosci!fcs280s.ncifcrf.gov!fcrdcnews.NCIFCRF.GOV!washdc3-snf1!crtntx1-snh1.gtei.net!su-news-hub1.bbnplanet.com!news.gtei.net!newsfeed.berkeley.edu!dispose.news.demon.net!demon!newsfeed.nacamar.de!newsfeed.nacamar.de!fu-berlin.de!pc207-60.biochem.uni-potsdam.DE!not-for-mail From: Frank =?iso-8859-1?Q?F=FCrst?= Newsgroups: bionet.molbio.proteins Subject: Re: Inclusion Bodies Date: Fri, 30 Jul 1999 18:30:38 +0200 Message-ID: <37A1D32E.517AB51A@rz.uni-potsdam.de> References: <37A1A4F8.29B8@uctgsh1.uct.ac.za> NNTP-Posting-Host: pc207-60.biochem.uni-potsdam.de (141.89.207.60) Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Access: 16 49 955 X-Trace: fu-berlin.de 933353641 19002 (none) 141.89.207.60 X-Mailer: Mozilla 4.51 [de]C-CCK-MCD QXW03200 (Win95; I) X-Accept-Language: de-DE,en Lines: 60 Dr Lester Davids wrote: > > I am currently trying to get proteins out of inclusion bodies in E.coli > after an overnight culture growth and am really battling. Anyone out > there with advice to get prots out of inclusion bodies without > destroying/denaturing the proteins and rendering them inactive? > I think what you want is impossible, because proteins in inclusion bodies aren't active, they are aggregated and in a conformation (partially) different to the native, functional conformation. Inclusion bodies are formed when bacteria are overexpressing a protein to such an extent that the bi-(or perhaps multi-)molecular aggregation reaction of folding intermediates gets faster than their rearrangement to the native conformation. Folding is a unimolecular reaction and thus independent of protein concentration, while aggregation as a bimolecular reaction is. The critical concentration of intermediates can be achieved simply by increasing the protein concentration, i.e. overexpression, or when aggregation-prone off-pathway folding intermediates are stabilized relative to on-pathway intermediates, e.g. by mutations or by increased temperature, or by both ways. If you're not familiar with my use of the term aggregation: In this context it means association of partially folded proteins, a process that does not rely on a specific interaction like the one governing oligomerization, but is also not completely unspecific, because one usually finds mainly one protein in aggregates (e.g. IB), even if there's two with similar high concentration. As it is folding intermediates that aggregate, there's often some native structure even in the IB, namely secondary structure, but often the beta-sheet content is higher. On the other hand, many proteins can fold to their native conformation /in vitro/ starting from an unfolded chain under apropriate conditions. Thus, there's two ways out of your problem: Either prevent the IBs from beeing formed at all, by: - lowering the level of expression (which you probably don't want to do...) - lowering temperature (_may_ help, or may not) - coexpressing chaperones (a technology I am not an expert in, so somebody else should comment on that) Or by denaturing and solubilizing your protein in the IBs and trying to renature it. If you're protein is of at most medium size and monomeric, I bet you can find conditions that allow refolding in sufficient yields. Hope this helps, Frank -- Frank Fuerst, Institute of Biochemistry of Potsdam University Im Biotechnologiepark, D-14943 Luckenwalde Tel.: +49-3371-681334; Fax: +49-3371-681339 I'm SignatureVirus 99! Copy me into your signature and join the fun! From owner-proteins@net.bio.net Sun Aug 01 09:23:00 1999 Path: biosci!cnn.nas.nasa.gov!news.sgi.com!howland.erols.net!portc02.blue.aol.com!audrey01.news.aol.com!not-for-mail From: stcaris@aol.com (Stcaris) Newsgroups: bionet.molbio.proteins Subject: Re: Inclusion Bodies Lines: 45 NNTP-Posting-Host: ladder07.news.aol.com X-Admin: news@aol.com Date: 31 Jul 1999 07:31:40 GMT References: <37A1D32E.517AB51A@rz.uni-potsdam.de> Organization: AOL Bertelsmann Online GmbH & Co. KG http://www.germany.aol.com X-Newsreader: AOL Offline Reader Mime-Version: 1.0 Content-Type: text/plain; charset=ISO-8859-1 Content-Transfer-Encoding: 8bit Message-ID: <19990731033140.13703.00002544@ngol03.aol.com> Im Artikel <37A1D32E.517AB51A@rz.uni-potsdam.de>, Frank =?iso-8859-1?Q?F=FCrst?= schreibt: >- coexpressing chaperones (a technology I am not an expert in, so >somebody else should comment on that) > That's just what I was about to suggest, because I have just finished reading a recent CELL paper about the role of the DnaK chaperone in preventing protein precipitation t very high local concentrations, as they occur at the polyribosome... I admit that I am not an expert either. >Or by denaturing and solubilizing your protein in the IBs and trying >to renature it. If you're protein is of at most medium size and >monomeric, I bet you can find conditions that allow refolding in >sufficient yields. Years ago, we were able to renature proteins within SDS-PAGE-Gels (!), up to ~100 kDa in size, by including BSA into the gel polymerization reaction (yes indeed, most batches of BSA contain poorly characterized "chaperonoids", such as prolyl isomerases and I-dunno-what-else) and soaking the gel for several days in buffers containing some "alchemy", mostly low and decreasing concentrations of mercaptoethanol. That was enough to regain lots of activity. >I'm SignatureVirus 99! Copy me into your signature and join the fun! HEY! Have you read Dawkins & Goodenough on "mind viruses", published a few years ago in either NATURE or SCIENCE, I do not remember which? Read that and have double fun! :-) Good luck, Rüdiger === Yours sincerely, Chevalier Dr. Ruediger Marcus Flaig University of Heidelberg Email: sanctacaris@bigfoot.com sanctacaris@unforgettable.com sanctacaris@rocketmail.com sanctacaris@hotmail.com stcaris@aol.com 01727652946@d2-message.de (mobile - short mails only) "Tell truth and shame the devil." (Shakespeare, Henry IV.) From owner-proteins@net.bio.net Sun Aug 01 10:19:00 1999 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newspump.monmouth.com!newspeer.monmouth.com!hermes.visi.com!news-out.visi.com!cam-news-hub1.bbnplanet.com!news.gtei.net!newscon02!prodigy.com!not-for-mail From: Newsgroups: bionet.molbio.proteins Subject: AutoExec2000.com Date: 31 Jul 1999 07:39:33 GMT Organization: Prodigy Internet http://www.prodigy.com Lines: 3 Message-ID: <7nu97l$3vf0$9578@newssvr04-int.news.prodigy.com> NNTP-Posting-Host: miamb109-23.splitrock.net X-Trace: newssvr04-int.news.prodigy.com 933406773 3767148 209.156.29.92 (31 Jul 1999 07:39:33 GMT) X-Complaints-To: abuse@prodigy.net NNTP-Posting-Date: 31 Jul 1999 07:39:33 GMT Just ran across this great web site, I thought you would be very interested in! http://www.AutoExec2000.com From owner-proteins@net.bio.net Sun Aug 01 20:20:00 1999 Path: biosci!news.stanford.edu!newsfeed.stanford.edu!newsfeed.berkeley.edu!news.maxwell.syr.edu!news.mel.connect.com.au!news.unimelb.edu.au!ludwignt-4 From: murphy_r@licre.ludwig.edu.au (Roger Murphy) Newsgroups: bionet.molbio.proteins Subject: Re: Inclusion Bodies Date: Mon, 02 Aug 99 03:16:23 GMT Organization: Ludwig Institute for Cancer Research Lines: 35 Message-ID: <7o35t5$hd$1@izvestia.its.unimelb.edu.au> References: <37A1A4F8.29B8@uctgsh1.uct.ac.za> NNTP-Posting-Host: ludwignt-4.austin.unimelb.edu.au X-Trace: izvestia.its.unimelb.edu.au 933567205 557 128.250.186.193 (2 Aug 1999 04:13:25 GMT) X-Complaints-To: news@news.unimelb.edu.au NNTP-Posting-Date: 2 Aug 1999 04:13:25 GMT X-Newsreader: News Xpress 2.0 Beta #0 In article <37A1A4F8.29B8@uctgsh1.uct.ac.za>, lester@uctgsh1.uct.ac.za wrote: >I am currently trying to get proteins out of inclusion bodies in E.coli >after an overnight culture growth and am really battling. Anyone out >there with advice to get prots out of inclusion bodies without >destroying/denaturing the proteins and rendering them inactive? > >Looking for help > >Dr L.Davids, Cape Town, South Africa If your protein is in an inclusion body it's already likely to be denatured as it's not in a soluble form. The only way to get it out is to solubilise appropriately (we use 6M Gd.HCl, Tris buffer, pH 7, 10mM 2-ME) and then slowly remove the denaturing extractants by dialysis. Fingers crossed that the protein then refolds as you want it! Cheers, Roger ------------------------------------------------------------ Roger Murphy, Ph.D. Biological Production Facility Ludwig Institute for Cancer Research Austin & Repatriation Medical Centre Studley Road, Heidelberg, Vic. 3084 Australia. Tel 61-3-94965463 Fax 61-3-94965436 Email Roger.Murphy@Ludwig.edu.au From owner-proteins@net.bio.net Mon Aug 02 02:15:00 1999 Path: biosci!newshost.lanl.gov!logbridge.uoregon.edu!dispose.news.demon.net!demon!ayres.ftech.net!news.ftech.net!colt.net!Pollux.Teleglobe.net!iafrica.com!news.adamastor.ac.za!not-for-mail From: Dr Lester Davids Newsgroups: bionet.molbio.proteins Subject: Inclusion bodies Date: Mon, 02 Aug 1999 12:05:22 +0200 Organization: University of Cape Town Message-ID: <37A56D62.1DBF@uctgsh1.uct.ac.za> Reply-To: lester@uctgsh1.uct.ac.za NNTP-Posting-Host: pc107.mdn.uct.ac.za Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Win95; I) Lines: 8 Hi All, Looking for advice as to how to PREVENT proteins from getting into these damn bacterial inclusion bodies! It seems out native proteins are not but our mutant proteins are. Any suggestions out there? Thanx, Lester Davids From owner-proteins@net.bio.net Mon Aug 02 02:46:00 1999 Path: biosci!news.stanford.edu!newsfeed.stanford.edu!skynet.be!remarQ-easT!supernews.com!remarQ.com!news.maxwell.syr.edu!nntp2.deja.com!nnrp1.deja.com!not-for-mail From: Kresten Newsgroups: bionet.molbio.proteins Subject: Re: Protease digestions Date: Mon, 02 Aug 1999 10:30:14 GMT Organization: Deja.com - Share what you know. Learn what you don't. Lines: 35 Message-ID: <7o3rvm$c91$1@nnrp1.deja.com> References: <379724c0.146635220@nntp.service.emory.edu> NNTP-Posting-Host: 10.5.192.244 X-Article-Creation-Date: Mon Aug 02 10:30:14 1999 GMT X-Http-User-Agent: Mozilla/4.5 [en] (Win95; I) X-Http-Proxy: 1.0 www2.crc.dk:3128 (Squid/2.1.RELEASE),NetCache@www-cache: Version 3.3.1, 1.0 x44.deja.com:80 (Squid/1.1.22) for client 130.226.182.217, 130.226.1.36 X-MyDeja-Info: XMYDJUIDkresten > Please send /or post your favorite protease digestion portocols to me I have recently used trypsin for detection of a post-translational modification. I used: 1% w/w trypsin in a 50 mM (NH4)(HCO3) pH 8.2 I stopped the reaction with a small amount of HCl and analysed directly on a MALDI mass-spectrometer (by taken out samples I did a time course analysis). This may of course not be the best way for you since your protein is approx. 3 times larger than mine and you modification a bit smaller but you could try. You could also try to purify the tryptic peptides on a HPLC. HTH Kresten > > Thanks in advance > > Dirk > -- The address kresten@my-dejanews.com is for spambots only. Please mail me at LysLeuLeu@crc.dk transforming the pre@-part into my initials. Kresten Lindorff Larsen, Dept. Yeast Genetics Sent via Deja.com http://www.deja.com/ Share what you know. Learn what you don't. From owner-proteins@net.bio.net Mon Aug 02 02:47:00 1999 Path: biosci!news.stanford.edu!newsfeed.stanford.edu!skynet.be!news-spur1.maxwell.syr.edu!news.maxwell.syr.edu!nntp2.deja.com!nnrp1.deja.com!not-for-mail From: Kresten Newsgroups: bionet.molbio.proteins Subject: Re: Protease digestions Date: Mon, 02 Aug 1999 10:34:25 GMT Organization: Deja.com - Share what you know. Learn what you don't. Lines: 54 Message-ID: <7o3s7h$c9o$1@nnrp1.deja.com> References: <379724c0.146635220@nntp.service.emory.edu> <7o3rvm$c91$1@nnrp1.deja.com> NNTP-Posting-Host: 10.5.192.244 X-Article-Creation-Date: Mon Aug 02 10:34:25 1999 GMT X-Http-User-Agent: Mozilla/4.5 [en] (Win95; I) X-Http-Proxy: 1.0 www2.crc.dk:3128 (Squid/2.1.RELEASE),NetCache@www-cache: Version 3.3.1, 1.0 x44.deja.com:80 (Squid/1.1.22) for client 130.226.182.217, 130.226.1.36 X-MyDeja-Info: XMYDJUIDkresten In article <7o3rvm$c91$1@nnrp1.deja.com>, Kresten wrote: > > > > Please send /or post your favorite protease digestion portocols to me > > I have recently used trypsin for detection of a post-translational > modification. > > I used: > 1% w/w trypsin in a 50 mM (NH4)(HCO3) pH 8.2 > > I stopped the reaction with a small amount of HCl and analysed directly > on a MALDI mass-spectrometer (by taken out samples I did a time course > analysis). This may of course not be the best way for you since your > protein is approx. 3 times larger sorry, that should have been *30* times larger kresten > than mine and you modification a bit > smaller but you could try. You could also try to purify the tryptic > peptides on a HPLC. > > HTH > Kresten > > > > > Thanks in advance > > > > Dirk > > > > -- > The address kresten@my-dejanews.com is for > spambots only. Please mail me at LysLeuLeu@crc.dk > transforming the pre@-part into my initials. > Kresten Lindorff Larsen, Dept. Yeast Genetics > > Sent via Deja.com http://www.deja.com/ > Share what you know. Learn what you don't. > -- The address kresten@my-dejanews.com is for spambots only. Please mail me at LysLeuLeu@crc.dk transforming the pre@-part into my initials. Kresten Lindorff Larsen, Dept. Yeast Genetics Sent via Deja.com http://www.deja.com/ Share what you know. Learn what you don't. 