From owner-proteins@net.bio.net Tue Apr 01 23:00:00 1997 Path: biosci!agate!spool.mu.edu!uwm.edu!newsfeeds.sol.net!europa.clark.net!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsxfer3.itd.umich.edu!rill.news.pipex.net!pipex!btnet!btnet-feed2!unlisys!blackbush.xlink.net!news-ge.switch.ch!news-zh.switch.ch!rzunews.unizh.ch!NewsWatcher!user From: hindges@vetbio.unizh.ch (Robert Hindges) Newsgroups: bionet.molbio.proteins Subject: Re: GST fusion protein of >100kDa Date: Thu, 03 Apr 1997 08:15:51 -0600 Organization: Universitat Zurich-Irchel Lines: 23 Distribution: world Message-ID: References: <33424E31.2EAA@rex.uokhsc.edu> NNTP-Posting-Host: 130.60.120.26 Hello I expressed and purified a 125kDa protein fused to GST. To get my protein soluble I had to lower the bacterial culture temperature to 30 celsius. The purification with the Glutathione Sepharose according to the Pharmacia protocol worked well and I got pure protein. However, cleavage with thrombin resulted in an precipitation of my protein. Somehow the GST keeps the protein soluble. Anyway, my protein was active even with the GST part. Robert In article <33424E31.2EAA@rex.uokhsc.edu>, spaul@REX.RE.uokhsc.edu wrote: > Does anyone have experience expressing and purifying proteins > more than 70kDa using the GST fusion system? So the fusion protein > would be 100kDa or more. > Any tips on purifying protein by cleaving the GST with thrombin would > be helpfull. > Thankyou very much > SPaul From owner-proteins@net.bio.net Tue Apr 01 23:00:00 1997 Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!metro!metro!news From: "Michael Bonifacio" Newsgroups: bionet.molbio.proteins Subject: CE IEF Date: 28 Mar 1997 22:35:41 GMT Organization: Home Lines: 7 Distribution: inet Message-ID: <01bc3bc8$8e157480$1d7b4e81@mbonifac.ucc.su.OZ.AU> NNTP-Posting-Host: modem-b-29.mp.usyd.edu.au X-Newsreader: Microsoft Internet News 4.70.1155 Has anyone had experience with IEF on CE systems, especially the BioRad CE3000. The main issue concerning me (above many others) is the high variation in run time, both intra- and inter- day. Any ideas would be appreciated. Regards Michael Bonifacio From owner-proteins@net.bio.net Tue Apr 01 23:00:00 1997 Path: biosci!agate!nature.berkeley.edu!lhom From: lhom@nature.berkeley.edu (Louis Hom) Newsgroups: bionet.cellbiol,bionet.molbio.proteins Subject: DEAD box proteins and CPP32 Date: 2 Apr 1997 18:40:12 GMT Organization: University of California, Berkeley Lines: 9 Message-ID: <5hu96c$1bd@agate.berkeley.edu> NNTP-Posting-Host: nature.berkeley.edu Xref: biosci bionet.cellbiol:7031 bionet.molbio.proteins:10397 I was struck by the similarity between the DEAD motif and the DEVD peptide inhibitor of the apoptosis-related protease CPP32. A keyword search on medline turned up no connection between the two. Does anyone know of any links here? (yes, a total shot in the dark . . .) -- _______________________________________________________________________________ Lou Hom >K '93 lhom@nature.berkeley.edu http://www.ocf.berkeley.edu/~lhom From owner-proteins@net.bio.net Tue Apr 01 23:00:00 1997 Path: biosci!REX.RE.UOKHSC.EDU!spaul From: spaul@REX.RE.UOKHSC.EDU ("spaul@rex.uokhsc.edu") Newsgroups: bionet.molbio.proteins Subject: GST fusion protein of >100kDa Date: 2 Apr 1997 08:59:35 -0800 Organization: uokhsc Lines: 7 Sender: daemon@net.bio.net Distribution: world Message-ID: <33424E31.2EAA@rex.uokhsc.edu> Reply-To: spaul@REX.RE.uokhsc.edu NNTP-Posting-Host: net.bio.net Does anyone have experience expressing and purifying proteins more than 70kDa using the GST fusion system? So the fusion protein would be 100kDa or more. Any tips on purifying protein by cleaving the GST with thrombin would be helpfull. Thankyou very much SPaul From owner-proteins@net.bio.net Tue Apr 01 23:00:00 1997 Path: biosci!agate!newsgate.cuhk.edu.hk!news-hk.gsl.net!news.gsl.net!cyclic.gsl.net!news.gsl.net!gsl-stkh-ns.gsl.net!news.gsl.net!uninett.no!nntp.uio.no!news.apfel.de!uni-erlangen.de!lrz-muenchen.de!not-for-mail From: Boris Steipe Newsgroups: bionet.molbio.proteins Subject: Re: histidine metal chelating Date: Wed, 02 Apr 1997 14:55:07 +0200 Organization: Genzentrum Lines: 19 Distribution: world Message-ID: <33425723.766F@LMB.uni-muenchen.de> References: <5h4je9$gj5@fridge-nf0.shore.net> Reply-To: steipe@LMB.uni-muenchen.de NNTP-Posting-Host: bs_boris.lmb.uni-muenchen.de Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Macintosh; I; PPC) Kathleen Blackwell wrote: > > Given a complex nitrogen source with various peptides in solution, would > digesting to various levels of free amino acids lead to a solutions with better > metal chelating properties as the levels of free amino acids rises? My thinking > is that higher levels of free histidine would chelate metals better than > histidine bound in peptides, mostly for steric reasons. Any thought? TIA You are probably thinking of divalent cations ? Actually, any amino-acid has very significant chelating properties for divalent cations, simply from the free amino and carboxylate group. So a solution of free amino acids will have a higher chelating capacity then any peptide or protein mixture, even though individual stability constants for specific sites in proteins may be higher. Best wishes, Boris From owner-proteins@net.bio.net Wed Apr 02 23:00:00 1997 Path: biosci!daresbury!bioftp.unibas.ch!infobiogen.fr!lovelace.infobiogen.fr!ahelme From: Alix Helme-Guizon Newsgroups: bionet.molbio.proteins Subject: How to improve sensitivity of immunocytochemistry? Date: Thu, 3 Apr 1997 12:20:30 +0200 Organization: "GIS INFOBIOGEN, 7 rue Guy Moquet BP8, 94801 VILLEJUIF, France" Lines: 20 Message-ID: NNTP-Posting-Host: lovelace.infobiogen.fr Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII I want to detect a membranous protein on slices (20 or 30 microns)in an area that express only few of this protein.(Of course this area express the protein, as this protein can be seen on western blot with extract from this area (first antibody 1/20 000 for 6 microgrammes of membrane preparation.) i've tried -DAB plus nickel with biotynilated secondary antibody (first antibody 1/5OO overnight RT in triton 0,3%+ serum 10% in PBS, preincubation 1h in serum10%+triton+PBS RT). this gave a very good labeling in another area (known to express well the protein) but not in the the place i want to study! -DAB without nickel (same protocol) -fluorescence with a secondary antibody (1/3oo) What can I do to improve sensitivity of antigen detection on slices without increasing too much the background? Thanks a lot for your help! Alix From owner-proteins@net.bio.net Wed Apr 02 23:00:00 1997 Path: biosci!webtv.net!uunet!in3.uu.net!128.230.129.106!news.maxwell.syr.edu!news.apfel.de!news-fra1.dfn.de!news-ge.switch.ch!news-zh.switch.ch!ubaclu.unibas.ch!bioftp.unibas.ch!infobiogen.fr!jussieu.fr!univ-lr.fr!u-bordeaux.fr!univ-rennes1.fr!melon.univ-brest.fr!news Newsgroups: bionet.molbio.proteins Subject: looking for list of protease site Message-ID: <5hvr58$et8@melon.univ-brest.fr> From: <> Date: 3 Apr 1997 08:52:56 GMT Organization: Universite de Brest , France NNTP-Posting-Host: pc-lbcm-5.univ-ubs.fr Lines: 2 I am looking for a site in the web where i can find most of protease site thank you From owner-proteins@net.bio.net Wed Apr 02 23:00:00 1997 Path: biosci!rutgers.rutgers.edu!gatech!csulb.edu!hammer.uoregon.edu!news-xfer.netaxs.com!cpk-news-hub1.bbnplanet.com!cam-news-hub1.bbnplanet.com!news.bbnplanet.com!howland.erols.net!vixen.cso.uiuc.edu!uwm.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!daemon From: klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: Urea-containing buffers: How long can I store them? Date: Fri, 04 Apr 1997 02:47:54 GMT Organization: UW-Madison Lines: 24 Message-ID: <5i1q87$4k80@news.doit.wisc.edu> References: <333073A4.736@acs-popmail.uchicago.edu> <333B7BF7.3EE@lilly.com> NNTP-Posting-Host: f182-149.net.wisc.edu X-Newsreader: News Xpress 2.01 X-No-Archive: Yes Xref: biosci bionet.molbio.methds-reagnts:56356 bionet.molbio.proteins:10413 In article <333B7BF7.3EE@lilly.com>, "David B. Flora" wrote: #Viraj Master wrote: #> 8M urea #> 0.1M NaH2PO4 #> 0.01M Tris #>I would like to be able to prepare batches of these #> buffers and store them. Does anybody know whether this can be done and #> not affect the protein prep? # #I am a biochemist at Eli Lilly and Co. and we use 7M urea quite a bit #and make up 10L batches. We store them in a cold room to reduce the #formation of a break-down product that results in the carbamylation of #primary amines in proteins we work with. We also pass the urea through #a column of a mixed-bed ion exchange media (Amberlite). The column #removes the urea break-down product and we see little carbamylation of #proteins. In other words, if you want to store urea-containing buffer, purify it every time before use... On a lab scale making fresh is _always_ better and cheaper. - Dima From owner-proteins@net.bio.net Wed Apr 02 23:00:00 1997 Path: biosci!rutgers.rutgers.edu!gatech!csulb.edu!hammer.uoregon.edu!news-peer.gsl.net!howland.erols.net!math.ohio-state.edu!jussieu.fr!univ-lyon1.fr!in2p3.fr!news-ge.switch.ch!news-zh.switch.ch!rzunews.unizh.ch!NewsWatcher!user From: zjons@vetbio.unizh.ch (Zophonias O. Jonsson) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Coating ELISA plates in Urea Date: 3 Apr 1997 22:37:42 GMT Organization: Universitat Zurich-Irchel Lines: 26 Message-ID: NNTP-Posting-Host: 130.60.120.19 Xref: biosci bionet.molbio.methds-reagnts:56354 bionet.molbio.proteins:10412 Dear friends We have a small problem here. We have a protein and want to couple it to ELISA plates. As you already guessed, the protein is rather insoluble so this has to be done in the presence of a solubilizing agent. Our first idea was to use Ni-NTA coated wells and bind the His-tagged protein in the presence of Urea. This should work, but those plates are very expen$ive. So I started thinking. Isn't it possible to couple the protein to standard ELISA plates in the presence of Urea or another solubilizing agent? I, not being a chemist, can't see why it should not work but maybe someone out there can prove me wrong. If someone has tried this or has a strong opinion, please don't hesitate to respond. We are dying to hear from you! Thank you ever so much Zophonias et al. _____________________________________________________________________ Zophonias O. Jonsson Institut fur Veterinarbiochemie Tel: (41-1)-257-54-75 Universitat Zurich-Irchel Fax: (41-1)-362-05-01 Winterthurerstrasse 190 CH-8057 Zurich Switzerland _____________________________________________________________________ From owner-proteins@net.bio.net Wed Apr 02 23:00:00 1997 Path: biosci!NAHUEL.BIOL.UNLP.EDU.AR!natalu From: natalu@NAHUEL.BIOL.UNLP.EDU.AR ("Dra. Claudia L. Natalucci") Newsgroups: bionet.molbio.proteins Subject: Re: looking for list of protease site Date: 3 Apr 1997 14:29:12 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 19 Sender: daemon@net.bio.net Distribution: world Message-ID: <199704032221.TAA06538@biol.unlp.edu.ar> NNTP-Posting-Host: net.bio.net I don't know your name and neither understand exactly your need, but anyway I send you some Web sites: http://specter.dcrt.nih.gov:8004/gbobj?id(['3.4.21.4',enzyme,'[]']) http://anon.psc.edu/general/software/packages/swiss/swiss.html as well as the address where you can subscribe to the protease list: Majordomo@mail.orst.edu You just shoud write: subscribe protease end (without subject) I'm working on plant proteases in Argentina Good luck, Claudia Claudia L. Natalucci LIPROVE, Argentina natalucci@biol.unlp.edu.ar From owner-proteins@net.bio.net Wed Apr 02 23:00:00 1997 Path: biosci!daresbury!uninett.no!nntp.uio.no!news.maxwell.syr.edu!