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Please call us at 1-888-309-5852 (Toll Free) and speak with Al Schweitzer directly (between the hours of 9:00am - 5:00pm, Mountain Time) Thank you for your time and interest. rem -ko987@excite.com em -ko From owner-proteins@net.bio.net Mon Aug 02 03:21:00 1999 Path: biosci!rutgers!uwm.edu!arclight.uoregon.edu!news.ibm.net.il!ibm.net!news.biu.ac.il!news.huji.ac.il!news!bgumail.bgu.ac.il!vyc From: Vyaches Ibragimov Newsgroups: bionet.molbio.proteins Subject: subscription Date: Mon, 2 Aug 1999 13:55:43 +0300 Organization: Ben-Gurion University, Beer Sheva, Israel Lines: 3 Message-ID: NNTP-Posting-Host: bgumail.bgu.ac.il Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII X-Trace: avdat2.bgu.ac.il 933623576 24562 132.72.143.1 (2 Aug 1999 19:52:56 GMT) X-Complaints-To: usenet@avdat2.bgu.ac.il NNTP-Posting-Date: 2 Aug 1999 19:52:56 GMT Hi I want to subscribe to this newsgroup From owner-proteins@net.bio.net Mon Aug 02 06:49:00 1999 Path: biosci!newshost.lanl.gov!arclight.uoregon.edu!hammer.uoregon.edu!newsfeed.direct.ca!dispose.news.demon.net!demon!newspeer.clara.net!news.clara.net!newsfeed.nacamar.de!fu-berlin.de!pc207-60.biochem.uni-potsdam.DE!not-for-mail From: Frank =?iso-8859-1?Q?F=FCrst?= Newsgroups: bionet.molbio.proteins Subject: Re: Inclusion bodies Date: Mon, 02 Aug 1999 16:01:52 +0200 Message-ID: <37A5A4D0.F31B810C@rz.uni-potsdam.de> References: <37A56D62.1DBF@uctgsh1.uct.ac.za> NNTP-Posting-Host: pc207-60.biochem.uni-potsdam.de (141.89.207.60) Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Access: 16 49 955 X-Trace: fu-berlin.de 933604961 4380 (none) 141.89.207.60 X-Mailer: Mozilla 4.51 [de]C-CCK-MCD QXW03200 (Win95; I) X-Accept-Language: de-DE,en Lines: 66 Dr Lester Davids wrote: > > Hi All, > > Looking for advice as to how to PREVENT proteins from getting into these > damn bacterial inclusion bodies! It seems out native proteins are not > but our mutant proteins are. Any suggestions out there? > Well, as I said in reply to your first posting: (Destabilizing) Mutations are a frequent reason for aggregation. In particular, there are two possibilities: 1. As described earlier, there is a folding intermediate that is susceptible to aggregation, either like this: a) U ---> I ---> F | ---> Aggregates, where U denotes unfolded and F native folded Protein, or like this: b) /--> F / U ---> I-on <---> I-off ---> Aggregates where <---> denotes an equilibrium between the on-pathway intermediate I-on and the off-pathway intermediate I-off The effect of the mutation could be (in a and b) to slow down folding from I (I-on, respectively) to F, so that I (I-on + I-off) accumulates and the aggregation reaction (n I ---> In) with reaction order n gets faster than first-order folding to F, or (in b) (which I think occurs more frequently) a shift in equilibrium between I-on and I-off. Thus the concentration of I-off increases, and aggregation occurs. In either two of these cases, my tips could help (decreasing expression level, or temperature, or coexpressing chaperones, or solubilising IBs and refolding). 2. The second possibility is that the mutations affect the stability of the (native structure of the) protein enough to prevent it from folding at all; what was the native structure in the wild-type is now energetically unfavorable compared to a compact intermediate with at least the tertiary structure beeing flexible (and this intermediate, in turn, is less stable than the aggregates formed out of it). If this is the case, you won't rescue your protein (but you've learned something on structural important residues...) If you only have single point mutations that do not involve Cys making disulfide bridges, the second case is very unlikely. But if you have, e.g., amber mutations, you should also think of that. hope this helps, Frank -- Frank Fuerst, Institute of Biochemistry of Potsdam University Im Biotechnologiepark, D-14943 Luckenwalde Tel.: +49-3371-681334; Fax: +49-3371-681339 I'm SignatureVirus 99! Copy me into your signature and join the fun! From owner-proteins@net.bio.net Mon Aug 02 11:37:00 1999 Path: biosci!MINDSPRING.COM!grizzlyan From: grizzlyan@MINDSPRING.COM (Michael Sherrell) Newsgroups: bionet.molbio.proteins Subject: 377 seqs, synths, LC/MSs, NMRs for sale Date: 2 Aug 1999 12:37:19 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 49 Sender: daemon@net.bio.net Distribution: world Message-ID: <01BEDCE3.1D0C8800@user-33qtgv3.dialup.mindspring.com> NNTP-Posting-Host: net.bio.net Sequencers, synthesizers, mass spectrometers, NMRs for sale: Peptide and oligo synthesizers and sequencers: ABI 392: $2,500 (electronics working; add $3,000 for rebuild/warranty) ABI 394: $12,500 (Valve blocks rebuilt; warranteed) ABI 390Z: $4,000 (50-100uM yields) ABI 430: $6,000 (electronics working; add $5,000 for rebuild/warranty) ABI 431: $12,500 (Rebuilt, warranteed) ABI 433: $19,000 (ABI upgrade) PerSeptive 9050+: $6,000 (As is/was working when decommissioned; add $3,500 for rebuild/warranty) ABI 373 stretch: $9,000 (Big dye upgrade; under ABI service contract) ABI 373 stretch: $7,000 (4-filter) ABI 377: $80,000 (96 lanes; under ABI service contract; quantity discounts for 2 or more) ABI 377: $60,000 (48-lane; under ABI contract) ABI Procise 492: $45,000 obo (ABI-certified) LC/Mass spectrometers: Sciex API III+ LC/MS/MS: $ 59,000 (ES, APCI, under PE service contract) Sciex API 300: $100,000 (365 upgrade, PE install/warranty available) HP 1100 benchtop LC/MSD: $120,000 (APCI & API-electrospray, autosampler, DAD detector, < 1 year old) Finnigan LCQ: $120,000 ("Classic" MS^n; 1.5 yrs old; site license included) PE Mariner: $140,000 (1998 model; includes refurb, install and 1 yr. warranty) Finnigan Navigator benchtop LC/MS: $ 75,000; 18 mos. old; factory refurb, install & 90-day warr. included Finnigan TSQ 7000 LC+GC/MS/MS: $ 97,500; current software; API-1 source; under Finnigan service contract Sciex 150 benchtop LC/MS: $ 98,000; 2 yrs old; includes Gilson 215 liquid handler, ELSD detector & HP 1100 HPLC. 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Michael Sherrell Grizzly Analytical 707 887 2919/fax 707 887 9834 www.grizzlyanalytical.com From owner-proteins@net.bio.net Tue Aug 03 09:43:00 1999 Path: biosci!news.stanford.edu!newsfeed.stanford.edu!logbridge.uoregon.edu!nntp.upenn.edu!mail2.sas.upenn.edu!okashlan From: okashlan@mail2.sas.upenn.edu (Ossama B Kashlan) Newsgroups: bionet.molbio.proteins Subject: electrophoresis advice Date: 3 Aug 1999 17:36:54 GMT Organization: University of Pennsylvania Lines: 18 Message-ID: <7o79bm$qlp$1@netnews.upenn.edu> NNTP-Posting-Host: mail2.sas.upenn.edu X-Newsreader: TIN [version 1.2 PL2-upenn1.3] I'm trying to visualize a crosslinked protein on a polyacrylamide gel, and am having some difficulty with sharpness with the heavy bands. My expected products have masses between 90 kD and 540 kD. I'm allowing the 90 kD monomer to run to the bottom of the gel. I've run a 4% 20 cm x 20 cm x 1.5 mm gel with a 4% stacking gel. Any suggestions as to how to sharpen an expected 540 kD band? Thank you, Ossama Kashlan University of Pennsylvania Department of Chemistry Box 233 231 S. 34th St. Philadelphia, PA 19104 okashlan@mail.sas.upenn.edu Lab: (215)898-2927 Fax: (215)898-2037 From owner-proteins@net.bio.net Tue Aug 03 23:26:00 1999 Path: biosci!news.stanford.edu!newsfeed.stanford.edu!cyclone.bc.net!sunqbc.risq.qc.ca!newsfeed.nacamar.de!newsfeed.nacamar.de!fu-berlin.de!pc207-60.biochem.uni-potsdam.DE!not-for-mail From: Frank =?iso-8859-1?Q?F=FCrst?= Newsgroups: bionet.molbio.proteins Subject: Re: Inclusion bodies Date: Wed, 04 Aug 1999 09:19:43 +0200 Lines: 32 Message-ID: <37A7E98F.7E2FCE15@rz.uni-potsdam.de> References: <37A56D62.1DBF@uctgsh1.uct.ac.za> <37A5A4D0.F31B810C@rz.uni-potsdam.de> NNTP-Posting-Host: pc207-60.biochem.uni-potsdam.de (141.89.207.60) Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: quoted-printable X-Access: 16 49 955 X-Trace: fu-berlin.de 933751203 28897 (none) 141.89.207.60 X-Mailer: Mozilla 4.51 [de]C-CCK-MCD QXW03200 (Win95; I) X-Accept-Language: de-DE,en Hi, I got a private reply from somebody whose newsserver didn't work: From: "L. Guan" To: Frank F=FCrst Subject: Re: Inclusion bodies Date sent: Tue, 3 Aug 1999 19:12:02 +0900 Dear Frank, Would you please let me know if inclusion body is lethal to cells? I found my mutants go to die if I use high concentration induction. And, whether still have some native comformation protein out of inclusion body when inclusion body forms? I restored the function of one of my mutants at 28 C. and also have partial function with low level induction. It seems the mutation do not attack the folding important structure. Since I can not post this, sorry to bother you. Thanks in advance! Sincerely, Lan Department of Molecular life Science Tokai University School of MedicineJapan From owner-proteins@net.bio.net Tue Aug 03 23:28:00 1999 Path: biosci!pravda.ucr.edu!ihnp4.ucsd.edu!gondor!newshub.sdsu.edu!newsfeed.berkeley.edu!newsfeed.tli.de!newsfeed.nacamar.de!fu-berlin.de!pc207-60.biochem.uni-potsdam.DE!not-for-mail From: Frank =?iso-8859-1?Q?F=FCrst?= Newsgroups: bionet.molbio.proteins Subject: Re: Inclusion bodies Date: Wed, 04 Aug 1999 09:21:50 +0200 Lines: 76 Message-ID: <37A7EA0E.4EF0B53D@rz.uni-potsdam.de> References: <37A56D62.1DBF@uctgsh1.uct.ac.za> <37A5A4D0.F31B810C@rz.uni-potsdam.de> <37A7E98F.7E2FCE15@rz.uni-potsdam.de> NNTP-Posting-Host: pc207-60.biochem.uni-potsdam.de (141.89.207.60) Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: quoted-printable X-Access: 16 49 955 X-Trace: fu-berlin.de 933751328 29140 (none) 141.89.207.60 X-Mailer: Mozilla 4.51 [de]C-CCK-MCD QXW03200 (Win95; I) X-Accept-Language: de-DE,en And this is what I mailed to him: Dear Lan, > Would you please let me know if inclusion body is lethal to cells? I > found my mutants go to die if I use high concentration induction. > = I think they are not lethal as long as they do not get big enough to = mechanically distort the cell. So some inclusion body formation = would be tolerable, but it is not astonishing that they die when the = IB grow too big. On the other hand, overproducing recombinant = protein is always stress to the cells, so some additional stress = might be harmful. I must admit that I am not an expert in these cellular things, because I've studied chemistry and am now in the protein folding field. > And, whether still have some native comformation protein out of inclusi= on > body when inclusion body forms? = Probably there's also native protein in solution. The on-pathway = intermediate is still there, though perhaps in lower concentration, = and is still able to form native protein. But usually under conditions = where inclusion bodies are formed, the amount of soluble protein is = too small to base a purification on that. I restored the function of one of my > mutants at 28 C. and also have partial function with low level inductio= n. = > It seems the mutation do not attack the folding important structure. > = Fine! Now you have to chose which way you try it. = BTW 28=B0C is not the lowest possible temperature. If you grow the = cells at 37=B0 to an OD of about 1 before induction and then shift to = 25 or even 20=B0, you might get more soluble protein. I observed that = the total amount of protein seemed to be higher at elevated = temperatures, but if your aggregation reaction is temperature- dependent (which is often the case), you might get more soluble = protein that way > Since I can not post this, sorry to bother you. No problem. Okay that I post your and my article? = Bye, Frank -- = Frank Fuerst, Institute of Biochemistry of Potsdam University Im Biotechnologiepark, D-14943 Luckenwalde Tel.: +49-3371-681334; Fax: +49-3371-681339 I'm SignatureVirus 99! Copy me into your signature and join the fun! From owner-proteins@net.bio.net Wed Aug 04 00:32:00 1999 Path: biosci!poyry.se!bihuusoo89 From: bihuusoo89@poyry.se (7u62m28s) Newsgroups: bionet.molbio.proteins Subject: confidential Date: 4 Aug 1999 01:31:17 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 31 Sender: daemon@net.bio.net Distribution: world Message-ID: <1999080451IAA38080@72u8there82m.protech.com.eg> NNTP-Posting-Host: net.bio.net I am looking for a special person with a good work Ethic and an extraordinary desire who wants to earn $3000-$5000 per week in 30-45 days "THIS IS NOT MULTI-LEVEL MARKETING" We will give you the training and personal support you will need to ensure your success. 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NOT MLM. *FREE INFO 24 HOUR MESSAGE CALL 1-416-410-7530 rem - mike_watters998.yahoo.com  From owner-proteins@net.bio.net Wed Aug 04 01:28:00 1999 Path: biosci!news.stanford.edu!newsfeed.stanford.edu!logbridge.uoregon.edu!dispose.news.demon.net!demon!easynet-tele!easynet.net!tank.news.pipex.net!pipex!server1.netnews.ja.net!pegasus.csx.cam.ac.uk!hgmp.mrc.ac.uk!not-for-mail From: Maria Jesus Martin Newsgroups: bionet.molbio.proteins Subject: Release 11 of TREMBL, a protein sequence database supplementing SWISS-PROT Date: Wed, 04 Aug 1999 10:21:18 +0100 Organization: EMBL-EBI Message-ID: <37A8060E.160D61E2@ebi.ac.uk> Reply-To: martin@ebi.ac.uk NNTP-Posting-Host: burns.ebi.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: niobium.hgmp.mrc.ac.uk 933758478 19210 193.62.196.82 (4 Aug 1999 09:21:18 GMT) X-Complaints-To: news@hgmp.mrc.ac.uk NNTP-Posting-Date: 4 Aug 1999 09:21:18 GMT X-Mailer: Mozilla 4.06 [en] (X11; U; Linux 2.0.36 i686) Lines: 98 INTRODUCTION ============ TrEMBL is a protein sequence database supplementing the SWISS-PROT Protein Sequence Data Bank. TrEMBL contains the translations of all coding sequences (CDS) present in the EMBL Nucleotide Sequence Database not yet integrated in SWISS-PROT. TrEMBL can be considered as a preliminary section of SWISS-PROT. For all TrEMBL entries which should finally be upgraded to the standard SWISS-PROT quality, SWISS-PROT accession numbers have been assigned. RELEASE 11.0 OF TrEMBL ===================== The goal of this TrEMBL release is to achieve synchronization with SWISS-PROT release 38.0. Therefore, all sequence entries present in SWISS-PROT release 38.0 have been removed from TrEMBL release 11, further upgrading of existing TrEMBL entries was achieved and only a very few new entries were incorporated. TrEMBL is split in two main sections; SP-TrEMBL and REM-TrEMBL: SP-TrEMBL (SWISS-PROT TrEMBL) contains the entries (199'794), which should be eventually incorporated into SWISS-PROT. SWISS-PROT accession numbers have been assigned for all SP-TrEMBL entries. SP-TrEMBL is organized in subsections: arc.dat (Archea): 7383 entries fun.dat (Fungi): 6656 entries hum.dat (Human): 7880 entries inv.dat (Invertebrates): 23594 entries mam.dat (Other Mammals): 3094 entries mhc.dat (MHC proteins): 4210 entries org.dat (Organelles): 16227 entries phg.dat (Bacteriophages): 1963 entries pln.dat (Plants): 17250 entries pro.dat (Prokaryotes): 45908 entries rod.dat (Rodents): 7348 entries unc.dat (Unclassified): 44 entries vrl.dat (Viruses): 53911 entries vrt.dat (Other Vertebrates): 4326 entries REM-TrEMBL (REMaining TrEMBL) contains the entries (45'967) that we do not want to include in SWISS-PROT. WEEKLY UPDATES OF TrEMBL AND NON-REDUNDANT DATA SETS ==================================================== Weekly cumulative updates of TrEMBL are available by anonymous FTP and from the EBI SRS server. We also produce every week a complete non-redundant protein sequence collection by providing three compressed files (these are in the directory /pub/databases/sp_tr_nrdb on the EBI FTP server): sprot.dat.Z, trembl.dat.Z and trembl_new.dat.Z. ACCESS/DATA DISTRIBUTION ======================== FTP server: ftp.ebi.ac.uk/pub/databases/trembl SRS server: http://srs.ebi.ac.uk/ TREMBL is also available on the SWISS-PROT CD-ROM. SWISS-PROT + TREMBL is searchable on the FASTA3, BLAST2 and Bic_sw servers of the EBI. TrEMBL HAS BEEN PREPARED BY: ============================ Rolf Apweiler, Kirsty Bates, Margaret Biswas, Sergio Contrino, Wolfgang Fleischmann, Gill Fraser, Henning Hermjakob, Vivien Junker, Youla Karavidopoulou, Fiona Lang, Minna Lehvaslaiho, Michele Magrane, Maria Jesus Martin, Steffen Moeller, Nicoletta Mitaritonna, Nicola Mulder, Claire O'Donovan, Lucia Rodriguez-Monge and Eleanor Whitfield at the EMBL Outstation - European Bioinformatics Institute (EBI) in Hinxton, UK; Amos Bairoch and Alain Gateau at the Swiss Institute of Bioinformatics in Geneva, Switzerland. ----------------------------------------------------------------- Maria Jesus Martin email:martin@ebi.ac.uk EMBL Outstation EBI (European Bioinformatics Institute) URL: http://www.ebi.ac.uk Wellcome Trust Genome Campus Tel: +44 (1223) 494408 Hinxton fax: +44 (1223) 494468 Cambridge CB10 1SD UK ----------------------------------------------------------------- From owner-proteins@net.bio.net Wed Aug 04 03:51:00 1999 Path: biosci!news.stanford.edu!newsfeed.stanford.edu!newsfeed.concentric.net!netnews.com!news-peer-europe.sprintlink.net!news.sprintlink.net!newsfeed1.swip.net!swipnet!newsfeed2.funet.fi!ousrvr3.oulu.fi!sun1.oulu.fi!pkursula From: Petri Kursula Newsgroups: bionet.molbio.proteins Subject: Re: electrophoresis advice Date: Wed, 4 Aug 1999 14:30:13 +0300 Organization: University of Oulu Lines: 49 Message-ID: References: <7o79bm$qlp$1@netnews.upenn.edu> NNTP-Posting-Host: sun1.oulu.fi Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII X-Trace: ousrvr3.oulu.fi 933766215 15105 130.231.240.11 (4 Aug 1999 11:30:15 GMT) X-Complaints-To: news@news.oulu.fi NNTP-Posting-Date: 4 Aug 1999 11:30:15 GMT X-Sender: pkursula@sun1.oulu.fi In-Reply-To: <7o79bm$qlp$1@netnews.upenn.edu> I think one problem here might be that the proteins may have been crosslinked at several different sites, resulting in conformations that are not fully denatured by SDS. The presence of differently covalently crosslinked complexes with the same MW then results in a diffuse band. Well, at least this is how I interpret my own results...but I really have not tried to find a way around it. Maybe changing the crosslinker or its concentration might help? Actually, even using a crosslinker on a protein that is not crosslinked to another protein makes the band diffuse, due to the different amounts of crosslinking agent reacting with each individual protein molecule. A crosslinked protein molecule population is no more homogenous, that is. Generally, though, protein bands are sharper on minigels, the running time being shorter and thus the proteins have less time to diffuse in the gel during the run. On 3 Aug 1999, Ossama B Kashlan wrote: > I'm trying to visualize a crosslinked protein on a polyacrylamide gel, and > am having some difficulty with sharpness with the heavy bands. My > expected products have masses between 90 kD and 540 kD. I'm allowing the > 90 kD monomer to run to the bottom of the gel. I've run a 4% 20 cm x 20 > cm x 1.5 mm gel with a 4% stacking gel. Any suggestions as to how to > sharpen an expected 540 kD band? > > Thank you, > Ossama Kashlan > University of Pennsylvania > Department of Chemistry > Box 233 > 231 S. 34th St. > Philadelphia, PA 19104 > okashlan@mail.sas.upenn.edu > Lab: (215)898-2927 > Fax: (215)898-2037 > > > ----------------------------------------------------------------------- Petri Kursula "I am somehow less interested in the weight and University of Oulu convolutions of Einstein's brain than in the near Petri.Kursula@oulu.fi certainty that people of equal talent have lived http://start.at/MAG and died in cotton fields and sweatshops." http://cc.oulu.fi/~pkursula -- Stephen Jay Gould ----------------------------------------------------------------------- From owner-proteins@net.bio.net Wed Aug 04 04:47:00 1999 Path: biosci!news.stanford.edu!newsfeed.stanford.edu!newsfeed.berkeley.edu!howland.erols.net!newsfeed.concentric.net!newsfeed.gol.com!wnoc-tyo-news!news.nc.u-tokyo.ac.jp!gray.ims.u-tokyo.ac.jp!not-for-mail From: GIW '99 cfp Newsgroups: bionet.molbio.proteins Subject: CFP: Genome Informatics Workshop (GIW99) Date: Wed, 04 Aug 1999 21:37:38 +0900 Organization: Human Genome Center, Inst. of Medical Science, Univ. of Tokyo, Japan. Lines: 52 Message-ID: <37A83412.1783AB83@lily.ims.u-tokyo.ac.jp> Reply-To: giw@ims.u-tokyo.ac.jp NNTP-Posting-Host: pine.ims.u-tokyo.ac.jp Mime-Version: 1.0 Content-Type: text/plain; charset=iso-2022-jp Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.6 [ja] (Win95; I) X-Accept-Language: ja Call for Papers The Tenth Workshop on Genome Informatics (GIW '99) December 14-15, 1999 Yebisu Garden Place, Tokyo, Japan ( http://www.hgc.ims.u-tokyo.ac.jp/giw ) [PC Chairman of GIW '99] Kiyoshi Asai (asai@etl.go.jp) [Scope] The Tenth Workshop on Genome Informatics (GIW '99) focuses on Genome Informatics, including, but not limited to, the following areas related to Genome Science: sequence analysis, motif extraction and search, multiple alignment, gene structure prediction, gene function prediction, phylogeny tree, systems for supporting experimental work (mapping, sequencing, primer design, etc.), comparative analysis of genomes, gene expression profile analysis, knowledge extraction from literature, knowledge discovery from databases, linkage analysis program, association analysis, protein structure and function prediction, genome database, pathway analysis, proteome analysis, simulation of biological system, DNA computing, artificial life, which are overall related to Genome Science. [Paper Submission Guidelines] Authors are requested to submit full papers or extended abstracts around 10 pages in one of the following form: - PS file or PDF file or Plain text - until SUBMISSION DEADLINE: August 31, 1999 - to the address: Electronic submission: giw@ims.u-tokyo.ac.jp Satoru Miyano Human Genome Center Institute of Medical Science, University of Tokyo 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. - The following information should be provided: paper title, author name(s), affiliation(s), postal and e-mail address(es), abstract, and the correspondence author name. [Poster/Software Demos Submission Guidelines] - Submission Deadline: October 10, 1999 - See the details at http://giw.ims.u-tokyo.ac.jp/giw99/ [Organizing Chairman of GIW '99] Satoru Miyano ( miyano@ims.u-tokyo.ac.jp ) Email: giw@ims.u-tokyo.ac.jp Tel: +81-3-5449-5615 Fax: +81-3-5449-5442 Human Genome Center, Institute of Medical Science, University of Tokyo 4-6-1 Shirokanedai, Minato-ku, 108-8639 Tokyo, Japan. From owner-proteins@net.bio.net Wed Aug 04 05:17:00 1999 Path: biosci!BRISTOL.AC.UK!K.Jandt From: K.Jandt@BRISTOL.AC.UK ("Dr Klaus D. Jandt") Newsgroups: bionet.molbio.proteins Subject: PhD Studentship Date: 4 Aug 1999 06:17:43 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 56 Sender: daemon@net.bio.net Distribution: world Message-ID: Reply-To: K.Jandt@bristol.ac.uk NNTP-Posting-Host: net.bio.net Dear Sir/Madam, Would you please be son kind to put the following PhD studentship vacancy on your web pages? Thanks+kind regards K Jandt ********************************************** The Biomaterials Science Group Department of Oral and Dental Science University of Bristol Lower Maudlin Street, Bristol, BS1 2LY, England For a PhD Studentship we are looking for Student with a background in either Physics, Chemistry, Biochemistry Food Science, Biology or Materials Science or a student of related areas. The project is concerned with the study of de- and remineralisation of biological materials, employing the exciting new technique of atomic force microscopy (AFM) to measure processes at nanometre resolution. This is an excellent opportunity to perform novel research within a multidisciplinary and international team and to interact strongly with an industrial partner to gain insight into commercial applications of such research. An attractive maintenance grant and additional training (computing, soft skills, statistics etc.) will be provided. The University of Bristol (http://www.bris.ac.uk./) is one of the leading research universities in Europe and is located in the attractive south-west of England. Applicants should have or expect to gain at least a 2.1 (good) Honours Degree in an appropriate subject. Applications are invited from now on until the position has been filled. A full application including two references should be sent to: Dr. Klaus D. Jandt Senior Lecturer Dental Materials Science and Biomaterials University of Bristol Department of Oral and Dental Science Lower Maudlin Street Bristol, BS1 2LY, UK Phone: ++ 44 (0)117 9 28 44 18 Fax: ++ 44 (0) 117 9 28 47 80 Internet: K.Jandt@bris.ac.uk -------------------------------------------------------------------------------- Dr. rer. nat. Klaus D. Jandt Senior Lecturer in Dental Materials Science and Biomaterials University of Bristol, Department of Oral and Dental Science Lower Maudlin Street, Bristol, BS1 2LY, UK Phone: ++ 44 (0) 117 9 28 44 18, Fax: ++ 44 (0) 117 9 28 47 80 Internet: K.Jandt@bris.ac.uk WWW: http://www.dent.bris.ac.uk/Biomaterials/kdj.htm "We make Biomaterials Science work!" From owner-proteins@net.bio.net Thu Aug 05 05:57:00 1999 Path: biosci!news.stanford.edu!newsfeed.stanford.edu!newsfeed.berkeley.edu!news.maxwell.syr.edu!newsfeed.icl.net!newsfeed.icl.net!nntp.news.xara.net!xara.net!server6.netnews.ja.net!news.shef.ac.uk!not-for-mail From: BOP96SJH@shef.ac.uk (S J Hunter) Newsgroups: bionet.molbio.proteins Subject: aminotransferase assays Date: 5 Aug 1999 13:46:19 GMT Organization: Your Organization Lines: 18 Message-ID: <7oc4jb$9qp$1@bignews.shef.ac.uk> NNTP-Posting-Host: pc110165.shef.ac.uk Mime-Version: 1.0 Content-Type: Text/Plain; charset=US-ASCII X-Newsreader: WinVN 0.99.9 (Released Version) (16bit) Ergent request! If anyone has protocols for alanine aminotransferase and/or aspartate aminotransferase assays could they please mail me at bop97at@sheffieldac.uk. I would be eternally grateful. Thanks very much Anna Taylor, University of Sheffield, Animal and Plant Sciences, Alfred Denny Building, Western Bank, Sheffield, S10 2TN England Tel: 0114 22 20115 From owner-proteins@net.bio.net Thu Aug 05 08:16:00 1999 Path: biosci!UIC.EDU!jhong1 From: jhong1@UIC.EDU (Joo Yun Hong) Newsgroups: bionet.molbio.proteins Subject: appratus for screening monoclonal antibodies??? Date: 5 Aug 1999 09:15:54 -0700 Organization: University of IL at Chicago Lines: 17 Sender: daemon@net.bio.net Distribution: world Message-ID: <37A9B736.1CDFD4CD@uic.edu> NNTP-Posting-Host: net.bio.net hello!! i'm using hydridoma supernatant to screen monoclonal antibody about protein on a membrane. since the amount of supernatant i can use is limited to a small volume, i used this appratus named 'multi-protean2' from the bio-rad. but it has been giving me a problem of leakage between the wells. so i was wondering if anyone could tell me about similar kind of appratus using a membrane to screen mutiple samples of hydridoma supernatant or have any idea of eliminating the leakage problem. thank you joo jhong1@uic.edu From owner-proteins@net.bio.net Thu Aug 05 08:30:00 1999 Path: biosci!news.stanford.edu!newsfeed.stanford.edu!newsfeed.berkeley.edu!newsfeed.germany.net!newsfeed.nacamar.de!univ-lyon1.fr!cri.ens-lyon.fr!news From: Stephane Ory Newsgroups: bionet.molbio.proteins Subject: anti-chimaerin antibody Date: Thu, 05 Aug 1999 18:08:47 +0100 Organization: Ecole Normale Superieure de Lyon, France Lines: 4 Message-ID: <37A9C51D.57FF@ens-lyon.fr> Reply-To: Stephane.Ory@ens-lyon.fr NNTP-Posting-Host: mac-lr5-139b.ens-lyon.fr Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: quoted-printable X-Mailer: Mozilla 3.01 [fr] (Macintosh; I; PPC) Does anybody know a commercial source of an anti-chimaerin antibody? Any answer would be appreciate. St=E9phane From owner-proteins@net.bio.net Sat Aug 07 14:58:00 1999 Path: biosci!NCBI.NLM.NIH.GOV!galperin From: galperin@NCBI.NLM.NIH.GOV (Michael Galperin) Newsgroups: bionet.molbio.proteins Subject: Position in genome analysis at the NCBI Date: 7 Aug 1999 15:58:43 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 41 Sender: daemon@net.bio.net Distribution: world Message-ID: <37ACB8A6.C8406338@ncbi.nlm.nih.gov> NNTP-Posting-Host: net.bio.net Biologist, Protein Classification Analyst Scientific data analyst to participate in a protein family classification project. The project is based on the database of a clusters of orthologous genes (COGs) from complete genomes recently developed at NCBI. For details see http://www.ncbi.nlm.nih.gov/COG/ or Tatusov et al. (1997) Science 278: 631-637; Koonin et al. (1998) Curr. Opin. Struct. Biol. 8: 355-363; Makarova et al. (1999) Genome Res. 9: 608-628. Responsibilities include review, analysis, and functional and phylogenetic annotation of protein sequence families, making extensive use of NCBI's specialized and general sequence analysis software tools. Excellent opportunity to use state-of-art computer systems and gain experience using bioinformatics tools developed by a leading computational biology research group. This is not a laboratory position, but molecular biology lab experience is needed to understand and interpret the information in the database records. The project involves a strong research component; the successful applicant will have opportunities to participate in scientific publications. This position is with ComputerCraft Corporation, working as a contractor on location at NCBI. MS or PhD in molecular biology or related field required. Excellent oral and written communication skills required. Experience with computers, UNIX operating system, and sequence analysis software also important. Please mail, fax or e-mail resume, cover letter, and references to: ComputerCraft Corporation Attn: Molecular Biology 6701 Democracy Boulevard Suite 401 Bethesda, MD 20817 fax (301) 530-0634 e-mail: ghill@computercraft-usa.com © 1999 The Washington Post. Date Posted: 07/25/99 From owner-proteins@net.bio.net Mon Aug 09 04:07:00 1999 Path: biosci!news.stanford.edu!newsfeed.stanford.edu!news-feed.inet.tele.dk!bofh.vszbr.cz!news.algonet.se!algonet!newsfeed.tli.de!do.de.uu.net!news-koe1.dfn.de!uni-muenster.de!expmac19.uni-muenster.de!user From: RNA.world@uni-muenster.de (Juergen Brosius) Newsgroups: bionet.molbio.proteins Subject: needed: additional dye markers (BP, XC) for native protein gels Date: Mon, 09 Aug 1999 13:59:14 +0100 Organization: University of Muenster Lines: 20 Sender: "Juergen Brosius" Message-ID: NNTP-Posting-Host: expmac19.uni-muenster.de X-Trace: redenix.uni-muenster.de 934200066 21790 128.176.61.232 (9 Aug 1999 12:01:06 GMT) X-Complaints-To: abuse@uni-muenster.de NNTP-Posting-Date: 9 Aug 1999 12:01:06 GMT For native acrylamide gels (run in Tris-glycine buffer) we are looking for dyes that would migrate significantly slower than bromophenolblue (BP) or xylene cyanol (XC). Stained proteins are OK but not as desirable. We'd like to exclude a lot of low MW material before we start collecting fractions on a preparative electrophoresis cell. Size of the RNP complexex to be isolated is ~150-350 kD Thanks for any hints, Juergen -- Juergen Brosius Inst. f. Xp. Path./Molecular Neurobiology University of Muenster Von-Esmarch-Str. 56 D-48149 Muenster, Germany Fax: +49 251 835 8512 Tel: +49 251 835 8511 From owner-proteins@net.bio.net Mon Aug 09 12:33:00 1999 Path: biosci!dfo-mpo.gc.ca!MorinY2 From: MorinY2@dfo-mpo.gc.ca ("Morin, Yves") Newsgroups: bionet.molbio.proteins Subject: AKTAFPLC Date: 9 Aug 1999 13:33:26 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 10 Sender: daemon@net.bio.net Distribution: world Message-ID: <3B675D1AC3E1D111BC1600805FEACC676C331C@lauimls05.qc.dfo.ca> NNTP-Posting-Host: net.bio.net We are thinking of purchasing a AKTA FPLC (chromatography system for proteins purification) from Amersham Pharmacia. Does anyone out there have experience or opinion about that system and is there any "bug" if we install the program (Unicorn) in our computer instead of the one the compagny want to sell us? My e-mail is : MorinY2@dfo-mpo.gc.ca Thank you! From owner-proteins@net.bio.net Tue Aug 10 06:30:00 1999 Path: biosci!igr.fr!cprivenz From: cprivenz@igr.fr ("Dr. Cyril V. Privezentzev") Newsgroups: bionet.molbio.proteins Subject: bacterial inclusion bodies Date: 10 Aug 1999 07:30:09 -0700 Organization: IGR Lines: 21 Sender: daemon@net.bio.net Distribution: world Message-ID: <37B0A370.18AB@igr.fr> NNTP-Posting-Host: net.bio.net Dear Netters: Here I've read a lot of info how to handle with bacterial inclusion bodies. However, I'd like to know how could one detect them? Any help will be highly appreciated. Thanx in advance, -- Dr. Cyril V. Privezentzev Group "Reparation des Lesions Radio- et Chimioinduites CNRS UMR 8532 Institut Gustave-Roussy Pavillon de Recherche II 39 rue Camille Desmoulins 94805 Villejuif Cedex FRANCE Tel. +33 (0)1 42 11 54 04 Fax +33 (0)1 42 11 52 76 From owner-proteins@net.bio.net Tue Aug 10 08:06:00 1999 Path: biosci!newshost.lanl.gov!logbridge.uoregon.edu!newsfeed.cwix.com!198.147.221.40!news.xnet.com!news.pprd.abbott.com!not-for-mail From: Owen McCall Newsgroups: bionet.molbio.proteins Subject: Re: bacterial inclusion bodies Date: Tue, 10 Aug 1999 10:57:40 -0500 Organization: Abbott Laboratories Lines: 29 Message-ID: <37B04BF4.E603CC28@intronabbott.com> References: <37B0A370.18AB@igr.fr> NNTP-Posting-Host: lc942596.dhcp.pprd.abbott.com Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: fizban.pprd.abbott.com 934300793 19207 10.251.241.77 (10 Aug 1999 15:59:53 GMT) X-Complaints-To: usenet@fizban.pprd.abott.com NNTP-Posting-Date: 10 Aug 1999 15:59:53 GMT X-Mailer: Mozilla 4.51 [en] (Win95; I) X-Accept-Language: en Look at the cells under the microscope; inclusion bodies are easily visible. Owen McCall "Dr. Cyril V. Privezentzev" wrote: > Dear Netters: > Here I've read a lot of info how to handle with bacterial inclusion > bodies. > However, I'd like to know how could one detect them? Any help will be > highly appreciated. > > Thanx in advance, > > -- > Dr. Cyril V. Privezentzev > Group "Reparation des Lesions > Radio- et Chimioinduites > CNRS UMR 8532 > Institut Gustave-Roussy > Pavillon de Recherche II > 39 rue Camille Desmoulins > 94805 Villejuif Cedex > FRANCE > > Tel. +33 (0)1 42 11 54 04 > Fax +33 (0)1 42 11 52 76 From owner-proteins@net.bio.net Tue Aug 10 17:02:00 1999 Path: biosci!UMICH.EDU!rozsa From: rozsa@UMICH.EDU ("frank rozsa") Newsgroups: bionet.molbio.proteins Subject: Date: 10 Aug 1999 18:02:10 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 35 Sender: daemon@net.bio.net Distribution: world Message-ID: <199908110055.UAA01245@runningman.rs.itd.umich.edu> NNTP-Posting-Host: net.bio.net Hi protein experts, I'm using Invitrogen's pTrcHis vector (without IPTG and overnight growth) to express a protein with an engineered His tag. I tried with and without IPTG and found better expression without it, so much for induction, though maybe this thing is toxic at too high expression levels. After trying to remove the His tag with dubious success, gave up and am just trying to elute the protein from the column using various imidazole concentrations. I need to optimize the protocol on a small scale (from 100 ml of E. coli) before moving up to larger scale. I'm using Western Breeze immunoblot system with rabbit polyclonals. Got alot of E.coli junk so increased the volume of pre-elution wash many fold and eluted with multiple volumes of increasing imidazole in 25mM increases in effort to elute off E.coli proteins and try to hit the "sweet spot" for this protein. Now my samples there but it's in imidazole and way too dilute. I'm a newbie in protein work so forgive the following naive questions: 1. Does imidazole interfere with SDS-PAGE? If not, can I use the blot for subsequent western blots? 2. Anyone have any idea if imidazole is toxic to Cos7 cells. I want to see if this protein binds to tissue culture cells. Just looking for a heads up answer here, I know this is easy to check. 3. I can dialyze the protein out of imidazole (vs 10 mM Tris, 0.5% Triton), but when I concentrate using an amicon filter, the sample precipitates. What is a good buffer to store protein samples in at -80? 4. What about an ice cold acetone precipitation? Is this considered "denaturing"? I've heard that detergents form a slimy, difficult to solubilize pellet in acetone. Any hints about maintaining biological integrity using acetone precipitation? Thanks in advance! From owner-proteins@net.bio.net Tue Aug 10 17:06:00 1999 Path: biosci!UMICH.EDU!rozsa From: rozsa@UMICH.EDU ("frank rozsa") Newsgroups: bionet.molbio.proteins Subject: Properties of proteins purified in imidazole Date: 10 Aug 1999 18:06:04 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 35 Sender: daemon@net.bio.net Distribution: world Message-ID: <199908110059.UAA01316@runningman.rs.itd.umich.edu> NNTP-Posting-Host: net.bio.net Hi protein experts, I'm using Invitrogen's pTrcHis vector (without IPTG and overnight growth) to express a protein with an engineered His tag. I tried with and without IPTG and found better expression without it, so much for induction, though maybe this thing is toxic at too high expression levels. After trying to remove the His tag with dubious success, gave up and am just trying to elute the protein from the column using various imidazole concentrations. I need to optimize the protocol on a small scale (from 100 ml of E. coli) before moving up to larger scale. I'm using Western Breeze immunoblot system with rabbit polyclonals. Got alot of E.coli junk so increased the volume of pre-elution wash many fold and eluted with multiple volumes of increasing imidazole in 25mM increases in effort to elute off E.coli proteins and try to hit the "sweet spot" for this protein. Now my samples there but it's in imidazole and way too dilute. I'm a newbie in protein work so forgive the following naive questions: 1. Does imidazole interfere with SDS-PAGE? If not, can I use the blot for subsequent western blots? 2. Anyone have any idea if imidazole is toxic to Cos7 cells. I want to see if this protein binds to tissue culture cells. Just looking for a heads up answer here, I know this is easy to check. 3. I can dialyze the protein out of imidazole (vs 10 mM Tris, 0.5% Triton), but when I concentrate using an amicon filter, the sample precipitates. What is a good buffer to store protein samples in at -80? 4. What about an ice cold acetone precipitation? Is this considered "denaturing"? I've heard that detergents form a slimy, difficult to solubilize pellet in acetone. Any hints about maintaining biological integrity using acetone precipitation? Thanks in advance! From owner-proteins@net.bio.net Tue Aug 10 17:08:00 1999 Path: biosci!rutgers!gatech!pitt.edu!portc02.blue.aol.com!howland.erols.net!newsfeed.fast.net!uunet!ffx.uu.net!news7-gui.server.ntli.net!ntli.net!not-for-mail From: Newsgroups: bionet.molbio.proteins Subject: Assistance required from anyone in the UK Date: 11 Aug 1999 00:45:54 GMT Organization: NTL Internet News Service Lines: 12 Message-ID: <7oqh42$l5k$2555@nclient7-gui.server.ntli.net> NNTP-Posting-Host: p44-castor-gui.tch.cableol.net X-Trace: nclient7-gui.server.ntli.net 934332354 21684 194.168.5.44 (11 Aug 1999 00:45:54 GMT) X-Complaints-To: postmaster@ntli.net NNTP-Posting-Date: 11 Aug 1999 00:45:54 GMT Hello, people . . . Has anyone in the UK tried Totalserve free access because Ive been asked to market it and I'd never heard of it before. Its new but I would like some feedback about it as I don't want to add a naff service to my range!! Me and a few people I know have tried it and it seems either very stable and fast or completely unable to connect depending what hardware you use!!!! If there is anyone who isn't on it who wants to give it a whirl and give some feedback I'd be grateful, you can access it through my site at http://www.puk.co.uk you can hook up direct from there without a disc. You can either reply to this with feedback or email me at web@puk.co.uk Many thanks for any assistance you can provide Adrian Paris 21st Century UK Internet and Telecoms Services www.puk.co.uk web@puk.co.uk I appolagise if this is a repost, my program crashed last time . . . From owner-proteins@net.bio.net Wed Aug 11 19:49:00 1999 Path: biosci!news.stanford.edu!newsfeed.stanford.edu!newsfeed.berkeley.edu!nntp.abs.net!newshub2.home.com!news.home.com!news1.gvcl1.bc.home.com.POSTED!not-for-mail Reply-To: "Achim Recktenwald" From: "Achim Recktenwald" Newsgroups: bionet.molbio.proteins References: <199908110059.UAA01316@runningman.rs.itd.umich.edu> Subject: Re: Properties of proteins purified in imidazole Lines: 48 X-Newsreader: Microsoft Outlook Express 5.00.2314.1300 X-MimeOLE: Produced By Microsoft MimeOLE V5.00.2314.1300 Message-ID: <%ors3.752$Kh.3722@news1.gvcl1.bc.home.com> Date: Thu, 12 Aug 1999 03:42:51 GMT NNTP-Posting-Host: 24.64.220.105 X-Complaints-To: abuse@home.net X-Trace: news1.gvcl1.bc.home.com 934429371 24.64.220.105 (Wed, 11 Aug 1999 20:42:51 PDT) NNTP-Posting-Date: Wed, 11 Aug 1999 20:42:51 PDT Organization: @Home Network Canada "frank rozsa" wrote in message news:199908110059.UAA01316@runningman.rs.itd.umich.edu... > Hi protein experts, > [snip] > 3. I can dialyze the protein out of imidazole (vs 10 mM Tris, 0.5% Triton), > but when I concentrate using an amicon filter, the sample precipitates. > What is a good buffer to store protein samples in at -80? > Your ion concentration is rather low; some proteins like it, others not. I don't think , anybody has found a rule yet. Try adding some salt (e.g., 150mM NaCl) or dialyze it agains PBS. You could also try adding