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsxfer3.itd.umich.edu!newsxfer.itd.umich.edu!yale!oitnews.harvard.edu!purdue!haven.umd.edu!cs.umd.edu!info.usuhs.mil!not-for-mail From: DTerBush Newsgroups: bionet.molbio.proteins Subject: Postdoctoral position available. Bethesda. Date: Thu, 03 Apr 1997 12:15:07 +0000 Organization: USUHS Lines: 14 Message-ID: <33439F4B.7A95@aol.com> NNTP-Posting-Host: 131.158.9.184 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Macintosh; I; PPC) Postdoctoral position. Available immediately. Located in Bethesda, MD. The laboratory is interested in understanding the biochemistry of proteins which mediate vesicle targeting and fusion in exocytosis. We have identified a multiprotein complex, the Exocyst (TerBush et. al, EMBO J, 1996, 23: 6483), which is uniquely required for exocytosis and want to recruit a talented individual to identify Exocyst recptor(s). Salary is $26K plus benefits. A postdoc who writes a successful NIH or private foundation fellowship will be supplemented to $32K. US citizenship or permanent residency status required. Please send C.V., a brief description of research experience and the names and contact information for three references to: Dr. Daniel TerBush, Uniformed Services University of the Health Sciences, Dept. of Biochemistry, 4301 Jones Bridge Rd., Bethesda, MD 20814-4799. E-mail: danter1@aol.com. Fax: 301-295-3512. Affirmative Action/Equal Opportunity Employer. From owner-proteins@net.bio.net Wed Apr 02 23:00:00 1997 Path: biosci!rutgers.rutgers.edu!gatech!csulb.edu!hammer.uoregon.edu!news-peer.gsl.net!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!mindspring!cssun.mathcs.emory.edu!news-feed-1.peachnet.edu!cronkite.cc.uga.edu!mac200.ccrc.uga.edu!user From: rmerkle@uga.cc.uga.edu (Roberta K. Merkle) Newsgroups: bionet.molbio.proteins Subject: CARBOHYDRATE COURSES Date: 3 Apr 1997 20:18:39 GMT Organization: CCRC, Univ. Georgia Lines: 16 Message-ID: NNTP-Posting-Host: mac200.ccrc.uga.edu Techniques for Characterization of Complex Carbohydrates Four courses will be offered in 1997 at the Complex Carbohydrate Research Center (CCRC) of the University of Georgia: 1. Separation and Characterization of Glycoprotein Oligosaccharides (June 9-13), 2. Structural Analysis of Oligosaccharides (June 16-20), 3. Mass Spectrometry and MS/MS Analysis of Glycoconjugates (June 23-27), 4 NMR of Carbohydrates (July 14-18). Courses will consist of hands-on laboratory work, demonstrations and lectures; lab manual including selected analytical techniques and references will be provided. For further information contact Dr. Roberta K. Merkle, CCRC, 220 Riverbend Road, The University of Georgia, Athens, Georgia 30602-4712. Phone: 706-542-4402. FAX: 706-542-4412. E-mail: rmerkle@uga.cc.uga.edu From owner-proteins@net.bio.net Wed Apr 02 23:00:00 1997 Path: biosci!rutgers.rutgers.edu!gatech!csulb.edu!hammer.uoregon.edu!news-peer.gsl.net!news-paris.gsl.net!news.gsl.net!rain.fr!pressimage!news1.isdnet.net!usenet From: "Christophe Guenin" Newsgroups: bionet.molbio.proteins Subject: Receptors and G-Proteins Date: 3 Apr 1997 19:25:12 GMT Lines: 11 Message-ID: <01bc406c$ecc04340$adb695c2@default> NNTP-Posting-Host: ppp34.rai.hol.fr X-Newsreader: Microsoft Internet News 4.70.1155 Dear netters I am looking for structures of the genes of the receptors coupled to G Proteins You can answer in this news group ou send a mail to christophe.guenin@hol.fr Best wishes and many thanks for your participation Sophie-Penelope Lechevallier - SERVIER Labs From owner-proteins@net.bio.net Wed Apr 02 23:00:00 1997 Path: biosci!daresbury!uninett.no!nntp.uio.no!news.maxwell.syr.edu!news.visi.net!grouper.exis.net!out2.nntp.cais.net!news2.cais.com!news From: Bill Van Antwerp Newsgroups: bionet.molbio.proteins Subject: Re: Circular Dichroism Date: 3 Apr 1997 17:30:57 GMT Organization: MiniMed Inc Lines: 19 Message-ID: <5i0pgh$nak@news2.cais.com> References: <33396308.2BED@mti-n.uni-jena.de> NNTP-Posting-Host: 207.171.18.139 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) X-URL: news:33396308.2BED@mti-n.uni-jena.de Gunter Maubach wrote: >Dear netters, > >If there anybody with experience in CD-measurement. I have the problem >to choice the buffer with the lowest background at wavelengths from 260 >to 180 (or 184)nm. In the buffer I must include any solubilizer (we use >Acetonitril 10%). Another question is, if NaCl has any effect on the >spectra.If there any suggestions, please mail me. > >Gunter In our experience with relatively small proteins, acetonitrile is a bad idea since it often leads to loss of the structure that you are trying to find with the CD measurements. Bill From owner-proteins@net.bio.net Wed Apr 02 23:00:00 1997 Path: biosci!daresbury!uninett.no!nntp.uio.no!news.maxwell.syr.edu!news.visi.net!out2.nntp.cais.net!news2.cais.com!news From: Bill Van Antwerp Newsgroups: bionet.molbio.proteins Subject: Re: Circular Dichroism Date: 3 Apr 1997 17:30:49 GMT Organization: MiniMed Inc Lines: 19 Message-ID: <5i0pg9$kmd@news2.cais.com> References: <33396308.2BED@mti-n.uni-jena.de> NNTP-Posting-Host: 207.171.18.139 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) X-URL: news:33396308.2BED@mti-n.uni-jena.de Gunter Maubach wrote: >Dear netters, > >If there anybody with experience in CD-measurement. I have the problem >to choice the buffer with the lowest background at wavelengths from 260 >to 180 (or 184)nm. In the buffer I must include any solubilizer (we use >Acetonitril 10%). Another question is, if NaCl has any effect on the >spectra.If there any suggestions, please mail me. > >Gunter In our experience with relatively small proteins, acetonitrile is a bad idea since it often leads to loss of the structure that you are trying to find with the CD measurements. Bill From owner-proteins@net.bio.net Wed Apr 02 23:00:00 1997 Path: biosci!rutgers.rutgers.edu!gatech!howland.erols.net!worldnet.att.net!news.sprintlink.net!news-peer.sprintlink.net!uunet!in2.uu.net!130.235.20.4!news.lth.se!merkurius.lu.se!not-for-mail From: anne.farewell@gmm.gu.se Newsgroups: bionet.molbio.proteins Subject: Re: Re. Sensitive Protein Stains Date: Thu, 03 Apr 1997 15:27:46 +0000 Organization: Lund University Lines: 18 Message-ID: <3343CC72.2F22@gmm.gu.se> References: <3336C4CB.6B8D@durham.ac.uk> <5hpab4$b8r@cronkite.ocis.temple.edu> NNTP-Posting-Host: mibi18.mikrbiol.lu.se Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Macintosh; I; PPC) Dr Ron Croy (r.r.d.croy@durham.ac.uk) wrote: : We need to use a sensitive protein stain to detect protein spots on 2D : gels with the intention of sequencing these from PVDF blots (automated : ABI 477A). Coomassie blue stains are out because they tend to interfere : with the sequencing and clog up the reaction vessel. Silver staining : methods are out due to the protein modification. We have tried a small : sample of a stain called BT blu (originally from BT Scientific : Technologies, Carlsbad, Ca) with promising results but cannot contact : this company for further supplies (it has been suggested we contact : Zolon or Zolan Research for a stain called 'Fast Stain'). Any comments : and further contacts would be appreciated. We use Amido Black to stain the membrane after blotting 2D's. From owner-proteins@net.bio.net Wed Apr 02 23:00:00 1997 Path: biosci!daresbury!uninett.no!sn.no!nntp.uio.no!newsfeeds.sol.net!uwm.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!daemon From: klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins Subject: Re: Zn / QIAGEN-NTA-column Date: Thu, 03 Apr 1997 15:36:03 GMT Organization: UW-Madison Lines: 17 Message-ID: <5i0isj$1lj8@news.doit.wisc.edu> References: <334017AB.167E@MIT.EDU> NNTP-Posting-Host: f182-066.net.wisc.edu X-Newsreader: News Xpress 2.01 X-No-Archive: Yes In article <334017AB.167E@MIT.EDU>, Thomas Schwartz wrote: #Hello, # #has anybody out there experience about charging Qiagen's NTA-column #with Zinc instead of Nickel ? #My problem is, that the protein I'm working on seems to be #nickel-sensitive, so I want to change his-tag-purification over zinc #instead. #I used IMAC with zinc already on other columns then Qiagen's NTA, but #the result was bad because I couldn't use beta-ME, and zinc didn't bind #very tightly to this particular column. I have done this with Zn and Cu. It works just fine. In both cases selectivity for (His)n decreases but it should not be a problem with well-behaving His-tagged protein that sticks tigthly. - Dima From owner-proteins@net.bio.net Wed Apr 02 23:00:00 1997 Path: biosci!agate!howland.erols.net!newsxfer.itd.umich.edu!newsxfer3.itd.umich.edu!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!cam-news-feed2.bbnplanet.com!sol.caps.maine.edu!discovery.umeres.maine.edu!Keith_Hutchison From: Keith_Hutchison@discovery.umeres.maine.edu (Keith Hutchison) Newsgroups: bionet.molbio.proteins Subject: Faculty Position - Protein Biochemist Date: Thu, 3 Apr 1997 08:59:23 -0500 Organization: University of Maine Lines: 18 Sender: Keith_Hutchison@discovery.umeres.maine.edu Message-ID: NNTP-Posting-Host: discovery.umeres.maine.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 8bit Content-ID: X-Gateway: NASTA Gate 1.18 for FirstClass(R) PROTEIN BIOCHEMIST: The Department of Biochemistry, Microbiology and Molecular Biology at the University of Maine seeks a biochemist with research interests in protein-protein or protein-nucleic acid interactions to fill a tenure track position at either the Assistant or Associate Professor level, available September 1, 1997. Additional research experience in the molecular genetics or developmental biology of aquatic organisms is desirable. A Ph.D. and postdoctoral experience are required. The successful candidate will be expected to maintain a vigorous, extramurally funded research program and contribute to teaching of the undergraduate and graduate curricula in Biochemistry. Minimum salary is $40,000 per academic year. Send a cover letter, description of research interests, curriculum vitae and three letters of recommendation to: Dr. Michael E. Vayda, Chair, Biochemistry Search Committee, 5735 Hitchner-BMMB, University of Maine, Orono, ME 04469-5735. Review of applications will commence June 1, 1997 and applications will be considered until a suitable candidate is located. Women and minorities are encouraged to apply. The University of Maine is an Affirmative Action/Equal Employment Opportunity Employer. From owner-proteins@net.bio.net Wed Apr 02 23:00:00 1997 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!cam-news-hub1.bbnplanet.com!news.bbnplanet.com!news-xfer.netaxs.com!hammer.uoregon.edu!csulb.edu!drivel.ics.uci.edu!news.service.uci.edu!dmthomas From: dmthomas@orion.oac.uci.edu (Didier Thomas) Newsgroups: bionet.molbio.proteins Subject: Re: How to improve sensitivity of immunocytochemistry? Date: Thu, 03 Apr 1997 18:55:39 +1000 Organization: UC Irvine Lines: 41 Message-ID: References: NNTP-Posting-Host: rablab.biochem.uci.edu Mime-Version: 1.0 Content-Type: text/plain; charset=ISO-8859-1 Content-Transfer-Encoding: 8bit X-Newsreader: Yet Another NewsWatcher 2.4.0 In article , Alix Helme-Guizon wrote: > I want to detect a membranous protein on slices (20 or 30 microns)in an > area that express only few of this protein.