up to 20% glycerol, it often helps keeping proteins in solution. In case your protein is not properly folded and/or contains free cysteins, you might want to add a reducing agent, like beta-mercaptoethanol, DTT. Tris is a very bad buffer for storage at -80C. Due to the dpKa/C value of -0.031 for Tris , your pH adjusted at 25C will rise by almost 3.3 pH-units at -80C. The dpKa/C for phosphate is only 1/10 of the value for Tris; therefore, the pH will rise by about 0.33 units. You can find dpKa/C-values in many buffer listings. > 4. What about an ice cold acetone precipitation? Is this considered > "denaturing"? I've heard that detergents form a slimy, difficult to > solubilize pellet in acetone. Any hints about maintaining biological > integrity using acetone precipitation? Acetone precipitation at low temperatures is very mild and not denaturing; it is in its treatment of proteins similar to ammonium sulfate precipitation. I would recommend cooling your stock of acetone to -20C, and if possible your centrifugation rotor, as well. Otherwise it is very difficult to keep the temperature sufficiently low during the whole procedure. Why do you need a detergent in your buffer? Are you purifying a membrane protein? If not, have you tried purifying it without detergent? Cheers, Achim From owner-proteins@net.bio.net Wed Aug 11 23:21:00 1999 Path: biosci!newshost.lanl.gov!logbridge.uoregon.edu!fu-berlin.de!news.rwth-aachen.de!post.student.mu-luebeck.de!not-for-mail From: "Lars Komorowski" Newsgroups: bionet.molbio.proteins Subject: Cytochromes Date: Thu, 12 Aug 1999 08:58:20 +0200 Organization: Med. Universitaet zu Luebeck Lines: 5 Message-ID: <7otqu1$n1t$1@post.student.mu-luebeck.de> NNTP-Posting-Host: wahnsinn.biochem.mu-luebeck.de X-Trace: post.student.mu-luebeck.de 934440705 23613 141.83.167.171 (12 Aug 1999 06:51:45 GMT) X-Complaints-To: usenet@student.mu-luebeck.de NNTP-Posting-Date: 12 Aug 1999 06:51:45 GMT X-Priority: 3 X-MSMail-Priority: Normal X-Newsreader: Microsoft Outlook Express 5.00.2014.211 X-MimeOLE: Produced By Microsoft MimeOLE V5.00.2014.211 Can anybody tell me of a good site with spectroscopic features of cytochromes ? Lars From owner-proteins@net.bio.net Thu Aug 12 03:36:00 1999 Path: biosci!news.stanford.edu!newsfeed.stanford.edu!newsfeed.berkeley.edu!news.maxwell.syr.edu!newsfeed.nacamar.de!blackbush.xlink.net!news-kar1.dfn.de!news-han1.dfn.de!news.gwdg.de!not-for-mail From: Marc Saric Newsgroups: bionet.molbio.proteins Subject: Re: Properties of proteins purified in imidazole Date: Thu, 12 Aug 1999 12:28:48 +0200 Organization: Max Planck Institut =?iso-8859-1?Q?f=FCr?= molekulare Physiologie Lines: 19 Message-ID: <37B2A1E0.BCB1D658@mpi-dortmund.mpg.de> References: <199908110059.UAA01316@runningman.rs.itd.umich.edu> NNTP-Posting-Host: m99106.mpi-dortmund.mpg.de Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Trace: gwdu67.gwdg.de 934453728 52589 141.5.193.151 (12 Aug 1999 10:28:48 GMT) X-Complaints-To: news@gwdg.de NNTP-Posting-Date: 12 Aug 1999 10:28:48 GMT X-Mailer: Mozilla 4.6 [de] (WinNT; I) X-Accept-Language: de frank rozsa schrieb: > 1. Does imidazole interfere with SDS-PAGE? If not, can I use the blot for > subsequent western blots? No experience with Western Blotting, but it should work for normal SDS-Gels (no problems here with a wider range of soluble proteins expressed in E.coli). -- Bye, Marc Saric Max-Planck-Institut für molekulare Physiologie Otto-Hahn-Strasse 11 44227 Dortmund phone: +49 231 133 2168 From owner-proteins@net.bio.net Thu Aug 12 06:06:00 1999 Path: biosci!NETSGO.COM!choibear From: choibear@NETSGO.COM ("=?euc-kr?B?w9aw5sij?=") Newsgroups: bionet.molbio.proteins Subject: about imidazole Date: 12 Aug 1999 07:06:17 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 61 Sender: daemon@net.bio.net Distribution: world Message-ID: <000701bee4c9$50a36ec0$afb96fd2@--------> Reply-To: "=?euc-kr?B?w9aw5sij?=" NNTP-Posting-Host: net.bio.net This is a multi-part message in MIME format. ------=_NextPart_000_0004_01BEE514.BFBA0B20 Content-Type: text/plain; charset="euc-kr" Content-Transfer-Encoding: base64 SGkgIQ0KSSBoYXZlIHNvbWUgcHJvYmxlbSBpbiBteSBleHBlcmltZW50IGFuZCBwbGVhc2UgaGVs cCBtZS4NCkkgZXhwcmVzc2VkICBhIGJhY3RlcmlhbCB0b3hpbiBwcm90ZWluIGluIEJMMjEgREUz IGNlbGxzICB1c2luZyAgcEVUMTViIGV4cHJlc3Npb24gdmVjdG9yLCBhbmQgcHVyaWZpZWQgIHRo cm91Z2ggTmktTlRBIHJlc2luKE5vdmFnZW4pLiBJIGZpbmFsbHkgZWx1dGVkIHRhcmdldCBwcm90 ZWluIGluIDFNIGltaWRhem9sZSBidWZmZXIsIGJ1dCBJIGNvdWxkIG5vdCBkaWFseXplIG91dCBp bWlkYXpvbGUuIFdoZW4gSSBkaWFseXplZCBpdCBpbnRvIFBCUywgaXQgd2VudCBpbnRvIG1hc3Np dmUgcHJlY2lwaXRhdGlvbi4gQWRkaXRpb24gb2YgaW1pZGF6b2xlIHRvIHRoZSBjb25jZW50cmF0 aW9uIG9mIDI1MG1NIGhhcyBtYWRlIGl0IHRvIGJlIHNvbHVibGUgYWdhaW4uDQpJIHdvbmRlciB3 aHkgdGhlIGhpZ2ggY29uY2VudHJhdGlvbiBvZiBpbWlkYXpvbGUgaXMgbmVjZXNzYXJ5IGZvciB0 aGUgc29sdWJpbGl0eSBvZiBwdXJpZmllZCBIaXMtdGFnZ2VkIHByb3RlaW4sIGJlY2F1c2UgaXQg d2FzIHN0aWxsIHNvbHVibGUgaW4gdGhlIGNydWRlIGx5c2F0ZSBpbiBiaW5kaW5nIGJ1ZmZlciB3 aGljaCBjb250YWlucyBvbmx5IDVtTSBpbWlkYXpvbGUuDQpIYXMgYW55b25lIGV4cGVyaWVuY2Vk IGFuZCBzb2x2ZWQgdGhpcyBzb2x1YmlsaXR5IHByb2JsZW0/IElmIHNvbWVvbmUgaGFzLCBwbGVh c2UgbGV0IG1lIGtub3cgaG93IHRvLg0KSSBzaG91bGQgZ2V0IHJpZCBvZiBpbWlkYXpvbGUgdG8g cHJvY2VlZCBmdXJ0aGVyLg0KDQpLeXVuZyBIbyBDaG9pLCBNRCwgUGhEDQpEZXBhcnRtZW50IG9m IGJpb2NoZW1pc3RyeSwgY29sbGVnZSBvZiBtZWRpY2luZSwgU2VvdWwgTmF0aW9uYWwgVW5pdmVy c2l0eQ0KRS1tYWlsIDogY2hvaWRvY0B5YWhvby5jb20NCiAgICAgICAgICAgOiBjaG9pYmVhckBu ZXRzZ28uY29tDQo= ------=_NextPart_000_0004_01BEE514.BFBA0B20 Content-Type: text/html; charset="euc-kr" Content-Transfer-Encoding: base64 PCFET0NUWVBFIEhUTUwgUFVCTElDICItLy9XM0MvL0RURCBXMyBIVE1MLy9FTiI+DQo8SFRNTD4N CjxIRUFEPg0KDQo8TUVUQSBjb250ZW50PSJ0ZXh0L2h0bWw7IGNoYXJzZXQ9a3NfY181NjAxLTE5 ODciIGh0dHAtZXF1aXY9Q29udGVudC1UeXBlPg0KPE1FVEEgY29udGVudD0nIk1TSFRNTCA0Ljcy LjMxMTAuNyInIG5hbWU9R0VORVJBVE9SPg0KPC9IRUFEPg0KPEJPRFkgYmdDb2xvcj0jZmZmZmZm Pg0KPERJVj48Rk9OVCBjb2xvcj0jMDAwMDAwIHNpemU9Mj5IaSAhPEJSPkkgaGF2ZSBzb21lIHBy b2JsZW0gaW4gbXkgZXhwZXJpbWVudCBhbmQgDQpwbGVhc2UgaGVscCBtZS48QlI+SSBleHByZXNz ZWQmbmJzcDsgYSBiYWN0ZXJpYWwgdG94aW4gcHJvdGVpbiBpbiBCTDIxIERFMyANCmNlbGxzJm5i c3A7IHVzaW5nJm5ic3A7IHBFVDE1YiBleHByZXNzaW9uIHZlY3RvciwgYW5kIHB1cmlmaWVkJm5i c3A7IHRocm91Z2ggDQpOaS1OVEEgcmVzaW4oTm92YWdlbikuIEkgZmluYWxseSBlbHV0ZWQgdGFy Z2V0IHByb3RlaW4gaW4gMU0gaW1pZGF6b2xlIGJ1ZmZlciwgDQpidXQgSSBjb3VsZCBub3QgZGlh bHl6ZSBvdXQgaW1pZGF6b2xlLiBXaGVuIEkgZGlhbHl6ZWQgaXQgaW50byBQQlMsIGl0IHdlbnQg aW50byANCm1hc3NpdmUgcHJlY2lwaXRhdGlvbi4gQWRkaXRpb24gb2YgaW1pZGF6b2xlIHRvIHRo ZSBjb25jZW50cmF0aW9uIG9mIDI1MG1NIGhhcyANCm1hZGUgaXQgdG8gYmUgc29sdWJsZSBhZ2Fp bi48QlI+SSB3b25kZXIgd2h5IHRoZSBoaWdoIGNvbmNlbnRyYXRpb24gb2YgaW1pZGF6b2xlIA0K aXMgbmVjZXNzYXJ5IGZvciB0aGUgc29sdWJpbGl0eSBvZiBwdXJpZmllZCBIaXMtdGFnZ2VkIHBy b3RlaW4sIGJlY2F1c2UgaXQgd2FzIA0Kc3RpbGwgc29sdWJsZSBpbiB0aGUgY3J1ZGUgbHlzYXRl IGluIGJpbmRpbmcgYnVmZmVyIHdoaWNoIGNvbnRhaW5zIG9ubHkgNW1NIA0KaW1pZGF6b2xlLjxC Uj5IYXMgYW55b25lIGV4cGVyaWVuY2VkIGFuZCBzb2x2ZWQgdGhpcyBzb2x1YmlsaXR5IHByb2Js ZW0/IElmIA0Kc29tZW9uZSBoYXMsIHBsZWFzZSBsZXQgbWUga25vdyBob3cgdG8uPEJSPkkgc2hv dWxkIGdldCByaWQgb2YgaW1pZGF6b2xlIHRvIA0KcHJvY2VlZCBmdXJ0aGVyLjxCUj48L0ZPTlQ+ PC9ESVY+DQo8RElWPjxGT05UIGNvbG9yPSMwMDAwMDAgc2l6ZT0yPkt5dW5nIEhvIENob2ksIE1E LCBQaEQ8QlI+RGVwYXJ0bWVudCBvZiANCmJpb2NoZW1pc3RyeSwgY29sbGVnZSBvZiBtZWRpY2lu ZSwgU2VvdWwgTmF0aW9uYWwgVW5pdmVyc2l0eTxCUj5FLW1haWwgOiA8QSANCmhyZWY9Im1haWx0 bzpjaG9pZG9jQHlhaG9vLmNvbSI+Y2hvaWRvY0B5YWhvby5jb208L0E+PEJSPiZuYnNwOyZuYnNw OyZuYnNwOyZuYnNwOyZuYnNwOyZuYnNwOyZuYnNwOyZuYnNwOyZuYnNwOyZuYnNwOyANCjogPEEg DQpocmVmPSJtYWlsdG86Y2hvaWJlYXJAbmV0c2dvLmNvbSI+Y2hvaWJlYXJAbmV0c2dvLmNvbTwv QT48L0ZPTlQ+PC9ESVY+PC9CT0RZPjwvSFRNTD4NCg== ------=_NextPart_000_0004_01BEE514.BFBA0B20-- From owner-proteins@net.bio.net Thu Aug 12 13:17:00 1999 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.berkeley.edu!logbridge.uoregon.edu!nntp.upenn.edu!ovh173.vetschool.upenn.edu!user From: hines@pharm.med.upenn.edu (John Hines) Newsgroups: bionet.molbio.proteins Subject: a question of sufficient n's Date: Thu, 12 Aug 1999 17:13:35 -0500 Organization: University of Pennsylvania Lines: 57 Message-ID: NNTP-Posting-Host: ovh173.vetschool.upenn.edu A friend posed a question to me last night. I have my own opinion and I gave it to him. I would be interested in hearing what other scientists might think on the matter: I will change the details a little just to protect the innocent. The experiment being conducted measure the effect of a particular drug on the levels of certain liver enzymes. The levels of the enzymes will be quantitated by western blotting. In the experiment, 8 mice are treated with the drug for the prescribed time and dosage and 8 mice are treated with vehicle. Their livers are pooled according to treatment group (drug or control) and homogenized. SDS-PAGE is done and the blots are performed. Now, if the scientist runs a 7 lane gel, where: lane 1 = MW markers lane 2, 4, 6 = control homogenate lane 3, 5, 7 = drug treated homogenate and then blots the entire piece of nitrocellulose for the level of target enzyme, is that 3 replicate measurements (comparing lane 2 vs. 3; 4 vs. 5; 6 vs. 7) ?? Or is that an n of 3? or second scenario: if the scientist runs a 3 lane gel, where: lane 1 = MW markers lane 2 = control homogenate lane 3 = drug treated homogenate and blots for the enzyme. And then runs a second identical gel next week and blots that; and then runs a third identical gel the following week and blots that; is that 3 replicate measurements (remember, the very same homogenates were used in all the blots), or is that an n of 3?? The answer to me seems pretty obvious in either scenario, but I'd like to hear other opinions too. John hines@pharm.med.upenn.edu From owner-proteins@net.bio.net Thu Aug 12 20:06:00 1999 Path: biosci!newshost.lanl.gov!logbridge.uoregon.edu!howland.erols.net!cpk-news-hub1.bbnplanet.com!news.gtei.net!newshub2.home.com!newshub1.home.com!news.home.com!news1.gvcl1.bc.home.com.POSTED!not-for-mail Reply-To: "Achim Recktenwald" From: "Achim Recktenwald" Newsgroups: bionet.molbio.proteins References: Subject: Re: a question of sufficient n's Lines: 81 X-Newsreader: Microsoft Outlook Express 5.00.2314.1300 X-MimeOLE: Produced By Microsoft MimeOLE V5.00.2314.1300 Message-ID: Date: Fri, 13 Aug 1999 03:59:24 GMT NNTP-Posting-Host: 24.64.220.105 X-Complaints-To: abuse@home.net X-Trace: news1.gvcl1.bc.home.com 934516764 24.64.220.105 (Thu, 12 Aug 1999 20:59:24 PDT) NNTP-Posting-Date: Thu, 12 Aug 1999 20:59:24 PDT Organization: @Home Network Canada In order for this experiment a complete repeat, the Western would have to be repeated , as well; especially, since protein quantification by Western is of questionable reliability. What he is doing is to measure a triplicate of the same pooled sample, n=1. As long as the livers are pooled into one homogenate, it will always be only one sample pooled from 8 mice; the two options described do not change this. The difference is only in the detection method. The first option allows to monitor the variability of the same sample in one Western blot. The 2nd option is slightly better, since the variability of the Western itself can be monitored, as well. It allows to obtain more information on the accuracy and precision of the detection method. Achim John Hines wrote in message news:hines-1208991713350001@ovh173.vetschool.upenn.edu... > > A friend posed a question to me last night. I have my own opinion and I > gave it to him. I would be interested in hearing what other scientists > might think on the matter: > > I will change the details a little just to protect the innocent. > > The experiment being conducted measure the effect of a particular drug on > the levels of certain liver enzymes. The levels of the enzymes will be > quantitated by western blotting. > > In the experiment, 8 mice are treated with the drug for the prescribed > time and dosage and 8 mice are treated with vehicle. Their livers are > pooled according to treatment group (drug or control) and homogenized. > SDS-PAGE is done and the > blots are performed. > > Now, if the scientist runs a 7 lane gel, where: > > lane 1 = MW markers > > lane 2, 4, 6 = control homogenate > > lane 3, 5, 7 = drug treated homogenate > > and then blots the entire piece of nitrocellulose for the level of target > enzyme, is that 3 replicate measurements (comparing lane 2 vs. 3; 4 vs. 5; > 6 vs. 7) ?? Or is that an n of 3? > > > > > > or second scenario: > > if the scientist runs a 3 lane gel, where: > > lane 1 = MW markers > > lane 2 = control homogenate > > lane 3 = drug treated homogenate > > and blots for the enzyme. And then runs a second identical gel next week and > blots that; and then runs a third identical gel the following week and > blots that; is that 3 replicate measurements (remember, the very same > homogenates > were used in all the blots), or is that an n of 3?? > > > > The answer to me seems pretty obvious in either scenario, but I'd like to > hear other opinions too. > > John > > hines@pharm.med.upenn.edu From owner-proteins@net.bio.net Fri Aug 13 01:00:00 1999 Path: biosci!internet!biosci!not-for-mail From: biohelp (BIOSCI Administrator) Newsgroups: bionet.molbio.proteins Subject: BIOSCI/bionet miniFAQ & Fundraiser Date: 13 Aug 1999 02:00:17 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 233 Sender: daemon@net.bio.net Distribution: world Message-ID: <199908130900.CAA09561@net.bio.net> NNTP-Posting-Host: net.bio.net (LAST REVISION: 30-JUL-95) This BIOSCI "miniFAQ" is designed to answer the questions that come up the *most frequently*. The main BIOSCI FAQ (Frequently Asked Questions) is accessible on the World Wide Web at URL http://www.bio.net/. If you can not find an answer to your question in this or other documentation, the BIOSCI technical support staff answers e-mail queries sent to biosci-help@net.bio.net We can only answer questions about the use of the newsgroups and mailing lists. We unfortunately do not have the staff to do Internet information searches or answer scientific questions. Please post those to the appropriate BIOSCI/bionet newsgroups. Contents: -------- 0) BIOSCI NEEDS YOUR SUPPORT!! 