(Of course this area express > the protein, as this protein can be seen on western blot with extract from > this area (first antibody 1/20 000 for 6 microgrammes of membrane > preparation.) > > i've tried > -DAB plus nickel with biotynilated secondary antibody (first > antibody 1/5OO overnight RT in triton 0,3%+ serum 10% in PBS, > preincubation 1h in serum10%+triton+PBS RT). this gave a very good > labeling in another area (known to express well the protein) but not in > the the place i want to study! > -DAB without nickel (same protocol) > -fluorescence with a secondary antibody (1/3oo) > > What can I do to improve sensitivity of antigen detection on slices > without increasing too much the background? > Thanks a lot for your help! > Alix Alix, I would stick to immunofluorescence labeling. It gives much sharper labeling than DAB (plus enhancer). 0.3% is definitely too much, especially when trying to detect a membranar protein. Actually, if you add 0.3% TX100 in all your buffers, I don't think membranes are preserved. I would recommend a short permeation step of 1 min with 0.1% TX and not to use it anymore for the remaining steps of the detection. Tell me how it works. Good luck -- Didier Thomas Dept. of Physiology and Biophysics Med. Sci. I, Room D340 University of California, Irvine Irvine, CA 92697 From owner-proteins@net.bio.net Thu Apr 03 23:00:00 1997 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news-peer.sprintlink.net!EU.net!main.Germany.EU.net!news-koe1.dfn.de!news.ruhr-uni-bochum.de!not-for-mail From: Bastian Zimmermann Newsgroups: bionet.molbio.proteins Subject: Re: Help! cyclic-AMP gene Date: Fri, 04 Apr 1997 17:41:21 -0800 Organization: Ruhr-Universitaet Bochum, Rechenzentrum Lines: 14 Message-ID: <3345ADC1.5D76@rz.ruhr-uni-bochum.de> References: <333DCCA0.2FC7@worldnet.att.net> NNTP-Posting-Host: r2-142.phc.ruhr-uni-bochum.de Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Win16; I) To: CHEE SENG LEE CHEE SENG LEE wrote: > > Does anyone know if it is known the gene for cyclic-AMP, CREB1, CREB2 > or kinase? Hi, the gene for the catalytic subunit c-alpha of the camp-dependent protein kinase localizes to human chromosome region 19p13.1. For further details look for Genomics 36(3):535-38, 1996 Sep15 Tasken k et al greetings from rub Bochum Bastian Zimmermann From owner-proteins@net.bio.net Thu Apr 03 23:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!news.maxwell.syr.edu!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news-peer.gsl.net!uwm.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!daemon From: klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins Subject: Re: Puirification of His-tag protein Date: Sat, 05 Apr 1997 02:18:46 GMT Organization: UW-Madison Lines: 23 Message-ID: <5i4ctn$n5i@news.doit.wisc.edu> References: <01bc412b$9a8778e0$a8a995c2@cmariecl.hol.fr> NNTP-Posting-Host: f183-033.net.wisc.edu X-Newsreader: News Xpress 2.01 X-No-Archive: Yes In article <01bc412b$9a8778e0$a8a995c2@cmariecl.hol.fr>, "MARIE-CLAIRE C" wrote: #We are working on a zinc metalloprotease. We expressed the enzyme as a 6 #His-Tag protein using the pRset system from invitrogen and the TALN Co2+ #resin from Clontech to purify the product. We have problems to dissociate #the His-Tag protein from the resin. #We've tried imidazole up to 0.5 M (slight dissociation) and pH 5 (no #dissociation). EDTA works well but it also inhibit our enzyme! #If anyone has experience in these problem please contact me #Thanks for your help and attention Such strong binding is very unusual. 1. Does EDTA inhibit reversibly? In other words, can you elute with EDTA, dialyze against Zn2+, then dialyze against working buffer and get active protein? If yes, you have no problem but a method to get very pure enzyme. 2. If EDTA kills your protein, try no tag at all. Chances are your protein sticks so tigthly NOT through 6His. Another thought k(off) is too slow - try to leave resin with imidazole for much longer. - Dima From owner-proteins@net.bio.net Thu Apr 03 23:00:00 1997 Newsgroups: bionet.molbio.proteins,bionet.molbio.methds-reagnts Path: biosci!rutgers.rutgers.edu!gatech!csulb.edu!hammer.uoregon.edu!leto.ou.edu!news.ou.edu!news.ecn.uoknor.edu!munnari.OZ.AU!metro!metro!news.nsw.CSIRO.AU!tastiger.dbe.csiro.au!user From: graham@dbe.csiro.au (Lloyd Graham) Subject: Labelling -COOH groups with fluorosceinyl-Gly-amide: help! X-Nntp-Posting-Host: tastiger.dbe.csiro.au Message-ID: Sender: news@news.nsw.CSIRO.AU Organization: CSIRO Date: Wed, 2 Apr 1997 07:04:37 GMT Lines: 21 Xref: biosci bionet.molbio.proteins:10415 bionet.molbio.methds-reagnts:56367 Does anyone have details of reaction conditions suitable for labelling the surface carboxylic acids of a protein with fluoresceinyl glycine amide in the presence of EDAC and NHSS? The reaction is apparently done under aqueous conditions, but the manufacturers supply the reagents without any hints as to buffers, pH, concentrations, reaction time, etc., and (incredibly) the literature references they cite don't supply this vital information either. Thanks, Lyn. ************************************ DIRECT EMAIL REPLIES TO: lyns@med.su.oz.au Lynette Schedlich Kolling Institute of Medical Research RNSH, St Leonards 2065, NSW, Australia Phone No 61 2 99268486; Fax No 61 2 99268484 ************************************ From owner-proteins@net.bio.net Thu Apr 03 23:00:00 1997 Path: biosci!rutgers.rutgers.edu!gatech!csulb.edu!hammer.uoregon.edu!news.oru.edu!news-spur1.maxwell.syr.edu!news.maxwell.syr.edu!worldnet.att.net!news.dra.com!news.inlink.com!news.starnet.net!news.starnet.net!axb.slu.edu!news From: Darren Tyson Newsgroups: bionet.molbio.proteins Subject: Re: Call for Links Date: Wed, 02 Apr 1997 10:42:14 -0600 Organization: Saint Louis University Lines: 17 Message-ID: <33428C66.33C8@slu.edu> References: NNTP-Posting-Host: 165.134.73.104 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Win95; I) JJ Miranda wrote: > > Hi all, > > I've set up a small protein science web site with links to major sites of > interest (i.e. Brookhaven database). Since I want to make this as > comprehensive as possible, does anyone have any sites of their own or > other important links that they would like to see on resource site? If > so, please email the links to me. Thanks in advance for everyone's help. > > Sincere regards, > JJ Miranda What's the URL? I'd like to see what you have already. Darren From owner-proteins@net.bio.net Thu Apr 03 23:00:00 1997 Path: biosci!daresbury!uninett.no!news-feed.inet.tele.dk!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news-peer.gsl.net!uwm.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!alpha.oncology.wisc.edu!user From: mcmahan@oncology.wisc.edu (Scott McMahan) Newsgroups: bionet.molbio.proteins Subject: Re: Puirification of His-tag protein Date: Fri, 04 Apr 1997 17:18:33 -0500 Organization: University of Wisconsin-Madison Lines: 25 Message-ID: References: <01bc412b$9a8778e0$a8a995c2@cmariecl.hol.fr> NNTP-Posting-Host: alpha.oncology.wisc.edu X-Newsreader: Yet Another NewsWatcher 2.1.8 In article <01bc412b$9a8778e0$a8a995c2@cmariecl.hol.fr>, "MARIE-CLAIRE C" wrote: :We are working on a zinc metalloprotease. We expressed the enzyme as a 6 :His-Tag protein using the pRset system from invitrogen and the TALN Co2+ :resin from Clontech to purify the product. We have problems to dissociate :the His-Tag protein from the resin. :We've tried imidazole up to 0.5 M (slight dissociation) and pH 5 (no :dissociation). EDTA works well but it also inhibit our enzyme! :If anyone has experience in these problem please contact me :Thanks for your help and attention : C. MARIE-CLAIRE : cmc@hol.fr I'm not surprised you're having problems like this. Your protein has two potential IMAC sites. The original Zn binding site and the engineered His-tag. The column may not be completely charged and chelating the Zn, the Zn may be dissociating some of the Co, or the chelated Co may be displacing the Zn. I'd try running a column without the His-tag or increasing the imidazole concentration to account for the 2 sites per molecule. Good luck. -- Scott McMahan mcmahan@oncology.wisc.edu From owner-proteins@net.bio.net Thu Apr 03 23:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!news.maxwell.syr.edu!news.apfel.de!fu-berlin.de!unlisys!blackbush.xlink.net!news-ge.switch.ch!in2p3.fr!univ-lyon1.fr!jussieu.fr!iway.fr!news1.isdnet.net!usenet From: "MARIE-CLAIRE C" Newsgroups: bionet.molbio.proteins Subject: Puirification of His-tag protein Date: 4 Apr 1997 19:15:11 GMT Lines: 11 Message-ID: <01bc412b$9a8778e0$a8a995c2@cmariecl.hol.fr> NNTP-Posting-Host: ppp29.par.hol.fr Mime-Version: 1.0 Content-Type: text/plain; charset=ISO-8859-1 Content-Transfer-Encoding: 7bit X-Newsreader: Microsoft Internet News 4.70.1157 We are working on a zinc metalloprotease. We expressed the enzyme as a 6 His-Tag protein using the pRset system from invitrogen and the TALN Co2+ resin from Clontech to purify the product. We have problems to dissociate the His-Tag protein from the resin. We've tried imidazole up to 0.5 M (slight dissociation) and pH 5 (no dissociation). EDTA works well but it also inhibit our enzyme! If anyone has experience in these problem please contact me Thanks for your help and attention C. MARIE-CLAIRE cmc@hol.fr From owner-proteins@net.bio.net Fri Apr 04 23:00:00 1997 Path: biosci!rutgers.rutgers.edu!gatech!csulb.edu!hammer.uoregon.edu!arclight.uoregon.edu!newsfeeds.sol.net!uwm.edu!news.cse.psu.edu!news3.cac.psu.edu!psuvm!jxl199 Organization: Penn State University Date: Sat, 5 Apr 1997 17:32:19 EST From: Li Jiaxu Message-ID: <97095.173219JXL199@psuvm.psu.edu> Newsgroups: bionet.molbio.proteins Subject: Antibody to ser/thr kinase Lines: 1 Hi, Does anybody out there know a sourceof antibodies against the highly conse rved catalytic domain of ser/thr protein kinases? From owner-proteins@net.bio.net Sat Apr 05 23:00:00 1997 Path: biosci!biosci!not-for-mail From: Jan Koster Newsgroups: bionet.cellbiol,bionet.molbio.proteins,bionet.immunology,bionet.general,sci.bio.misc,sci.bio.microbiology,sci.misc Subject: The Integrin Page Date: 6 Apr 1997 08:53:43 -0700 Organization: Netherlands Cancer Institute, Amsterdam Lines: 14 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Xref: biosci bionet.cellbiol:7045 bionet.molbio.proteins:10422 bionet.immunology:11416 bionet.general:26334 sci.bio.misc:8278 sci.bio.microbiology:5654 sci.misc:22389 I'd like to bring the existence of a homepage about integrins to your attention. The site features: > general information about these interesting transmembrane glycoproteins. > Antibody data and references (74 are already present) > Easy access to genbank sequences from the subunits > and much more The site can be accessed on: http://www.geocities.com/CapeCanaveral/9629 From owner-proteins@net.bio.net Sat Apr 05 23:00:00 1997 Path: biosci!rutgers.rutgers.edu!gatech!arclight.uoregon.edu!ais.net!ameritech.net!uunet!in3.uu.net!205.198.116.242!kiki.bway.net!not-for-mail From: Thomas Weber Newsgroups: bionet.molbio.proteins Subject: Re: Puirification of His-tag protein Date: Sat, 05 Apr 1997 23:50:59 -0500 Organization: bway.net, part of Outernet Inc. in New York City Lines: 21 Message-ID: <33472BB2.4C45@bway.net> References: <01bc412b$9a8778e0$a8a995c2@cmariecl.hol.fr> <5i4ctn$n5i@news.doit.wisc.edu> Reply-To: tomweber@bway.net NNTP-Posting-Host: 205.198.117.76 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Macintosh; I; 68K) To: Dima Klenchin Dima Klenchin wrote: > > In article <01bc412b$9a8778e0$a8a995c2@cmariecl.hol.fr>, "MARIE-CLAIRE C" wrote: > #We are working on a zinc metalloprotease. We expressed the enzyme as a 6 > #His-Tag protein using the pRset system from invitrogen and the TALN Co2+ > #resin from Clontech to purify the product. We have problems to dissociate > #the His-Tag protein from the resin. > #We've tried imidazole up to 0.5 M (slight dissociation) and pH 5 (no > #dissociation). EDTA works well but it also inhibit our enzyme! > #If anyone has experience in these problem please contact me > #Thanks for your help and attention > A) tru Ni-NTA resin from say Qiagen, it seems to bind his tagged proteins less strongly B) try pH 5 AND 0.5M Imidazole. hope this helps Thomas From owner-proteins@net.bio.net Sat Apr 05 23:00:00 1997 Path: biosci!agate!howland.erols.net!worldnet.att.net!arclight.uoregon.edu!leto.ou.edu!news.ou.edu!not-for-mail From: "Kiley R. Prilliman" Newsgroups: bionet.molbio.proteins Subject: HELP--affinity chromatography nightmare Date: Sun, 06 Apr 1997 22:14:44 -0600 Organization: University of Oklahoma Health Sciences Center Lines: 52 Message-ID: <3348828F.44E2@rex.re.uokhsc.edu> Reply-To: kprillim@rex.re.uokhsc.edu NNTP-Posting-Host: 157.142.8.172 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0 (Macintosh; I; 68K) To Whom It May Concern: (Yes, this sounds like the beginning of a Last Will & Testament, but it very well COULD be for me if I don't get this problem solved, for which I am seeking YOUR HELP!)... This time last year I poured a large (100mL) affinity column for purifying a soluble protein of interest from mammalian cell culture supernatants (the volumes of supernatant I work from are rather large...I currently have ~30L sitting in a cold room waiting to be processed, hence the "nightmare" status of the problem I will describe). The affinity column matrix consists of a monoclonal antibody coupled to CNBr-activated Sepharose 4B (Pharmacia) --- I performed last year's coupling at room temperature precisely according to the manufacturer's instructions, and the column worked terrifically...all was good and life in the lab was happy, etc. etc. HOWEVER.......last weekend, in an attempt to make life EVEN BETTER (with the old column, I could process 2L of supernatant at a time...I wanted to be able to process up to 5-6L, or hopefully more, at a time), I took 700mg of our monoclonal (which took 5 months to produce and purify "in-house" in this amount) and a load of newly-purchased CNBR-activated Sepharose 4B and perfomred another coupling (again, exactly as I had done it before). To my extreme horror, when I tested the "new stuff" out last Monday, NONE OF MY PROTEIN FROM THE SUPERNATANT WAS BOUND! A setback indeed...however, I felt that not all was lost, for I still had the "old" matrix (which, fortunately, I had not been dumb enough to mix with the "new" matrix yet). I don't think I really need to tell you the rest of the story, but I will: I put the "old" matrix back in the column (it has been moved in/out of its column several times in the past year with no ill affect), ran some supernatant over it, and IT FAILED AS WELL. I am at a loss to explain this. The only "leads" I have at the moment are slim, but are as follows: (i) the supernatants I had been passing over the column up until this time were pH 7.0, but I have made note that the "more recent" supernatants are pH 7.5-7.6 (surely this minor discrepancy wouldn't affect the Ag-Ab interaction so severely though, would they?), and (ii) the "older" supernatants I have been using until now contained approximately 1.5mg/L of my protein of interest, while the "newer" supernatants contain about 2.5mg/L of the protein. ...does ANYONE have some insight into what could be going on here? Any assistance you might provide would be sincerely appreciated! Thank you, Kiley R. Prilliman Department of Microbiology & Immunology University of Oklahoma Health Sciences Center phone: 405-271-1203 fax: 405-271-3117 From owner-proteins@net.bio.net Sat Apr 05 23:00:00 1997 Path: biosci!biosci!not-for-mail From: yasuhito@m.ehime-u.ac.jp (Yasuhito Abe) Newsgroups: bionet.molbio.proteins,bionet.immunology,bionet.general,bionet.cellbiol,bionet.molbio.methds-reagnts,sci.med,jpmed.clinical.surgery,sci.engr.biomed,jpmed.basic.misc,sci.med.informatics,tnn.medical,jpmed.basic.molecular,sci.med.immunology,sci.med.pathology,bionet.biology.cardiovascular Subject: Research cooperation Date: 6 Apr 1997 09:08:11 -0700 Organization: 2nd Dept of Surgery, Ehime Univ Lines: 19 Sender: biohelp@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Xref: biosci bionet.molbio.proteins:10424 bionet.immunology:11417 bionet.general:26335 bionet.cellbiol:7046 bionet.molbio.methds-reagnts:56445 sci.med:155582 sci.engr.biomed:7977 sci.med.informatics:8983 sci.med.immunology:9378 sci.med.pathology:5030 bionet.biology.cardiovascular:1610 Dear Sir, We are now seeking research cooperators on the antibody therapy using anti-neutrophil MAb Urge-8. If you were interested in this matter, please take a look at my home page, Urge-8 home page. URL is: http://www.m.ehime-u.ac.jp/~yasuhito/Urghome.html Yours sincerely, Yasuhito Abe, M.D. The Second Dept of Surgery, Ehime Univ School of Med, Shigenobu, Ehime 7910202 Japan e-mail: yasuhito@m.ehime-u.ac.jp www: http://www.m.ehime-u.ac.jp/~yasuhito/Home.html From owner-proteins@net.bio.net Sat Apr 05 23:00:00 1997 Path: biosci!biosci!not-for-mail From: biodigm@dial.pipex.com (M J Geisow) Newsgroups: bionet.molbio.proteins,bionet.structural-nmr Subject: ANNOUNCE: POPE '97 Genome-Proteome-Function - Conference Date: 6 Apr 1997 09:03:43 -0700 Organization: UUNet PIPEX server (post doesn't reflect views of UUNet PIPEX) Lines: 36 Sender: daemon@net.bio.net Distribution: world Message-ID: <3342faa9.56116982@news.dial.pipex.com> NNTP-Posting-Host: net.bio.net Xref: biosci bionet.molbio.proteins:10423 bionet.structural-nmr:1850 The Programme for the Perspectives on Protein Engineering Conference (Norwich Norfolk, UK 28 June - 1 July 1997) is now published, with speaker abstracts received to date at the Web site: http://www.biodigm.com/pope/pope6.htm A new conference rate for non-profit organisations and further reductions for pre-doctoral students is posted at the site Day 1 Genome - Proteome Opening speakers: J Craig Venter (TIGR) Amos Bairoch (Geneva) Mike Bevan (Norwich) Iain Campbell (Oxford) Minisymposia Membrane protein expression & structure Collaboration in bioinformatics via WWW Protein characterisation Metal Centres in Proteins Day 2 Protein Design & Expression Please See site for speakers Day 3 Structure-function: biocatalysis & plant protein engineering Please See site for speakers POPE 6 Secretariat biodigm@dial.pipex.com 64, Langdale Grove Bingham, NG13 8SS, UK Fax +44 1 949 876 156 M Geisow(UK Structural Biology CEC Contact Group representative) M Geisow(UK Structural Biology CEC Contact Group representative) From owner-proteins@net.bio.net Sun Apr 06 23:00:00 1997 Path: biosci!agate!nntpfeed.doc.ic.ac.uk!sunsite.doc.ic.ac.uk!charlie.lif.icnet.uk!mac052034.lif.icnet.uk!user From: I.McFarlane@icrf.icnet.uk (Ian McFarlane) Newsgroups: bionet.molbio.proteins Subject: Re: Help! Triton X114 blues.... Date: Mon, 07 Apr 1997 12:28:37 +0000 Organization: Imperial Cancer Research Fund Lines: 36 Message-ID: References: <5fkaul$jrd@is.bbsrc.ac.uk> <331e7b2b.2169592@news.univie.ac.at> NNTP-Posting-Host: mac052034.lif.icnet.uk X-Newsreader: Value-Added NewsWatcher 2.0b27+ In article <331e7b2b.2169592@news.univie.ac.at>, a8803349@unet.univie.ac.at (Martin Offterdinger) wrote: > On 5 Mar 1997 17:36:21 GMT, dunnsm@bbsrc.ac.uk (Steve Dunn) wrote: > > >Dear netters, > > > >I've been dabbling with Triton X114 phase-partitioning of my microsomal > >fraction. After washing the detergent-rich phase a few times with PBS, > >I (following a procedure in Methods In Enzymology...) precipitate the > >protein with 10 volumes of ice-cold acetone. This, according to my method, > >is supposed to get rid of the detergent so that it doesn't interfere with > >SDS-PAGE gels. After pelleting the protein and removing all the > >acetone, I resuspend the pellet in 5%SDS and loading buffer for SDS-PAGE. > >However, after staining the gel with coomassie, there's an artifactual > >smear obscuring my protein bands which I understand is symptomatic of > >X114 contamination. Even after processing the acetone pellet through a > >chloroform/MeOH cleanup (Wessel & Flugge, Anal. Biochem.,1984) the smear > >persists! If I run just SDS-solubilised microsomes, there's no smear. > >Any ideas how to successfully combine X114 extraction with SDS-PAGE?? > >All suggestions gratefully received... > > > >Steve Dunn > >dunnsm@bbsrc.ac.uk > Dear Steve > Triton is know to be able to form peroxides in solution , which might > partially react with your protein and therefore cause this smear. > Check if your Triton is expired, store in the dark(!) , use reducing > agents to reduce the peroxides(e.g.:DTT) ... > Hope this helps > Martin It's also possible to extract the peroxides from the X-114 before you start using it. Sorry I don't have a reference tho. Ian Mc From owner-proteins@net.bio.net Sun Apr 06 23:00:00 1997 Path: biosci!agate!nntpfeed.doc.ic.ac.uk!sunsite.doc.ic.ac.uk!charlie.lif.icnet.uk!mac052034.lif.icnet.uk!user From: I.McFarlane@icrf.icnet.uk (Ian McFarlane) Newsgroups: bionet.molbio.proteins Subject: Re: seek any methods determine and purify mutimeric protein Date: Mon, 07 Apr 1997 12:31:30 +0000 Organization: Imperial Cancer Research Fund Lines: 19 Message-ID: References: <331FC465.7F75@donald.hanho.co.kr> NNTP-Posting-Host: mac052034.lif.icnet.uk X-Newsreader: Value-Added NewsWatcher 2.0b27+ In article <331FC465.7F75@donald.hanho.co.kr>, compania@donald.hanhyo.co.kr wrote: > Hello.. > > i'm trying to find out that my recombinant protein which is secreted by > S. cerevisiae, is multimer or monomer in culture medium. > > would you show my applicable methods? > and if it will be mutimer, is it possible to purify it as it is? > > i hope your help. Try running some non-denaturing (no bME), non-dissociating (no SDS) PAGE gels. If your multimer is fairly stable it should stay associated throughout the run. You could also try running a gel filtration column which would give you an idea of the size. Ian Mc From owner-proteins@net.bio.net Sun Apr 06 23:00:00 1997 Path: biosci!rutgers.rutgers.edu!gatech!csulb.edu!hammer.uoregon.edu!xfer.kren.nm.kr!mr.net!news.idt.net!cam-news-hub1.bbnplanet.com!news.bbnplanet.com!uunet!in2.uu.net!128.6.21.17!dziuxsolim.rutgers.edu!er7.rutgers.edu!not-for-mail From: paco@eden.rutgers.edu (Indian Blues Box) Newsgroups: bionet.molbio.proteins Subject: Methylase vs Methyltransferase question Date: 7 Apr 1997 16:41:08 -0400 Organization: Rutgers University Lines: 11 Message-ID: <5ibm54$979@er7.rutgers.edu> NNTP-Posting-Host: er7.rutgers.edu Can anyone tell me the real difference (if any exists) between a Methylase and a methyltransferase? There doesn't seem to be a clear distinction (dam methylase, O6-Methylguanyltransferase, etc... all seem to have overlapping/similar functions). Please do not post the answer. Send mail to edelstlc@umdnj.edu and/or paco@ruphys.rutgers.edu thank you -gautam malhotra len edelstein From owner-proteins@net.bio.net Sun Apr 06 23:00:00 1997 Path: biosci!VINES2.WAU.NL!Joachim=Goedhart%Laser%BC.WAU From: Joachim=Goedhart%Laser%BC.WAU@VINES2.WAU.NL (joachim goedhart) Newsgroups: bionet.molbio.proteins Subject: Disulfide bonds Date: 7 Apr 1997 04:13:28 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 10 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Hello, I'm looking for a method to seperate the L and H chains of an IgG molecule. These chains are linked by disulfide bonds. DTT and mercaptaethanol probably destroy all disulfide bonds?! Does anybody know a method to disrupt only the INTERmolecular disulfide bonds without destroying the INTRAmolecular bridges? Thanx, Joachim. From owner-proteins@net.bio.net Sun Apr 06 23:00:00 1997 Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!news.ecn.uoknor.edu!feed1.news.erols.com!newsfeeds.sol.net!uwm.