1) Using the WWW to access the BIOSCI/bionet newsgroups. 2) What to do about "spams," i.e., junk mail, ads, etc. 3) Examples of subscribing and unsubscribing to the mailing lists. 4) The BIOSCI user address and research interest directory. 0) BIOSCI NEEDS YOUR SUPPORT!! ------------------------------ BIOSCI's government funding has been expended, and we are now operating solely from advertising revenue that we have raised from our Web site at http://www.bio.net/. We need just a few minutes of your time to help us serve you. You can do two important things which will take very little time for you individually and will immensely help us continue to help you. First, please use our WWW system at http://www.bio.net/ to access the archives. You can post or reply to messages via your Web browser as described in item #1 below. Your usage helps attract sponsors. If you contact any of our sponsors, please be sure to thank them for supporting BIOSCI. It is critical for them to get this feedback if they are to continue their sponsorship for the long term. Second, if you work for a company or organization that provides products or services of interest to the biology community, please pass this message on to your marketing or marketing communications department or other appropriate group. Please ask them to help support BIOSCI by sponsoring our Web site and explain the uses and benefits of the system to the biology community. If they are interested, they can then contact us for further information at our tech support address, biosci-help@net.bio.net. 1) Using the WWW to access the BIOSCI/bionet newsgroups. -------------------------------------------------------- As of 10 December 1995, all BIOSCI/bionet full newsgroups are accessible through the World Wide Web (WWW) at URL http://www.bio.net. One can read and reply publicly or privately to both recent postings and archived messages through one's Web browser if it is configured properly to send e-mail. Each newsgroup is equipped with its own WAIS index. The main BIOSCI home page also has access to the BIO-JOURNALS Table of Contents database WAIS index and the BIOSCI user address database described in another item further below. 2) What to do about "spams," i.e., junk mail, ads, etc. ------------------------------------------------------- BIOSCI is a set of parallel USENET newsgroups (the "bionet" groups), mailing lists, and a hypermail archive at URL http://www.bio.net/. The same postings are distributed on all media (except for a small number of mailing-list-only groups at net.bio.net). Unfortunately it is becoming a despicable practice on the Internet (by a few people out to make a fast buck) to do automated mass postings to thousands of newsgroups and mailing lists. These attempts to grab free advertising are refered to as "spams" in the usual, somewhat boneheaded, net terminology. USENET is more susceptible to this practice, and many spams originate on the USENET groups and then are passed on to the mailing lists. However, spammers also get lists of mailing addresses and hit these too, so neither medium is immune. What should you do personally if you get junk mail? --------------------------------------------------- Just delete it and move on without reading it further. Filing a protest is becoming increasingly useless because spammers are often disguising the addresses where the messages are sent from. Unless you really understand Internet mail systems, your attempt at protest by sending replies to the message will often end up being sent to the address of an innocent person that the spammer is victimizing. 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From owner-proteins@net.bio.net Fri Aug 13 02:03:00 1999 Path: biosci!news.stanford.edu!newsfeed.stanford.edu!newsfeed.berkeley.edu!newspeer.monmouth.com!news.belnet.be!news.uia.ac.be!treecon2.uia.ac.be From: Jeroen Raes Newsgroups: bionet.molbio.proteins,bionet.general Subject: developmental control genes Date: Fri, 13 Aug 1999 11:55:38 +0200 Organization: University of Antwerp - UIA Lines: 22 Message-ID: <37B3EB9A.1348DD00@uia.ua.ac.be> NNTP-Posting-Host: hnets.uia.ac.be Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: naxos.belnet.be 934538240 24615 143.169.254.1 (13 Aug 1999 09:57:20 GMT) X-Complaints-To: abuse@belnet.be NNTP-Posting-Date: 13 Aug 1999 09:57:20 GMT X-Mailer: Mozilla 4.61 [en] (Win98; I) X-Accept-Language: en X-NNTP-Posting-Host: treecon2.uia.ac.be Xref: biosci bionet.molbio.proteins:14679 bionet.general:33570 Hi all, Does anyone have a good general definition of developmental control genes ? Jeroen ________________________________________________________________ Jeroen Raes Research Group of Molecular Biology Dept. of Biochemistry University of Antwerp (UIA) Universiteitsplein 1 B-2620 Wilrijk BELGIUM Email: jraes@uia.ua.ac.be From owner-proteins@net.bio.net Fri Aug 13 06:26:00 1999 Path: biosci!news.stanford.edu!newsfeed.stanford.edu!logbridge.uoregon.edu!pln-w!spln!extra.newsguy.com!newsp.newsguy.com!enews4 From: "KuS" Newsgroups: bionet.molbio.proteins Subject: Freeze-drying Date: Fri, 13 Aug 1999 15:43:44 +0200 Organization: AMIS.NET Lines: 10 Message-ID: <934552090.460720@olympic.amis.net> NNTP-Posting-Host: olympic.amis.net X-Newsreader: Microsoft Outlook Express 4.72.3110.5 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3110.3 Cache-Post-Path: olympic.amis.net!unknown@mb4-25.dialup.amis.net X-Cache: nntpcache 2.3.3 (see http://www.nntpcache.org/) Hello, I would just like to know if the principles of the freeze-drying in a lyophilizator and in a speed-vac machine are the same? Thanks, Barbara (info@kos-computers.si) From owner-proteins@net.bio.net Fri Aug 13 07:36:00 1999 Path: biosci!news.stanford.edu!newsfeed.stanford.edu!paloalto-snf1.gtei.net!su-news-hub1.bbnplanet.com!washdc3-snf1!news.gtei.net!news.ums.edu!gamera.cbl.umces.edu!news.umces.edu!cbl.umces.edu!reno From: "Reno T. Nguyen" Newsgroups: bionet.molbio.proteins Subject: Re: Freeze-drying Date: Fri, 13 Aug 1999 11:22:06 -0400 Organization: University of Maryland Chesapeake Biological Laboratory Lines: 38 Message-ID: References: <934552090.460720@olympic.amis.net> NNTP-Posting-Host: cbl.umces.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII X-Trace: gamera.cbl.umces.edu 934557727 18704 131.118.224.6 (13 Aug 1999 15:22:07 GMT) X-Complaints-To: feedback@cbl.umces.edu NNTP-Posting-Date: 13 Aug 1999 15:22:07 GMT To: KuS In-Reply-To: <934552090.460720@olympic.amis.net> On Fri, 13 Aug 1999, KuS wrote: > Hello, > > I would just like to know if the principles of the freeze-drying in a > lyophilizator and in a speed-vac machine are the same? > > Thanks, > > Barbara (info@kos-computers.si) Hi Barbara, Well, although both freeze-drying (lyophilization) and speed-vac drying involve pulling a vacuum on your samples, the process of removal of water in each instrument is different. With freeze-drying, the samples are frozen, and water is removed through the process of sublimation- solid water is lost as a gas. With a speed-vac, the sample is not frozen but is usually heated in a chamber while being spun at high rpm's; liquid water is lost as a gas. Reno ************************************************************************* Reno T. Nguyen Chesapeake Biological Laboratory /----\ /-----\ / Univ. MD Center for Environmental Science / / / / P.O. Box 38 / /______/ / Solomons, MD 20688 / / \ / / / // Tel: (410) 326-7261 \_______/ \_______/ \_______/ (410) 326-7409 Fax: (410) 326-7341 E-mail: reno@cbl.umces.edu ************************************************************************ From owner-proteins@net.bio.net Fri Aug 13 08:04:00 1999 Path: biosci!newshost.lanl.gov!logbridge.uoregon.edu!remarQ73!supernews.com!remarQ.com!remarQ69!WReNclone!WReNphoon3.POSTED!WReN!not-for-mail From: Nick Theodorakis Subject: Re: Freeze-drying Newsgroups: bionet.molbio.proteins Message-ID: <17599f0b.f4b92069@usw-ex0102-016.remarq.com> Lines: 38 Bytes: 1148 X-Originating-Host: 128.151.74.69 Organization: http://www.remarq.com: The World's Usenet/Discussions Start Here References: <934552090.460720@olympic.amis.net> X-Wren-Trace: eBQxGRgBRgxHWgkfGVsZHBU4EBkUH1ANAxkQHg4gGEYWERoHFVcJHwsBFQNUUQMWVUlKQ00aISU6I1w6Ii0= Date: Fri, 13 Aug 1999 08:55:20 -0700 NNTP-Posting-Host: 10.0.2.16 X-Complaints-To: wrenabuse@remarq.com X-Trace: WReNphoon3 934559600 10.0.2.16 (Fri, 13 Aug 1999 08:53:20 PDT) NNTP-Posting-Date: Fri, 13 Aug 1999 08:53:20 PDT In article , "Reno T. Nguyen" wrote: >On Fri, 13 Aug 1999, KuS wrote: > >> Hello, >> >> I would just like to know if the principles of the freeze-drying in a >> lyophilizator and in a speed-vac machine are the same? >> >> Thanks, >> >> Barbara (info@kos-computers.si) > > >Hi Barbara, > >Well, although both freeze-drying (lyophilization) and speed-vac drying >involve pulling a vacuum on your samples, the process of removal of water >in each instrument is different. With freeze-drying, the samples are >frozen, and water is removed through the process of sublimation- solid >water is lost as a gas. With a speed-vac, the sample is not frozen but is >usually heated in a chamber while being spun at high rpm's; liquid water >is lost as a gas. > >Reno > Nothing prevents you from using the speed-vac as a lyophilizer, however, by freezing your samples first (as I do). Nick * Sent from RemarQ http://www.remarq.com The Internet's Discussion Network * The fastest and easiest way to search and participate in Usenet - Free! From owner-proteins@net.bio.net Fri Aug 13 23:31:00 1999 Path: biosci!news.stanford.edu!newsfeed.stanford.edu!newsfeed.berkeley.edu!ihnp4.ucsd.edu!news.scripps.edu!not-for-mail From: "Antonin Tutter" Newsgroups: bionet.molbio.proteins,bionet.general Subject: Re: developmental control genes Date: Fri, 13 Aug 1999 15:08:38 -0700 Organization: University of California, San Deigo Lines: 26 Message-ID: <7p2517$64s$1@hermes.scripps.edu> References: <37B3EB9A.1348DD00@uia.ua.ac.be> NNTP-Posting-Host: rbio05.salk.edu Mime-Version: 1.0 Content-Type: text/plain; charset="US-ASCII" Content-Transfer-Encoding: 7bit X-Newsreader: Microsoft Outlook Express Macintosh Edition - 4.5 (0410) Xref: biosci bionet.molbio.proteins:14684 bionet.general:33576 > Does anyone have a good general definition of developmental control > genes ? i don't know if an 'official' definition exists, but my own definition, stemming from my own work on a 'developmental control' gene, is that it is a gene that regulates certain other downstream genes (usually in trans in the form of a transcription factor)) to ensure they only express at precisely defined times during the development of an organism, such as during mesoderm induction, segment polarity, limb development, etc. these control genes usually get switched on as a result of a signalling pathway, and in turn regulate other downstream genes that will determine a particular fate for the cell in which they are being expressed. examples are the homeobox genes and the wnt genes. hope this sheds some light... _______________________________________ Antonin Tutter Salk Institute for Biological Studies RBIO-J 10010 N. Torrey Pines Rd. La Jolla, CA 92037 email: tutter@salk.edu web: http://www-biology.ucsd.edu/~atutter/ From owner-proteins@net.bio.net Sat Aug 14 08:12:00 1999 Path: biosci!igr.fr!cprivenz From: cprivenz@igr.fr ("Dr. Cyril V. Privezentzev") Newsgroups: bionet.molbio.proteins Subject: inclusion bodies Date: 14 Aug 1999 09:11:55 -0700 Organization: IGR Lines: 20 Sender: daemon@net.bio.net Distribution: world Message-ID: <37B60141.4153@igr.fr> NNTP-Posting-Host: net.bio.net > To: protein-analysis@net.bio.net > > From: Owen McCall > > Subject: Re: bacterial inclusion bodies > > Date: Tue, 10 Aug 1999 10:57:40 -0500 > > Look at the cells under the microscope; inclusion bodies are easily > visible. > > Owen McCall > OK. But shoul cells be fixed and stained or just put cell suspension on the glass? Cyril From owner-proteins@net.bio.net Sat Aug 14 14:20:00 1999 From: wcmziv@yahoo.com Newsgroups: bionet.molbio.proteins Subject: XXX Fucking Pics NNTP-Posting-Host: 207233.aebc.com Message-ID: <37b5e9c4$2@news.vphos.net> Date: 14 Aug 1999 15:12:20 -0800 X-Trace: 14 Aug 1999 15:12:20 -0800, 207233.aebc.com Lines: 3 Path: biosci!bloom-beacon.mit.edu!eecs-usenet-02.mit.edu!netnews.com!newspeer.monmouth.com!news.vphos.net!207233.aebc.com XXX PICS XXX PICS http://www.nettaxi.com/fcitizens/adult168/dp/p3.htm From owner-proteins@net.bio.net Mon Aug 16 09:09:00 1999 Path: biosci!newshost.lanl.gov!logbridge.uoregon.edu!news.maxwell.syr.edu!easynet-tele!easynet.net!news.vas-net.net!server2.netnews.ja.net!leicester!usenet From: "A.F. Simpson" Newsgroups: bionet.molbio.proteins Subject: Re: a question of sufficient n's Date: Mon, 16 Aug 1999 18:05:12 -0700 Organization: University of Leicester Lines: 51 Message-ID: <37B8B548.5DEC@le.ac.uk> References: NNTP-Posting-Host: pc42.cmht.le.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: rook.le.ac.uk 934822900 255933 143.210.176.54 (16 Aug 1999 17:01:40 GMT) X-Complaints-To: usenet@le.ac.uk NNTP-Posting-Date: 16 Aug 1999 17:01:40 GMT X-Mailer: Mozilla 3.01 (Win16; I) John Hines wrote: > In the experiment, 8 mice are treated with the drug for the prescribed > time and dosage and 8 mice are treated with vehicle. Their livers are > pooled according to treatment group (drug or control) and homogenized. > SDS-PAGE is done and the blots are performed. > > Now, if the scientist runs a 7 lane gel, where: > lane 1 = MW markers > lane 2, 4, 6 = control homogenate > lane 3, 5, 7 = drug treated homogenate > and then blots the entire piece of nitrocellulose for the level of target > enzyme, is that 3 replicate measurements (comparing lane 2 vs. 3; 4 vs. 5; > 6 vs. 7) ?? Or is that an n of 3? > > or second scenario: > if the scientist runs a 3 lane gel, where: > lane 1 = MW markers > lane 2 = control homogenate > lane 3 = drug treated homogenate > and blots for the enzyme. And then runs a second identical gel next week and > blots that; and then runs a third identical gel the following week and > blots that; is that 3 replicate measurements (remember, the very same > homogenates were used in all the blots), or is that an n of 3?? (sorry for the extensive quote, but I couldn't find a way of trimming it and still have it make sense for people who hadn't seen the firth post.) In both cases I would say n=1, because you only have one sample for each condition - the homogenised liver pools for treament and control. You have no replication because you only performed the actual treatment experiment once. What the two proceedures do give you is an estimte of the repeatability of the measurement ("3 replicate measurements"). The first protocol control for variation of loading etc within a single western, the second for the repeatability of the western. I would say that triplicate measurements are the minimum, especially for quantitating by Western which is not especially accurate. Personally, I would do at least three separate westerns, and I would load a dilution series of treatment and control on each one to make sure I wasn't overloading. But at the end of the day, n=1. > John > hines@pharm.med.upenn.edu love Anna From owner-proteins@net.bio.net Mon Aug 16 22:54:00 1999 Path: biosci!rutgers!news.sgi.com!howland.erols.net!newsfeed.cwix.com!216.12.0.50!news.cfw.com!paxfeed.eni.net!aol.com From: rbbadboy1@aol.com Newsgroups: bionet.molbio.proteins Subject: FREE Phonesex 90449 Date: Monday, 16 Aug 1999 23:50:29 -0600 Organization: More services than all the others Lines: 24 Message-ID: <16089923.5029@aol.com> Reply-To: jennyr@aol.com NNTP-Posting-Host: 14.pao900.pool.eni.net Want to talk with REAL horny girls? This is something VERY new in phone sex. The women on the line are NOT paid. They call for free phone sex with guys like YOU! Guys: 50 cents per minute Girls: TOTALLY FREE =====================>1-888-335-HOTT (toll-free) =====================>1-888-302-ORGY (toll-free) You can be billed discreetly directly to your phone bill. ----------------------------------------------------------------- For gay talk call: 1-888-800-GUYS (toll-free) ------------------------------------------------------------------- For Dominance & Submission Call: 888-700-WHIP --------------------------------------------------------------------- ;PiIgOV) From owner-proteins@net.bio.net Tue Aug 17 06:31:00 1999 Path: biosci!pravda.ucr.edu!ihnp4.ucsd.edu!gondor!newshub.sdsu.edu!newsfeed.berkeley.edu!newsfeed.acns.nwu.edu!news.cc.uic.edu!not-for-mail From: Joo Yun Hong Newsgroups: bionet.molbio.proteins Subject: about 'mini-protean 2' by bio-rad Date: Tue, 17 Aug 1999 09:10:47 -0500 Organization: University of IL at Chicago Lines: 22 Message-ID: <37B96D67.113EA058@uic.edu> NNTP-Posting-Host: a069.bios.uic.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.6 [en] (WinNT; I) X-Accept-Language: en hello!! i'm using hydridoma supernatant to screen monoclonal antibody about protein on a membrane. since the amount of supernatant i can use is limited to a small volume, i used this apparatus named 'mini-protean2' from the bio-rad. but it has been giving me a problem of leakage between the wells. so i was wondering if anyone could tell me about similar kind of apparatus using a membrane to screen multiple samples of hydridoma supernatant or have any idea of eliminating the leakage problem. thank you joo jhong1@uic.edu From owner-proteins@net.bio.net Tue Aug 17 07:14:00 1999 Path: biosci!NMSU.EDU!hroychow From: hroychow@NMSU.EDU ("Hiranya S. Roychowdhury") Newsgroups: bionet.molbio.proteins Subject: Re: about 'mini-protean 2' by bio-rad Date: 17 Aug 1999 08:14:17 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 43 Sender: daemon@net.bio.net Distribution: world Message-ID: <199908171507.JAA20982@nestor.NMSU.Edu> NNTP-Posting-Host: net.bio.net At 09:10 AM 8/17/99 -0500, Joo Yun Hong wrote: >hello!! > >i'm using hydridoma supernatant to screen monoclonal antibody about >protein on a >membrane. since the amount of supernatant i can use is limited to a >small volume, i >used this apparatus named 'mini-protean2' from the bio-rad. but it has >been giving >me a problem of leakage between the wells. so i was wondering if anyone >could tell >me about similar kind of apparatus using a membrane to screen multiple >samples of >hydridoma supernatant or have any idea of eliminating the leakage >problem. > > thank >you > >joo jhong1@uic.edu > I am not too impressed with the set up of the mini protean 2. However, leakage between the wells is not something that I have ever encountered in a protein gel where the lanes are 'preformed' in the stack. I am not sure why you should experience this. The only reason I can come up with is that if the stacking gel is set too much in advance it is drying up. If the stacking gel dries up, it pulls away from the glass plates, causing the sample to 'leak' out horizontally (it has to be poured several hours in advance for this to happen!). The stacking layer should not be poured unless you are ready with your samples, since it only takes about 30 min to polymerize. BTW, if you are just screening monoclonals with hyb. sup., why not use a dot blot? Dr. Hiranya Sankar Roychowdhury GENE LAB/ EPPWS New Mexico State University Las Cruces, NM 88003 Ph. (505) 646-5785 hroychow@nmsu.edu From owner-proteins@net.bio.net Wed Aug 18 02:43:00 1999 Path: biosci!pravda.ucr.edu!ihnp4.ucsd.edu!newsfeed.berkeley.edu!netnews.com!howland.erols.net!portc02.blue.aol.com!audrey01.news.aol.com!not-for-mail From: yathin95@aol.com (YATHIN95) Newsgroups: bionet.molbio.proteins Subject: metalloprotinases Lines: 9 NNTP-Posting-Host: ladder07.news.aol.com X-Admin: news@aol.com Date: 18 Aug 1999 10:36:23 GMT Organization: AOL http://www.aol.com Message-ID: <19990818063623.00341.00000569@ng-ck1.aol.com> I have made a recombinant of the substrate that a probable metalloprotinase acts on.I have the choice to in vitro label this substrate with alpha P32.Now I want to try different fractions of a particular tissue for enzyme clevage of the substrate. I have no real hand experience with metalloprotinases. If you are an expert please advice on a good protocol, buffer systems and possible tips. I will appreciate your help. You can e.mail me at rlatif@smtplink.mssm.edu From owner-proteins@net.bio.net Wed Aug 18 04:43:00 1999 Path: biosci!newshost.lanl.gov!logbridge.uoregon.edu!cyclone.mbnet.mb.ca!canopus.cc.umanitoba.ca!newsflash.concordia.ca!pitt.edu!not-for-mail From: Rich Dudley Newsgroups: bionet.molbio.proteins,bionet.cellbiol Subject: Re: Immunoprecipitations??? Date: Wed, 18 Aug 1999 08:42:32 -0400 Organization: University of Pittsburgh, Dept. of Cell Biology and Physiology Lines: 24 Message-ID: <37BAAA37.3C7F0ADF@pitt.edu> References: <37BA531E.E23E0F0B@home.com> Reply-To: rdudley+@pitt.edu NNTP-Posting-Host: whill.cbp.pitt.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.61 [en] (Win95; I) X-Accept-Language: en Xref: biosci bionet.molbio.proteins:14693 bionet.cellbiol:12324 The beads won't run through the gel--they're too big, and will remain in the well of the stack. The proteins not covalently bound will electrophorese off them, though. I just wash them, boil them in SDS-PAGE buffer, load and run. rich Neal Melvin wrote: > After performing an IP, do you run the actual beads themselves through > the gel?? Or is there a step that strips the protein A/G and bound > antibody/antigen off the beads, and the resulting supernatant is run > through the gel??? -- --- --- --- -- -- -- --- --- --- Richard J. Dudley (rdudley+@pitt.edu) Research Specialist V Dept. of Cell Biology and Physiology University of Pittsburgh http://www.cbp.pitt.edu ---> search BIONET archives at http://www.bio.net <--- From owner-proteins@net.bio.net Wed Aug 18 20:14:00 1999 Path: biosci!med.monash.edu.au!Zhonglin.Chai From: Zhonglin.Chai@med.monash.edu.au (Zhonglin Chai) Newsgroups: bionet.molbio.proteins Subject: leucine zipper Date: 18 Aug 1999 21:14:14 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 26 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Dear all, As we know that the "leucine zipper" has a pattern as L-x(6)-L-x(6)- L-x(6)-L, a novel protein I am working on has two regions similar to this pattern, reading as: region 1, L-x(6)-I-x(6)-L-x(6)-I-x(6)-L-x(6)-I (some I - L replacements), and region 2, I-x(6)-V-x(6)-L-x(6)-I-x(6)-M (I, V, M, L replacements). I wonder if the leucine residue in the "leucine zipper" can be replaced by I, V, and M and the functions (dimerization?) remain with the replacement? Cheers. ZhongLin Chai, PhD __________________________________________________________ Department of Pathology and Immunology Monash University Medical School Alfred Hospital Commercial Rd, Prahran, VIC 3181, AUSTRALIA Telephone: (61 3) 9903 0698 (lab) (61 3) 9903 0696 (office) Mobile: 0413 58 1940 or International: +61 413 58 1940 Fax: (61 3) 9903 0731 email: zhonglin.chai@med.monash.edu.au ___________________________________________________________ From owner-proteins@net.bio.net Thu Aug 19 05:29:00 1999 Path: biosci!news.stanford.edu!newsfeed.stanford.edu!arclight.uoregon.edu!wn4feed!worldnet.att.net!207.172.3.37!feed1.news.rcn.net!rcn!nntp.abs.net!newshub2.home.com!news.home.com!news.rdc1.tn.home.com.POSTED!not-for-mail Message-ID: <37BC0536.66CAAB2A@uab.edu> From: Peter Prevelige Reply-To: prevelig@uab.edu Organization: Dept. of Microbiology, UAB X-Mailer: Mozilla 4.5 C-AtHome0405(Macintosh; U; PPC) X-Accept-Language: en MIME-Version: 1.0 Newsgroups: bionet.molbio.proteins Subject: Postdoctoral Positions, Protein Folding and Assembly Content-Type: multipart/mixed; boundary="------------5B0412A54DF6711E08B567A7" Lines: 80 Date: Thu, 19 Aug 1999 13:23:03 GMT NNTP-Posting-Host: 24.2.24.8 X-Complaints-To: abuse@home.net X-Trace: news.rdc1.tn.home.com 935068983 24.2.24.8 (Thu, 19 Aug 1999 06:23:03 PDT) NNTP-Posting-Date: Thu, 19 Aug 1999 06:23:03 PDT This is a multi-part message in MIME format. --------------5B0412A54DF6711E08B567A7 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit Two postdoctoral positions are available immediately in the Dept of Microbiology at the University of Alabama at Birmingham (UAB). These positions are in the laboratory of Dr. Peter Prevelige. 1) A postdoctoral position is available to study the interactions between the structural domains of the HIV Gag polyprotein in polymers assembled in vitro, and in non-infectious budded particles. Amide H/D exchange as detected by mass spectrometry will be employed to detect intersubunit interfaces, and dynamic flexibility within the immature and mature virions, and relate these determinations to the available high resolution structures. This position will afford the applicant the opportunity to develop skills in state of the art mass spectrometry, protein purification, and eukaryotic molecular biology. The project is collaborative with members of the UAB Center for AIDS Research (CFAR) The UAB CFAR is a top ranked research center, with an outstanding group of interdisciplinary researchers. A Ph.D. degree in Biochemistry or Biophysics with experience in the purification and handling of proteins is preferred. This position is open immediately. 