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!martinlab From: klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins Subject: Re: Receptors and G-Proteins Date: Mon, 07 Apr 97 21:04:02 GMT Organization: UW-Madison Lines: 31 Message-ID: <5ibur1$11fg@news.doit.wisc.edu> References: <01bc406c$ecc04340$adb695c2@default> NNTP-Posting-Host: martinlab.biochem.wisc.edu X-Newsreader: News Xpress Version 1.0 Beta #4 X-No-Archive: Yes In article <01bc406c$ecc04340$adb695c2@default>, "Christophe Guenin" wrote: ->Dear netters -> ->I am looking for structures of the genes of the receptors coupled to G ->Proteins -> -> ->You can answer in this news group ou send a mail to ->christophe.guenin@hol.fr ->Best wishes and many thanks for your participation -> ->Sophie-Penelope Lechevallier - SERVIER Labs None has been crystallized => no structure. Seven helix structure, though, could be considered proven, and at least for rhodopsin sites of interactions with G-protein has been mapped. - Dima __ / /\ Dima Klenchin / / \ / / /\ \ klenchin@facstaff.wisc.edu / / /\ \ \ tel. (608)263-1163 / /_/__\ \ \ FAX (608)262-3453 /________\ \ \ \___________\/ From owner-proteins@net.bio.net Sun Apr 06 23:00:00 1997 Path: biosci!aol.com!ANATRAC434 From: ANATRAC434@aol.com Newsgroups: bionet.molbio.proteins Subject: Triton X-114 Date: 7 Apr 1997 11:51:31 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 41 Sender: daemon@net.bio.net Distribution: world Message-ID: <970407145155_774663084@emout10.mail.aol.com> NNTP-Posting-Host: net.bio.net >Dear netters, > > > >I've been dabbling with Triton X114 phase-partitioning of my microsomal > >fraction. After washing the detergent-rich phase a few times with PBS, > >I (following a procedure in Methods In Enzymology...) precipitate the > >protein with 10 volumes of ice-cold acetone. This, according to my method, > >is supposed to get rid of the detergent so that it doesn't interfere with > >SDS-PAGE gels. After pelleting the protein and removing all the > >acetone, I resuspend the pellet in 5%SDS and loading buffer for SDS-PAGE. > >However, after staining the gel with coomassie, there's an artifactual > >smear obscuring my protein bands which I understand is symptomatic of > >X114 contamination. Even after processing the acetone pellet through a > >chloroform/MeOH cleanup (Wessel & Flugge, Anal. Biochem.,1984) the smear > >persists! If I run just SDS-solubilised microsomes, there's no smear. > >Any ideas how to successfully combine X114 extraction with SDS-PAGE?? > >All suggestions gratefully received... > > > >Steve Dunn > >dunnsm@bbsrc.ac.uk > Dear Steve > Triton is know to be able to form peroxides in solution , which might > partially react with your protein and therefore cause this smear. > Check if your Triton is expired, store in the dark(!) , use reducing > agents to reduce the peroxides(e.g.:DTT) ... > Hope this helps > Martin >It's also possible to extract the peroxides from the X-114 before you >start using it. Sorry I don't have a reference tho. >Ian Mc Anatrace, Inc offers a purified, low peroxide grade of Triton X-114 if anyon is interested. Stored under argon. Mel Keyes Anatrace, Inc. ---------- From owner-proteins@net.bio.net Sun Apr 06 23:00:00 1997 Path: biosci!rutgers.rutgers.edu!gatech!csulb.edu!hammer.uoregon.edu!news-peer.gsl.net!howland.erols.net!cam-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!ais.net!ameritech.net!uunet!in2.uu.net!138.133.17.7!amgen!usenet From: John Philo Newsgroups: bionet.molbio.proteins Subject: Re: HELP--affinity chromatography nightmare Date: Mon, 07 Apr 1997 09:18:10 -0700 Organization: Amgen Inc. Lines: 26 Message-ID: <33491E41.ED2@amgen.com> References: <3348828F.44E2@rex.re.uokhsc.edu> Reply-To: jphilo@amgen.com Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01Gold (Win95; I) To: kprillim@rex.re.uokhsc.edu Kiley R. Prilliman wrote: > > This time last year I poured a large (100mL) affinity column for > purifying a soluble protein of interest from mammalian cell culture > > (snip) > > Any > assistance you might provide would be sincerely appreciated! > It seems to me your results strongly suggest that the problem is with your new batch of protein to be purified rather than with your affinity column. Are you certain your protein is actually still being expressed? Even if you can see it on a gel, are you certain there hasn't been some change (e.g. proteolysis) that would affect it binding to the antibody column. Proteolysis is certainly one thing that commonly varies from batch to batch. Can you see your protein in the conditioned media using this same antibody to do a Western? (presuming, of course, that this antibody work in a Western format). If it no longer shows up in a Wester, that tells you that indeed your epitope is gone or no longer available (possibly tied up by binding to a third component). John Philo, Protein Chemistry, Amgen *** Disclaimer: These are the opinions of the poster not Amgen Inc.*** From owner-proteins@net.bio.net Sun Apr 06 23:00:00 1997 Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!news.ecn.uoknor.edu!feed1.news.erols.com!news-xfer.netaxs.com!newsfeeds.sol.net!uwm.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!martinlab From: klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins Subject: Re: HELP--affinity chromatography nightmare Date: Mon, 07 Apr 97 20:57:54 GMT Organization: UW-Madison Lines: 72 Message-ID: <5ibufi$11fg@news.doit.wisc.edu> References: <3348828F.44E2@rex.re.uokhsc.edu> NNTP-Posting-Host: martinlab.biochem.wisc.edu X-Newsreader: News Xpress Version 1.0 Beta #4 X-No-Archive: Yes In article <3348828F.44E2@rex.re.uokhsc.edu>, "Kiley R. Prilliman" wrote: ->To Whom It May Concern: ->(Yes, this sounds like the beginning of a Last Will & Testament, but it ->very well COULD be for me if I don't get this problem solved, for which ->I am seeking YOUR HELP!)... -> ->This time last year I poured a large (100mL) affinity column for ->purifying a soluble protein of interest from mammalian cell culture ->supernatants (the volumes of supernatant I work from are rather ->large...I currently have ~30L sitting in a cold room waiting to be ->processed, hence the "nightmare" status of the problem I will ->describe). The affinity column matrix consists of a monoclonal antibody ->coupled to CNBr-activated Sepharose 4B (Pharmacia) --- I performed last ->year's coupling at room temperature precisely according to the ->manufacturer's instructions, and the column worked terrifically...all ->was good and life in the lab was happy, etc. etc. -> ->HOWEVER.......last weekend, in an attempt to make life EVEN BETTER (with ->the old column, I could process 2L of supernatant at a time...I wanted ->to be able to process up to 5-6L, or hopefully more, at a time), I took ->700mg of our monoclonal (which took 5 months to produce and purify ->"in-house" in this amount) and a load of newly-purchased CNBR-activated ->Sepharose 4B and perfomred another coupling (again, exactly as I had ->done it before). To my extreme horror, when I tested the "new stuff" ->out last Monday, NONE OF MY PROTEIN FROM THE SUPERNATANT WAS BOUND! A ->setback indeed...however, I felt that not all was lost, for I still had ->the "old" matrix (which, fortunately, I had not been dumb enough to mix ->with the "new" matrix yet). I don't think I really need to tell you the ->rest of the story, but I will: I put the "old" matrix back in the column ->(it has been moved in/out of its column several times in the past year ->with no ill affect), ran some supernatant over it, and IT FAILED AS ->WELL. -> ->I am at a loss to explain this. The only "leads" I have at the moment ->are slim, but are as follows: (i) the supernatants I had been passing ->over the column up until this time were pH 7.0, but I have made note ->that the "more recent" supernatants are pH 7.5-7.6 (surely this minor ->discrepancy wouldn't affect the Ag-Ab interaction so severely though, ->would they?), and (ii) the "older" supernatants I have been using until ->now contained approximately 1.5mg/L of my protein of interest, while the ->"newer" supernatants contain about 2.5mg/L of the protein. -> ->....does ANYONE have some insight into what could be going on here? Any ->assistance you might provide would be sincerely appreciated! Difficult case indeed. Unlikely anyone will come up with magic trick. Myself, I can only ask 2 things: 1. Have you checked coupling of the second batch of Ab? (ALWAYS so!) - there is a slim chance that your 2nd time prep didn't couple (whatever...), while 1st one died peasful death (whatever...) 2. Can you be sure the protein to be retained is the same in 2 cases? I can imagine a number of possibilities... How do you express/detect it? 3. In SOME cases mAb binding is extremely pH dependent. Rarely but happens. No refs, but I recall smth about anti-His-tag mAbs that fail to bind protonated His (in other words, they work at 7.5 and don't at 7.0) - Dima __ / /\ Dima Klenchin / / \ / / /\ \ klenchin@facstaff.wisc.edu / / /\ \ \ tel. (608)263-1163 / /_/__\ \ \ FAX (608)262-3453 /________\ \ \ \___________\/ From owner-proteins@net.bio.net Mon Apr 07 23:00:00 1997 Path: biosci!agate!howland.erols.net!europa.clark.net!news.maxwell.syr.edu!news.apfel.de!news-fra1.dfn.de!news-ge.switch.ch!serra.unipi.it!newsserver.cilea.it!oracle.csi.unimi.it!usenet From: clsmteam@imiucca.csi.unimi.it (Paolo Castano) Newsgroups: bionet.molbio.proteins Subject: IMMUNOFLUORESCENCE COURSE Date: Tue, 08 Apr 1997 14:28:25 GMT Organization: Istitute of Human Anatomy Lines: 15 Message-ID: <334a5600.28476785@news.csi.unimi.it> NNTP-Posting-Host: modem12.csi.unimi.it X-Newsreader: Forte Free Agent 1.1/32.230 Advanced International Immunofluorescence Course Gargnano '97 (Italy) The Advanced International Immunofluorescence Course is a post-doctorate theoretical/practical course, with propedeutical lectures and practical stages on traditional and confocal immunofluorescence microscopy and image and ion analysis. The course will take place in Gargnano (Lake of Garda) from 7 to 10 October 1997. Further information and registration details will be found at the following Web address http://imiucca.csi.unimi.it/endomi/ACIF.html Thank you Paolo Castano From owner-proteins@net.bio.net Mon Apr 07 23:00:00 1997 Newsgroups: bionet.molbio.proteins Path: biosci!daresbury!yama.mcc.ac.uk!liv!news From: lgbell@liverpool.ac.uk Subject: Re: Disulfide bonds Content-Type: text/plain; charset=us-ascii Message-ID: <334A132A.3805@liverpool.ac.uk> Sender: news@liverpool.ac.uk (News System) Nntp-Posting-Host: pc028010.med.liv.ac.uk Content-Transfer-Encoding: 7bit Organization: The University of Liverpool References: Mime-Version: 1.0 Date: Tue, 8 Apr 1997 09:43:06 GMT X-Mailer: Mozilla 2.02 (Win16; I) Lines: 26 joachim goedhart wrote: > > Hello, > > I'm looking for a method to seperate the L and H chains of an IgG molecule. These chains are linked by > disulfide bonds. DTT and mercaptaethanol probably destroy all disulfide bonds?! > Does anybody know a method to disrupt only the INTERmolecular disulfide bonds without destroying the > INTRAmolecular bridges? > > Thanx, > > Joachim.I would of thought, the way round this would be to break all the disulphides with say DTT. Seperate the two chains then re-form their respective intramolecular disulphides, by atmospheric oxidation at alkali pH. As I cant see how you would distinquish between inter and intraM disulphides. Though I guess if the protein structure is such that the interM bridges are more readily solvent, and hence DTT, accessible than the intraM bridges a kinetic differenc emay exist between the two forms. I hope this helps, Regards, Len Bell. lgbell@liv.ac.uk From owner-proteins@net.bio.net Mon Apr 07 23:00:00 1997 Path: biosci!rutgers.rutgers.edu!gatech!www.nntp.primenet.com!nntp.primenet.com!news.maxwell.syr.edu!news.apfel.de!news-fra1.dfn.de!news-ge.switch.ch!news-zh.switch.ch!rzunews.unizh.ch!newsadm From: Rolf Kocherhans Newsgroups: bionet.molbio.proteins Subject: Free practical programs for molecular biologists !!! Date: Tue, 08 Apr 1997 15:21:22 +0100 Organization: University of Zurich Lines: 49 Message-ID: <334A5462.7854@vetvir.unizh.ch> Reply-To: rolfk@vetvir.unizh.ch NNTP-Posting-Host: 130.60.22.38 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Macintosh; I; PPC) I wrote a few practical and simple to use programs which facilitate your daily work in the lab such as predicting the size of DNA fragments after digestion (graphical) with restriction enzymes. I would like to make these programs accessible to a broad user group by the Internet. All programs have been tested on MacOS and Windows95. My programs are accessible over the WWW and made functional using Netscape 2.x or Internet Explorer 2.x or higher in association with a free plugin which you have to download and install first. This is what you do: - Download the Roadster plugin http://www.unizh.ch/vetvir/plugin.html) install it on your computer. - Then connect to: http://www.unizh.ch/vetvir/programs.html That's it !! These are my programs which make your live as a molecular biologist easier ! Find here a few more examples or my programs: a. Digest Preview: Enter the size(s) of your DNA fragment(s) and see their migration pattern in a virtual gel in comparison to a 1 kb ladder. b. Adapter Design: Helps to create in frame adapters in order to link incompatible DNA ends together. c. Dilution Calculator: Does all the calculations when you have to make up solutions There are many other programs such as Oligo Tm, Compatible ends etc. Please have a look, comments are welcome! Have fun Rolf Kocherhans mailto:rolfk@vetvir.unizh.ch From owner-proteins@net.bio.net Mon Apr 07 23:00:00 1997 Path: biosci!daresbury!yama.mcc.ac.uk!news.york.ac.uk!not-for-mail From: David Scott Newsgroups: bionet.molbio.proteins Subject: FPLC controller Date: Tue, 08 Apr 1997 13:31:42 -0700 Organization: University of York. York. Y01 5DD Lines: 13 Sender: djs17@biolpc227.york.ac.uk Message-ID: <334AAB2E.2C2D@york.ac.uk> NNTP-Posting-Host: biolpc227.york.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0 (Win16; I) Hello I don't suppose anyone out there is getting rid of a Pharmacia LCC-500 FPLC controller?? Will pay carriage, etc.... David Scott Dept. Biology University of York YORK YO2 1JR UK + 44 1904 432867 From owner-proteins@net.bio.net Mon Apr 07 23:00:00 1997 Path: biosci!agate!spool.mu.edu!uwm.edu!news.cse.psu.edu!news3.cac.psu.edu!howland.erols.net!cam-news-hub1.bbnplanet.com!news.bbnplanet.com!news.maxwell.syr.edu!nntp.uio.no!uninett.no!news.uni-c.dk!news-inn.uni-c.dk!news.uni-c.dk!balder.adm.ku.dk!not-for-mail From: Claus Lundegaard Newsgroups: bionet.biophysics,bionet.molbio.proteins,bionet.biophysics Subject: Meeting on Enzyme Families in Nucleotide Metabolism, new deadline Date: Fri, 04 Apr 1997 13:38:18 +3500 Organization: Copenhagen University Lines: 19 Message-ID: <3349AF9A.167E@mermaid.molbio.ku.dk> NNTP-Posting-Host: cef.molbio.ku.dk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (X11; I; IRIX 6.2 IP22) Xref: biosci bionet.biophysics:2806 bionet.molbio.proteins:10437 Please see new deadline for attending this meeting in copenhagen: MAY 1 For further details see: http://cef.molbio.ku.dk/moede.html -- ___________________________________________________________________________ ******************************************************* Claus Lundegaard Center For Enzyme Research Soelvgade 83 H 1307 Koebenhavn K phone: (+45) 35 32 20 18 fax: (+45) 35 32 20 40 e-mail: mailto:lundegaard@mermaid.molbio.ku.dk www: http://cef.molbio.ku.dk/~lunde/Claus.html ******************************************************* From owner-proteins@net.bio.net Mon Apr 07 23:00:00 1997 Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!metro!metro!sbe From: sbe@biochem.usyd.edu.au (Simon Easterbrook-Smith) Newsgroups: bionet.molbio.proteins Subject: Re: Disulfide bonds Date: Tue, 08 Apr 1997 09:22:31 +1100 Organization: Department of Biochemistry, University of Sydney Lines: 42 Distribution: world Message-ID: References: NNTP-Posting-Host: sbepb.biochem.usyd.edu.au Mime-Version: 1.0 Content-Type: text/plain; charset=ISO-8859-1 Content-Transfer-Encoding: 8bit X-Newsreader: Yet Another NewsWatcher 2.3.4 I assume that you mean intra/inter-**chain**, rather than **molecular** disulfide bonds. In that case, you should be able to do it by using suitably low concns of the reducing agent - eg, for rabbit IgG it is possible to cleave the inter-chain -SS- bonds, leaving the intra-chain bonds largely intact using about 0.1 - 0.5 mM DTT at pH 8. I have never done this for other Igs but it should be possible. It is best to do an analytical run first (vary [DTT] and time, monitor what is happening by SDS/PAGE) to get the conditions right for prep-scale work. In article , Joachim=Goedhart%Laser%BC.WAU@VINES2.WAU.NL (joachim goedhart) wrote: || Hello, || || I'm looking for a method to seperate the L and H chains of an IgG molecule. These chains are linked by || disulfide bonds. DTT and mercaptaethanol probably destroy all disulfide bonds?! || Does anybody know a method to disrupt only the INTERmolecular disulfide bonds without destroying the || INTRAmolecular bridges? || || Thanx, || || Joachim. Simon -- _______________________________________________________________ Dr Simon B Easterbrook-Smith Voice: (+) 612 9351 3905 Department of Biochemistry FAX: (+) 612 9351 4726 University of Sydney Email: sbe@biochem.usyd.edu.au Sydney NSW 2006 AUSTRALIA _______________________________________________________________ Never attribute to malice that which is adequately explained by stupidity. _______________________________________________________________ From owner-proteins@net.bio.net Mon Apr 07 23:00:00 1997 Path: biosci!daresbury!uninett.no!sn.no!nntp.uio.no!news.apfel.de!univ-lyon1.fr!pasteur.fr!jussieu.fr!univ-lille1.fr!crihan.fr!u-psud.fr!usenet From: bruno Collinet <"bruno"@epcm.u-psud .fr> Newsgroups: bionet.molbio.proteins Subject: Stocking proteins in glycerol Date: Tue, 08 Apr 1997 17:38:55 +0200 Organization: Laboratoire de Modelistion et d'Ingenierie des Proteines Lines: 10 Message-ID: <5ido92$ia5@upsn6.u-psud.fr> Reply-To: bruno@epcm.u-psud.fr NNTP-Posting-Host: wagner.epcm.u-psud.fr Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Mailer: Mozilla 3.01 [fr] (Win95; I) Hi, I use to stock my purified protin in ammoniumsulfat but recently I tried to store it at -20°C in 20%glycerol. I think when I desalt an aliquot on a gel filtration column, there is still some glycerol stuck to the protein... Did anyone have an answer to this problem? I am also looking for a simple test to check the presence of glycerol after desalting... Many thanks for your answers. Bruno From owner-proteins@net.bio.net Mon Apr 07 23:00:00 1997 Path: biosci!daresbury!uninett.no!nntp.uio.no!newsfeeds.sol.net!newspump.sol.net!howland.erols.net!cam-news-hub1.bbnplanet.com!cpk-news-feed1.bbnplanet.com!news.bbnplanet.com!nih.gov!usenet From: bernard@elsie.nci.nih.gov (Bernard Murray) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: Coating ELISA plates in Urea Date: 8 Apr 1997 19:42:43 GMT Organization: National Institutes of Health, Bethesda, MD Lines: 34 Message-ID: <5ie73j$dpv@light.nih.gov> References: NNTP-Posting-Host: ermintrude.nci.nih.gov X-Newsreader: WinVN 0.99.7 Xref: biosci bionet.molbio.methds-reagnts:56518 bionet.molbio.proteins:10443 In article , zjons@vetbio.unizh.ch says... > >Dear friends > We have a small problem here. We have a protein and want to couple it >to ELISA plates. As you already guessed, the protein is rather insoluble >so this has to be done in the presence of a solubilizing agent. Our first >idea was to use Ni-NTA coated wells and bind the His-tagged protein in the >presence of Urea. This should work, but those plates are very expen$ive. >So I started thinking. Isn't it possible to couple the protein to >standard ELISA plates in the presence of Urea or another solubilizing >agent? I, not being a chemist, can't see why it should not work but maybe >someone out there can prove me wrong. > If someone has tried this or has a strong opinion, please don't hesitate >to respond. We are dying to hear from you! >Thank you ever so much >Zophonias et al. Zophonias, With most of the current "high capacity" ELISA plates there *is* binding in the presence of urea but I believe that the efficiency is somewhat lower (but don't ask me to put a figure on it) so your detection limit may not be very good. Low (eg. 0.1%) concentrations of SDS do not seem to interfere too much. My temptation is to suggest you switch to dot or slot blotting as attachment to membranes is much more efficient and definitely occurs in the presence of strong denaturants. You can also stain the membranes for total protein (eg. amido black) to ensure they are loaded well. I'm afraid that the bottom line is "try it and see". Bernard Bernard Murray, Ph.D. bernard@elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA) From owner-proteins@net.bio.net Mon Apr 07 23:00:00 1997 Path: biosci!daresbury!uninett.no!nntp.uio.no!newsfeeds.sol.net!worldnet.att.net!cpk-news-hub1.bbnplanet.com!cam-news-hub1.bbnplanet.com!news.bbnplanet.com!howland.erols.net!usc!usc!not-for-mail From: william@neuro.usc.edu (William Sun) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: in situ hybridization Date: 8 Apr 1997 13:25:32 -0700 Organization: University of Southern California, Los Angeles, CA Lines: 20 Sender: william@neuro.usc.edu Message-ID: <5ie9js$d0e@neuro.usc.edu> References: <5ie73j$dpv@light.nih.gov> NNTP-Posting-Host: neuro.usc.edu Xref: biosci bionet.molbio.methds-reagnts:56520 bionet.molbio.proteins:10444 Hello all, I'm doing in situ hybridization with brain sections. I use dextran sulfate in my hybe solution. I'm having problems getting bubbles on the slide. The hybe solution has a difficult time spreading across the tissue. In fact, when I put the solution on the tissue directly, the liquid beads up as if it were repelled. I treat the tissue with proteinase K to permeablize the tissue. Could the proteinase K treatment cause such repulsion? Another problem I have is that I'm using DPX to seal the coverslip onto the slide, which is both messy and time consuming. Is there a better way? Comments from experieced people are appreciated. -William -- William Sun, Ph.D Phone: (213)740-3406 Neuroscience Program FAX: (213)740-5687 University of Southern California Pager: (310)243-9878 Los Angeles, CA 90089-2520 http://www-bcf.usc.edu/~wisun/ From owner-proteins@net.bio.net Mon Apr 07 23:00:00 1997 Path: biosci!agate!howland.erols.net!rill.news.pipex.net!pipex!warwick!lyra.csx.cam.ac.uk!news From: Peter Wang Newsgroups: bionet.molbio.proteins,bionet.molbio.methds-reagnts Subject: H64A subtilisin (Genease I) source/experience? Date: Tue, 08 Apr 1997 14:39:55 +0000 Organization: University of Cambridge, England Lines: 13 Message-ID: <334A58BB.773F@mrc-lmb.cam.ac.uk> NNTP-Posting-Host: macx45.mrc-lmb.cam.