2) A postdoctoral position is available to study the kinetics and thermodynamics of protein/protein interactions during the assembly of the bacteriophage P22. Bacteriophage P22 is a model system for the assembly of dsDNA containing viruses, as well as for the control of form determination in the assembly of supramolecular assemblies. The project will involve the use of calorimetry, surface plasmon resonance, small angle X-ray scattering and and spectroscopic techniques (CD, fluoresence) to characterize the interactions between the coat and scaffolding protein molecules during capsid assembly. This project involves a number of active and productive collaborations including cyro-electron microscopy, Raman spectroscopy, and NMR. A Ph.D in Biochemistry or Biophysics with experience in purification and handling of proteins is preferred. The position is open immediately. Birmingham is a metropolitan region with a population of approximately 1 million and a moderate cost of living. UAB is a major research university and medical center located within the city. for more information, please visit: http://www.microbio.uab.edu/faculty/prevelige/prevelige-p.htm. Interested candidates should contact: Peter Prevelige Dept of Microbiology, BBRB 416/6 Univ. of Alabama at Birmingham Birmingham AL. 35294 FAX 205 975-5479 email: prevelig@uab.edu --------------5B0412A54DF6711E08B567A7 Content-Type: text/x-vcard; charset=us-ascii; name="prevelig.vcf" Content-Transfer-Encoding: 7bit Content-Description: Card for Peter Prevelige Content-Disposition: attachment; filename="prevelig.vcf" begin:vcard n:Prevelige;Peter tel;fax:205 975-5479 x-mozilla-html:FALSE url:http://www.microbio.uab.edu/faculty/Prevelige/prevelige-p.htm org:Univ. of Alabama at Birmingham;Microbiology version:2.1 email;internet:prevelig@uab.edu title:Associate Professor adr;quoted-printable:;;BBRB 416/6=0D=0A845 19th St. South;Birmingham;AL;35294;USA x-mozilla-cpt:;3 fn:Peter Prevelige end:vcard --------------5B0412A54DF6711E08B567A7-- From owner-proteins@net.bio.net Thu Aug 19 13:15:00 1999 Path: biosci!aecom.yu.edu!almo From: almo@aecom.yu.edu (Steven Almo) Newsgroups: bionet.molbio.proteins Subject: Position for Protein Chemist Date: 19 Aug 1999 14:15:15 -0700 Organization: Dept. of Biochem., Albert Einstein Coll. of Med. Lines: 16 Sender: daemon@net.bio.net Distribution: world Message-ID: <37BC7204.F842AB1@aecom.yu.edu> NNTP-Posting-Host: net.bio.net Research position in structural genomics available. The project involves interacting with an inter-disciplinary team of scientists developing a high-throughput expression and purification system for structural analysis by X-ray and NMR techniques. The successful candidate will have extensive experience in protein purification techniques. Experience with molecular biology techniques a plus. Salary commensurate with qualifications and experience. Please contact Dr. Steven Almo Department of Biochemistry Albert Einstein College of Medicine 1300 Morris Park Avenue Bronx, NY 10461 almo@aecom.yu.edu From owner-proteins@net.bio.net Thu Aug 19 20:07:00 1999 Path: biosci!news.stanford.edu!newsfeed.stanford.edu!logbridge.uoregon.edu!newshub.tc.umn.edu!mayonews.mayo.edu!NewsWatcher!user From: al@mayo.edu (RCF 7600) Newsgroups: bionet.molbio.proteins,bionet.cellbiol Subject: Re: Immunoprecipitations??? Date: 20 Aug 1999 03:58:04 GMT Organization: rcf Lines: 19 Message-ID: References: <37BA531E.E23E0F0B@home.com> NNTP-Posting-Host: 172.22.235.165 Xref: biosci bionet.molbio.proteins:14697 bionet.cellbiol:12333 In article <37BA531E.E23E0F0B@home.com>, Neal Melvin wrote: > After performing an IP, do you run the actual beads themselves through > the gel?? Or is there a step that strips the protein A/G and bound > antibody/antigen off the beads, and the resulting supernatant is run > through the gel??? The Ab/Ag complex will dissociate from protein A/G agarose beads after you add SDS-PAGE sample buffer. Incubate at RT for 15 min and spin for 1 min at 6000 rpm. Load only the supernatant. However, I suggest you load everything including the beads (with a long thin micro-pippet) after 15 min incubation if the well volume is allowed, as this reduces the loss of your protein. Rinse the tube with 10ul sample buffer when necessary. The beads will remain in the well during electrophoresis. Hope this helps. Mao Dong Dongm@mayo.edu From owner-proteins@net.bio.net Fri Aug 20 17:21:00 1999 Path: biosci!newshost.lanl.gov!awabi.library.ucla.edu!128.230.129.106!news.maxwell.syr.edu!nntp.abs.net!newshub2.home.com!news.home.com!news1.rdc1.ab.home.com.POSTED!not-for-mail Message-ID: <37BDFD72.D691D6F8@home.com> From: Neal Melvin X-Mailer: Mozilla 4.51 [en] (Win98; U) X-Accept-Language: en MIME-Version: 1.0 Newsgroups: bionet.molbio.proteins Subject: Immunoprecipitation advice?? Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit Lines: 6 Date: Sat, 21 Aug 1999 01:14:31 GMT NNTP-Posting-Host: 24.64.67.125 X-Complaints-To: abuse@home.net X-Trace: news1.rdc1.ab.home.com 935198071 24.64.67.125 (Fri, 20 Aug 1999 18:14:31 PDT) NNTP-Posting-Date: Fri, 20 Aug 1999 18:14:31 PDT Organization: @Home Network Canada I just read your reply to my IP question... and I have another one. Why do you suggest incubating for 15 min at RT... is it not better to boil the beads/sample just as in regular reducing SDS-PAGE? By the way, have you ever used the protein A-agarose from Santa Cruz?? From owner-proteins@net.bio.net Mon Aug 23 06:18:00 1999 Path: biosci!MAIL.MT.NET.AU!baimuanu11 From: baimuanu11@MAIL.MT.NET.AU (jyooscur) Newsgroups: bionet.molbio.proteins Subject: The Key West General Store--$1.00 Off shipping Date: 23 Aug 1999 07:18:09 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 16 Sender: daemon@net.bio.net Distribution: world Message-ID: <199908231312JAA49115@biuolindo.jiahau.com.tw> NNTP-Posting-Host: net.bio.net Click to visit the site A very small inventory of WONDERFUL and AFFORDABLE gifts from the island city of Key West. Nellie and Joe's Famous Key Lime juice (make your own REAL Key Lime Pie) or have a Key Lime Pie delivered directly to your door overnight express delivery. Check out some wonderful photography, a cookbook, or a very nice Key West poster. This fast loading site has a small inventory of inexpensive treasures from Key West. A little store with GREAT things! 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(Does not apply for Key Lime Pie Deliveries) Click to visit the site To be removed: email 13milesup@bigfoot.com Put "rem From owner-proteins@net.bio.net Mon Aug 23 07:37:00 1999 Path: biosci!bloom-beacon.mit.edu!howland.erols.net!news-out-b.news.pipex.net.MISMATCH!tank.news.pipex.net!pipex!do.de.uu.net!news-koe1.dfn.de!news-han1.dfn.de!news.gwdg.de!not-for-mail From: Masoud Bahrami Newsgroups: bionet.molbio.proteins Subject: antibody staining Date: Mon, 23 Aug 1999 17:05:45 +0200 Organization: GWDG, Goettingen Lines: 18 Message-ID: <37C16349.365C270F@gwdg.de> NNTP-Posting-Host: avidin.uni-zoo3.gwdg.de Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: gwdu67.gwdg.de 935420392 12394 134.76.141.136 (23 Aug 1999 14:59:52 GMT) X-Complaints-To: news@gwdg.de NNTP-Posting-Date: 23 Aug 1999 14:59:52 GMT X-Mailer: Mozilla 4.51 [de] (Win98; I) X-Accept-Language: de Hi all: I got a problem recently. I have to localise the gfp (green fluorecence protein) in the nucleus of cells from c.elegans with antibody which is conjugated with nanogold. Now I want to ask evryone. Does anyone who have the experience to labeling the gfp in the nucleus with nanogold before? If so, do you have any good ideas on how to optimise and enhance the results. Does anyone know of a worker who could help me out? Or can somebody please point me to a resource or give me some information on each protocol? Thank you in advance, Masoud mbahram@gwdg.de From owner-proteins@net.bio.net Mon Aug 23 17:27:00 1999 Path: biosci!news.stanford.edu!newsfeed.stanford.edu!newsfeed.berkeley.edu!dispose.news.demon.net!demon!newsfeed.nacamar.de!bignews.mediaways.net!news.tvd.be!uunet!ams.uu.net!ffx.uu.net!news.unired.net.pe!not-for-mail From: "Carlos Nostas A." Newsgroups: bionet.molbio.proteins Subject: Sell Protein Date: Mon, 23 Aug 1999 20:27:12 -0500 Organization: UniRed - TdP Message-ID: <7prott$lp5$1@ucayali.unired.net.pe> NNTP-Posting-Host: infovia86.lared.net.pe X-Trace: ucayali.unired.net.pe 935421693 22309 200.37.202.215 (23 Aug 1999 15:21:33 GMT) X-Complaints-To: abuse@unired.net.pe NNTP-Posting-Date: 23 Aug 1999 15:21:33 GMT X-Newsreader: Microsoft Outlook Express 4.72.2106.4 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.2106.4 Lines: 6 We produce Protein UpGrade. email me at: lajoyave@ucsm.edu.pe From owner-proteins@net.bio.net Mon Aug 23 17:41:00 1999 Path: biosci!news.stanford.edu!newsfeed.stanford.edu!news-feed.inet.tele.dk!bofh.vszbr.cz!newsfeed.tli.de!do.de.uu.net!uunet!ams.uu.net!ffx.uu.net!news.unired.net.pe!not-for-mail From: "Carlos Nostas A." 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If You are interested, email me at: lajoyave@ucsm.edu.pe From owner-proteins@net.bio.net Tue Aug 24 12:00:00 1999 Path: biosci!POP.HORT.PURDUE.EDU!hernandez From: hernandez@POP.HORT.PURDUE.EDU Newsgroups: bionet.molbio.proteins Subject: Signals for proteolysis Date: 24 Aug 1999 13:00:33 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 28 Sender: daemon@net.bio.net Distribution: world Message-ID: <1276615766-31353615@hort.purdue.edu> NNTP-Posting-Host: net.bio.net Dear all, Is there a way of finding if a certain protein is likely to be short-lived in a cell just by looking at the amino acid sequence? When making a literature search, I have found many papers that mention an "N-end rule" but I haven't been able to figure out what that is beyond knowing that a Lys residue is needed for binding of ubiquitin. Besides, I got the impresion that these signalling domains can be both at the N or the C-terminus in a protein but I don't know if they are different. By the way, I work in yeast, in case this makes a great difference in the way the proteins are processed (as you can see, I haven't got much idea about all these processes, sorry!) Could someone point me to a good www site for analysing my protein sequence and find if its turnover is likely to be rapid? Alternatively, anybody knows of a review that deals with domains in a protein that signal it for degradation? I haven't got direct access to this newsgroup, so I'd appreciate if you could send any answers to my mail address: hernandez@pop.hort.purdue.edu Thanks! Agustin Hernandez From owner-proteins@net.bio.net Wed Aug 25 04:04:00 1999 Path: biosci!newshost.lanl.gov!logbridge.uoregon.edu!dispose.news.demon.net!demon!newsfeed.icl.net!newsfeed.icl.net!isdnet!news-ge.switch.ch!news.grnet.gr!not-for-mail From: "Evangelos Christodoulou" Newsgroups: bionet.molbio.proteins Subject: Re: Signals for proteolysis Date: Wed, 25 Aug 1999 14:55:30 +0300 Organization: University of Athens Distribution: world Message-ID: <7q0lni$15t$1@foo.grnet.gr> References: <1276615766-31353615@hort.purdue.edu> NNTP-Posting-Host: imac.db.uoa.gr Mime-Version: 1.0 Content-Type: text/plain; charset="US-ASCII" Content-Transfer-Encoding: 7bit X-Newsreader: Microsoft Outlook Express Macintosh Edition - 4.5 (0410) Lines: 24 > Is there a way of finding if a certain protein is likely to be short-lived > in a cell just by looking at the amino acid sequence? When making a > literature search, I have found many papers that mention an "N-end rule" > but I haven't been able to figure out what that is beyond knowing that a > Lys residue is needed for binding of ubiquitin. Besides, I got the > impresion that these signalling domains can be both at the N or the > C-terminus in a protein but I don't know if they are different. By the way, > I work in yeast, in case this makes a great difference in the way the > proteins are processed (as you can see, I haven't got much idea about all > these processes, sorry!) > Could someone point me to a good www site for analysing my protein sequence > and find if its turnover is likely to be rapid? Alternatively, anybody > knows of a review that deals with domains in a protein that signal it for > degradation? http://genome.cbs.dtu.dk/services/SignalP/index.html -- Evangelos Christodoulou University of Athens, Greece If Che Guevara was alive he'd use a Mac -the only revolutionary-friendly platform From owner-proteins@net.bio.net Wed Aug 25 09:01:00 1999 Path: biosci!news.stanford.edu!newsfeed.stanford.edu!remarQ73!rQdQ!supernews.com!remarQ.com!corp.supernews.com!not-for-mail From: meyerdj@phibred.com (Dr. David J. Meyer) Newsgroups: bionet.molbio.proteins Subject: Re: Signals for proteolysis Date: Wed, 25 Aug 1999 16:56:44 GMT Organization: Pioneer Hi-Bred International, Inc. Lines: 52 Message-ID: References: <1276615766-31353615@hort.purdue.edu> <7q0lni$15t$1@foo.grnet.gr> X-Complaints-To: newsabuse@supernews.com X-Newsreader: Forte Free Agent v0.55 "Evangelos Christodoulou" wrote: >> Is there a way of finding if a certain protein is likely to be short-lived >> in a cell just by looking at the amino acid sequence? When making a >> literature search, I have found many papers that mention an "N-end rule" >> but I haven't been able to figure out what that is beyond knowing that a >> Lys residue is needed for binding of ubiquitin. Besides, I got the >> impresion that these signalling domains can be both at the N or the >> C-terminus in a protein but I don't know if they are different. By the way, >> I work in yeast, in case this makes a great difference in the way the >> proteins are processed (as you can see, I haven't got much idea about all >> these processes, sorry!) >> Could someone point me to a good www site for analysing my protein sequence >> and find if its turnover is likely to be rapid? Alternatively, anybody >> knows of a review that deals with domains in a protein that signal it for >> degradation? >http://genome.cbs.dtu.dk/services/SignalP/index.html >-- >Evangelos Christodoulou >University of Athens, Greece >If Che Guevara was alive he'd use a Mac >-the only revolutionary-friendly platform SignalP is used to predict N-terminal ER-targeting signal sequences, not what the original poster was looking for. If you look at Varshavsky's papers carefully, you will learn that the N-end rule has to do with what the N-terminal residue is. Some residues were determined to confer short half-lives, others long half-lives. The degradation appears to be via the ubiquitin-proteasome pathway. "Stabilizing" and "destabilizing" residues have been determined in both E. coli and S. cerevisiae. Hope this helps, although the world of protein folding and turnover is much more complicated than any algorithm to predict expression levels might indicate! David J. Meyer, Ph.D. Quality Traits Pioneer Hi-Bred International, Inc. 7300 NW 62nd Ave. Johnston, IA 50131-1004 Ph. 515/254-2639 FAX 515/254-2619 Email: meyerdj@phibred.com From owner-proteins@net.bio.net Thu Aug 26 04:04:00 1999 Path: biosci!AOL.COM!48don1500 From: 48don1500@AOL.COM Newsgroups: bionet.molbio.proteins Subject: Increase Merchant Sales Date: 26 Aug 1999 05:04:49 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 35 Sender: daemon@net.bio.net Distribution: world Message-ID: <199908261204.FAA15151@net.bio.net> NNTP-Posting-Host: net.bio.net Substantially increase your Business Profits! Would your business explode if you could accept credit cards? 99% of all Internet Transactions last year were in plastic. Stop losing valuable sales! Merchant Status will help you INCREASE SALES by an incredible 50% to 400%. Stop losing valuable sales! With one phon