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.01 (Macintosh; I; 68K) Xref: biosci bionet.molbio.proteins:10440 bionet.molbio.methds-reagnts:56503 I am looking for a source of GeneaseI, a variant of subtilisin engineered by Paul Carter and Jim Wells. Anyone know who sells it, and has anyone had any experiences they can share about using it? Please reply by email as well as posting. Thanks! --------------------------------------------------------- Peter Wang, M.D., Ph.D. MRC Centre for Protein Engineering, Hills Road, Cambridge, CB2 2QH, England Tel (01223) 402104 (international calls +44-1223-402104) Fax (01223) 402140 ( " " +44-1223-402140) --------------------------------------------------------- From owner-proteins@net.bio.net Mon Apr 07 23:00:00 1997 Path: biosci!daresbury!uninett.no!sn.no!www.nntp.primenet.com!nntp.primenet.com!news.maxwell.syr.edu!news.apfel.de!fu-berlin.de!wuff.mayn.de!wuff.franken.de!winx03!wpxx02!not-for-mail From: krasel@wpxx02.toxi.uni-wuerzburg.de (Cornelius Krasel) Newsgroups: bionet.molbio.proteins Subject: Re: Receptors and G-Proteins Date: Tue, 8 Apr 1997 12:38:51 +0200 Organization: University of Wuerzburg, Germany Lines: 25 Message-ID: References: <01bc406c$ecc04340$adb695c2@default> <5ibur1$11fg@news.doit.wisc.edu> NNTP-Posting-Host: wpxx02.toxi.uni-wuerzburg.de X-Newsreader: TIN [UNIX 1.3 950824BETA PL0] Dima Klenchin (klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu) wrote: > In article <01bc406c$ecc04340$adb695c2@default>, > "Christophe Guenin" wrote: > >I am looking for structures of the genes of the receptors coupled to G > >Proteins > > None has been crystallized => no structure. He was looking for gene structures. However, since there are several hundred G-protein coupled receptors, this will be a hard task (although it is likely that the genomic structure of only a fraction is known). Good places to start might be: - The G protein-coupled Receptor DataBase (GCRDb) http://receptor.mgh.harvard.edu/GCRDBHOME.html - GPCRDB http://www.sander.embl-heidelberg.de/7tm/ - http://mgddk1.niddk.nih.gov:8000/GPCR.html --Cornelius. -- /* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */ /* D-97078 Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de SP3 */ /* "Science is the game we play with God to find out what His rules are." */ From owner-proteins@net.bio.net Tue Apr 08 23:00:00 1997 Path: biosci!daresbury!uninett.no!nntp.uio.no!news.maxwell.syr.edu!news-peer.sprintlink.net!news-peer.sprintlink.net!news.sprintlink.net!sprint!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news.ums.edu!news.ab.umd.edu!not-for-mail From: Richard Anderson Newsgroups: bionet.molbio.proteins Subject: Termination Date: Wed, 09 Apr 1997 23:28:26 -0400 Organization: UMAB Lines: 6 Message-ID: <334C5E5A.6D72@umabnet.ab.umd.edu> NNTP-Posting-Host: umabnet.ab.umd.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.01 (Win95; I) I am trying to find out about transcription termination in eukaryotes. Does anybody know if there are any prokaryotic terminators that do NOT terminate transcription in eukaryotes. In particular, is there any evidence that a rho- homolog exists in eukaryotes and are there any reports of rho-dependent prokaryotic terminators functioning in eukaryotes? From owner-proteins@net.bio.net Tue Apr 08 23:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!news.maxwell.syr.edu!news-peer.sprintlink.net!news-peer.sprintlink.net!news.sprintlink.net!sprint!uunet!in1.uu.net!136.142.185.26!newsfeed.pitt.edu!mv29.pathology.pitt.edu!user From: pxpst2@vms.cis.pitt.edu (THE GREEK) Newsgroups: bionet.molbio.proteins Subject: Re: factor Xa Date: 9 Apr 1997 23:16:13 GMT Organization: USA Lines: 14 Distribution: world Message-ID: References: <9704092048.AA01569@mani.cbs.umn.edu> NNTP-Posting-Host: mv29.pathology.pitt.edu In article <9704092048.AA01569@mani.cbs.umn.edu>, nora@biosci.cbs.umn.edu wrote: -> We have had poor luck trying cleavage of fusion proteins with factor -> Xa. It cuts the target site very slowly and inefficiently, and during -> this time, other sites in the protein get cut as well. -> -> Nora Plesofsky-Vig -> nora@biosci.cbs.umn.edu I work extensivly with Xa but not in relation to fusion proteins. Here are some tips to enhancing the activity of Xa: 1. 5% PEG 8000 2. phospholipid micelles. I too find that Xa is an inefficent protease except in the body, in vivo it is very efficient. Peter Pediaditakis pxpst2@vms.cis.pitt.edu From owner-proteins@net.bio.net Tue Apr 08 23:00:00 1997 Path: biosci!bmg.bhs.uab.edu!BMOORE From: BMOORE@bmg.bhs.uab.edu ("Bryan Moore") Newsgroups: bionet.molbio.proteins Subject: I am looking for 3 proteins... Date: 9 Apr 1997 14:20:54 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 10 Sender: daemon@net.bio.net Distribution: world Message-ID: <109482354400@bmg.bhs.uab.edu> Reply-To: bmoore@bmg.bhs.uab.edu NNTP-Posting-Host: net.bio.net of molecular weights of less than 10 KDa, 20 KDa, and 30 KDa that bind a specific cell surface receptor. It would be nice if these proteins have been previously expressed from cDNA and possibly as a fusion protein. It is important for these proteins to contain an epitope that binds to a measurable degree to some cell surface receptor. Any suggestions are welcome. Bryan Moore Department of Biochemistry and Molecular Genetics University of Alabama at Birmingham Engler Lab (205) 975-8824 From owner-proteins@net.bio.net Tue Apr 08 23:00:00 1997 Path: biosci!biochem.umass.edu!bscott From: bscott@biochem.umass.edu (Brian Scott) Newsgroups: bionet.molbio.proteins Subject: Free molecular visualization workshops for profs Date: 9 Apr 1997 17:58:31 -0700 Organization: University of Massachusetts Lines: 53 Sender: daemon@net.bio.net Distribution: world Message-ID: <334C3AF7.6EA7@biochem.umass.edu> Reply-To: bscott@biochem.umass.edu NNTP-Posting-Host: net.bio.net Free Workshops on Molecular Visualization for Undergraduate Bioscience Teaching Amherst MA -- Summer, 1997-8 Understanding the three-dimensional structures of proteins, DNA, RNA, and their interactions is difficult from flat pictures, yet grasping structure is important to understanding function. Free software is now available which displays attention-grabbing 3D animated images of biological molecules in depth-cued spacefilling color. RasMol (http://www.umass.edu/microbio/rasmol), which works on Windows or Macs, encourages self-directed exploration. Chime (http://www.umass.edu/microbio/chime) allows annotated preset views of molecules to be delivered as web tutorials. Free, hands-on workshops will be held this summer at the University of Massachusetts in Amherst to prepare college faculty with no prior experience to use molecular visualization in their classes. Participants will travel to the Amherst campus on three separate days (two this summer, one the following summer), and are responsible for their own travel expenses (and therefore are expected primarily from the Northeastern USA). Overnight accomodations will be provided free to those needing them (based on distance traveled). Participants will be expected to incorporate molecular visualization into their teaching, and to mentor two faculty colleagues at their home institution. The workshops will be led by Eric Martz, a Professor in the Department of Microbiology. Martz has innovated molecular structure tutorials which are in use throughout the USA and in dozens of other countries. The web site he created (see above) was visited by 40,000 people in 1996. 1997 dates are June 17 and 30 (workshop A), or June 19 and July 2 (workshop B). Workshops A and B also meet for one day in late June 1998. To obtain more detailed information and a REGISTRATION FORM, visit http://www.umass.edu/microbio/rasmol/workshop.htm or FAX a request to 413-545-1578, or email a request to emartz@microbio.umass.edu. Supported by the National Science Foundation (Undergraduate Faculty Enhancement, Division of Undergraduate Education) and the University of Massachusetts. /* - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Eric Martz, Professor (Immunology), Dept Microbiology, Univ Massachusetts, Amherst MA US 01003-5720 413-545-2325 FAX:545-1578. RasMol Home Page, http://www.umass.edu/microbio/rasmol Other web projects listed at http://www.umass.edu/microbio/rasmol/em-web.htm - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -*/ From owner-proteins@net.bio.net Tue Apr 08 23:00:00 1997 Path: biosci!MOLE.BIO.CAM.AC.UK!tcg24 From: tcg24@MOLE.BIO.CAM.AC.UK (Teca Galvao) Newsgroups: bionet.molbio.proteins Subject: expressing small insoluble protein in e coli Date: 9 Apr 1997 12:27:13 -0700 Organization: SPAMMERS'R'US Lines: 9 Sender: daemon@net.bio.net Distribution: world Message-ID: <334BEDA4.4A2D@mole.bio.cam.ac.uk> Reply-To: tcg24@mole.bio.cam.ac.uk NNTP-Posting-Host: net.bio.net would you let me know: 1) if you have had problems expressing small insoluble proteins in bacteria, and tips on how you tackled that. 2) if you haven't had this problem but know of a good 'expression of recombinant proteins' handbook. thanks in advance teca galvao From owner-proteins@net.bio.net Tue Apr 08 23:00:00 1997 Path: biosci!bcm.tmc.edu!cs.utexas.edu!swrinde!howland.erols.net!vixen.cso.uiuc.edu!ais.net!uunet!in1.uu.net!192.89.123.24!nntp.inet.fi!news.funet.fi!news.utu.fi!newsmaster From: elebae@sara.cc.utu.fi Newsgroups: bionet.molbio.proteins Subject: digitonin Date: 9 Apr 1997 16:35:48 GMT Organization: Turun yliopisto Lines: 27 Message-ID: <5iggh4$ivp@news.utu.fi> NNTP-Posting-Host: kessu.ktb.utu.fi X-Newsreader: WinVN 0.92.6+ Hello! I am having tremendous problems with digitonin. I use it for solubilizing thylakoid membranes into grana stacks and stroma thylakoids. Everything worked perfectly as long as Merck manufactured the crystallized form of this detergent. Now that this is not available anymore, we can't find the way to solubilize the membranes. We have tried everything, all the digitonins in the market as well as their crystallized forms (heat 1 g in 20 ml of abs. ethanol, let it cold down at 4 C and crystallize and then filtrate the crystals), but NOTHING works. What is the explanation for the fact that the crystallized detergent might be more "efficient" than the uncrystallized on? One could understand this in the case that you get rid of putative impurities during the crystallization process, but this doesn't seem to be the case here, because everything gets dissolved very well in ethanol during heating...(?!) i am really lost in this matter, so I would really appreciate ANY comments or hints. Thank you very much! Cheers Elena Elena Baena Gonzalez Lab. Plant Physiology and Molecular Biology Department of Biology University of Turku FINLAND From owner-proteins@net.bio.net Tue Apr 08 23:00:00 1997 Path: biosci!uthct.edu!wu From: wu@uthct.edu Newsgroups: bionet.molbio.proteins Subject: ANNOUNCE: GeneFIND Server for protein family identification Date: 9 Apr 1997 08:49:29 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 40 Sender: daemon@net.bio.net Distribution: world Message-ID: <9704091550.AA26267@diana.uthct.edu> NNTP-Posting-Host: net.bio.net The Bioinformatics Research Group of the University of Texas Health Center at Tyler is pleased to announce the availability of its GeneFIND Server for protein family identification at: http://diana.uthct.edu/genefind.html GeneFIND (Gene Family Identification Network Design) is an integrated database search system that combines several search/alignment tools and ProClass database (http://diana.uthct.edu/proclass.html) to provide rapid and sensitive search results with enriched family information. Multi-level filters are used, starting with the fastest MOTIFIND neural networks, followed by BLAST search, SSEARCH (Smith-Waterman) sequence alignment, and motif pattern search. The server currently provides large-scale on-line identification of query sequences for 942 protein families. Search results are returned as HTML documents showing global and motif scores, alignment to best matched members of all possible ProSite protein groups and PIR superfamilies, motif pattern match, as well as links to corresponding ProClass family records. Please cite the following reference in publications that benefit from the GeneFIND family identification system: Wu, C. H., Zhao, S., Chen, H. L., Lo, C. J. and McLarty, J. (1996). Motif identification neural design for rapid and sensitive protein family search. CABIOS, 12(2), 109-118. Please visit our site and send suggestions and comments to me at wu@uthct.edu. Also contact me directly if you are interested in obtaining a copy of the GeneFIND software program. I am looking forward to hearing from you! Cathy Wu, Ph.D. Associate Professor of Biomathematics University of Texas Health Center at Tyler P. O. Box 2003, Tyler, TX 75710 E-Mail : wu@uthct.edu Fax : (903) 877-5914 Phone : (903) 877-7962 WWW URL : http://diana.uthct.edu/~wu From owner-proteins@net.bio.net Tue Apr 08 23:00:00 1997 Path: biosci!daresbury!uninett.no!sn.no!nntp.uio.no!news.maxwell.syr.edu!rill.news.pipex.net!pipex!warwick!news.nott.ac.uk!pmbfjd0.nottingham.ac.uk!user From: mbzrl@mbn1.biochem.nottingham.ac.uk (Rob) Newsgroups: bionet.molbio.proteins Subject: factor Xa Followup-To: bionet.molbio.proteins Date: 9 Apr 1997 14:50:53 GMT Organization: University of Nottingham Lines: 6 Message-ID: NNTP-Posting-Host: pmbfjd0.nottingham.ac.uk Help Has anyone got experience of using factor Xa for cleaving e.g. recombinant fusion proteins with Xa sites in. Is there any common problems - does it matter what residues are before or after the Xa site ???? Thanks in advance Rob From owner-proteins@net.bio.net Tue Apr 08 23:00:00 1997 Path: biosci!uthct.edu!wu From: wu@uthct.edu Newsgroups: bionet.molbio.proteins Subject: ANNOUNCE: ProClass Database Server for protein family information retrieval Date: 9 Apr 1997 08:21:31 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 39 Sender: daemon@net.bio.net Distribution: world Message-ID: <9704091522.AA26521@diana.uthct.edu> NNTP-Posting-Host: net.bio.net The Bioinformatics Research Group of the University of Texas Health Center at Tyler is pleased to announce the availability of its ProClass Database Server for protein family information retrieval at: http://diana.uthct.edu/proclass.html The ProClass database is a non-redundant protein database organized according to family relationships as defined collectively by ProSite patterns and PIR superfamilies. The ProClass database can facilitate protein family information retrieval, unveil domain and family relationships, and classify multi-domained proteins, by combining global and motif similarities into a single family organization scheme. The server provides on-line ID/keyword search for ProClass record retrieval, with hypertext links to SwissProt, ProSite and PIR databases. Free copies of the ProClass database can be obtained via anonymous FTP to: ftp://diana.uthct.edu/pub/ProClass/ Please cite the following reference in publications that benefit from the ProClass database: Wu, C. H., Zhao, S. and Chen, H. L. (1996). A protein class database organized with ProSite protein groups and PIR superfamilies. Journal of Computational Biology, 3(4), 547-561. Please visit our site and send suggestions and comments to me at wu@uthct.edu. I am looking forward to hearing from you! Cathy Wu, Ph.D. Associate Professor of Biomathematics University of Texas Health Center at Tyler P. O. Box 2003, Tyler, TX 75710 E-Mail : wu@uthct.edu Fax : (903) 877-5914 Phone : (903) 877-7962 WWW URL : http://diana.uthct.edu/~wu From owner-proteins@net.bio.net Tue Apr 08 23:00:00 1997 Path: biosci!MANI.CBS.UMN.EDU!npv From: npv@MANI.CBS.UMN.EDU (Nora Plesofsky-Vig) Newsgroups: bionet.molbio.proteins Subject: re:factor Xa Date: 9 Apr 1997 12:45:26 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 6 Sender: daemon@net.bio.net Distribution: world Message-ID: <9704092048.AA01569@mani.cbs.umn.edu> Reply-To: nora@biosci.cbs.umn.edu NNTP-Posting-Host: net.bio.net We have had poor luck trying cleavage of fusion proteins with factor Xa. It cuts the target site very slowly and inefficiently, and during this time, other sites in the protein get cut as well. Nora Plesofsky-Vig nora@biosci.cbs.umn.edu From owner-proteins@net.bio.net Wed Apr 09 23:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!europa.clark.net!worldnet.att.net!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!rill.news.pipex.net!pipex!warwick!news.nott.ac.uk!Fergus.Doherty From: Fergus.Doherty@nottingham.ac.uk (Fergus Doherty) Newsgroups: bionet.molbio.proteins Subject: Anti-HA (haemagglutinin epitope)? Date: Thu, 10 Apr 1997 11:54:06 +0000 Organization: Nottingham University Lines: 10 Message-ID: NNTP-Posting-Host: wmbpm8.nottingham.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=ISO-8859-1 Content-Transfer-Encoding: 8bit X-Newsreader: Yet Another NewsWatcher 2.4.0 Anyone know where to get an antihaemaglutinin antibody (MCYPYDVPDYASLG), called 12CA5? -- Fergus Doherty, Dept Biochemistry, Nottingham University, Fergus.Doherty@nottingham.ac.uk 0115 970 9366 (74-41366 internal) From owner-proteins@net.bio.net Wed Apr 09 23:00:00 1997 Path: biosci!daresbury!not-for-mail From: l.kempster@ic.ac.uk (L.Kempster) Newsgroups: bionet.molbio.proteins Subject: CFTR Date: 10 Apr 1997 12:57:58 +0100 Lines: 12 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <5iikk6$4rv@mserv1.dl.ac.uk> X-Sender: lkhl@tolstoy.nhli.ic.ac.uk Original-To: proteins@dl.ac.uk Does anyone know if any work has been carried out on quantifying the amount of CFTR in the apical membrane of airway epithelial cells (i.e. pg/fg or number of molecules per cell)? I know that there are typically 1-2 copies of CFTR mRNA per cell but it is possible to predict how many CFTR molecules would be translated from the message? Thanks in advance Lee Kempster, Imperial College. From owner-proteins@net.bio.net Wed Apr 09 23:00:00 1997 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.nacamar.de!dispatch.news.demon.net!demon!annaghman.demon.co.uk!tom From: tom@annaghman.demon.co.uk Newsgroups: bionet.molbio.proteins Subject: Help! Protein C isolation Date: Thu, 10 Apr 1997 23:27:06 +0000 Message-ID: NNTP-Posting-Host: annaghman.demon.co.uk X-NNTP-Posting-Host: annaghman.demon.co.uk X-Newsreader: Yet Another NewsWatcher 2.2.0b7 Lines: 10 I'm trying to isolate protein C from plasma without using immunoaffinity methods but am having real problems getting rid of other Ca-binding Vit. K-dependent proteins. Can anyone suggest a useful protocol? Also, the plasma contains EDTA (of unknown concentration) when I get it, does this stop me using barium citrate adsorption as a first step or is there a way around this? Thanks, Tom From owner-proteins@net.bio.net Wed Apr 09 23:00:00 1997 Path: biosci!bcm.tmc.edu!cs.utexas.edu!news-peer.sprintlink.net!news.sprintlink.net!sprint!news-peer.gsl.net!ix.netcom.com!news From: babco@ix.netcom.com(Thomas R. Anderson) Newsgroups: bionet.molbio.proteins Subject: Re: Anti-HA (haemagglutinin epitope)? Date: 11 Apr 1997 03:59:06 GMT Organization: Netcom Lines: 26 Message-ID: <5ikcua$obu@dfw-ixnews12.ix.netcom.com> References: NNTP-Posting-Host: wck-ca16-03.ix.netcom.com X-NETCOM-Date: Thu Apr 10 10:59:06 PM CDT 1997 In Fergus.Doherty@nottingham.ac.uk (Fergus Doherty) writes: > >Anyone know where to get an antihaemaglutinin antibody (MCYPYDVPDYASLG), >called 12CA5? Hello Fergus (and others) 12CA5, the first monoclonal antibody directed against the HA epitope, is available from Boehringer Mannheim. However, a second generation monoclonal antibody directed agaisnt the same epitope is available from Berkeley Antibody Company (BAbCO). If you would like more information about the antibody, or any of the other epitope tag antibodies, or about our distributors in other parts of the world, please let me know. Thanks in advance. Tom Anderson Berkeley Antibody Company (BAbCO) tel: 1-800-922-2226 (in the USA) tel: 1-510-412-8930 (anywhere) fax: 1-510-412-8940 email: tanders@babco.com on the internet at www.babco.com From owner-proteins@net.bio.net Wed Apr 09 23:00:00 1997 Path: biosci!ACPUB.DUKE.EDU!TSL From: TSL@ACPUB.DUKE.EDU ("T. S. Lai") Newsgroups: bionet.molbio.proteins Subject: kinetic question Date: 10 Apr 1997 19:38:02 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 10 Sender: daemon@net.bio.net Distribution: world Message-ID: <334D5CF3.4F15@acpub.duke.edu> NNTP-Posting-Host: net.bio.net Hi there, I wonder if anyone has experience studying the kinetics of metal-nucleotide complex. I am working with an enzyme that can use GTP as well as ATP as substrate for hydrolysis and I am trying to find out what is the substrate of this enzyme. Is it the magnesium-nucleotide or free nucleotdes ? Thank you for your help. John From owner-proteins@net.bio.net Wed Apr 09 23:00:00 1997 Path: biosci!WH.BAYER.COM!joanna From: joanna@WH.BAYER.COM (Joanna de Bear) Newsgroups: bionet.molbio.proteins Subject: Re: Anti-HA (haemagglutinin epitope)? Date: 10 Apr 1997 10:58:51 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 6 Sender: daemon@net.bio.net Distribution: world Message-ID: <199704101756.AA17473@miwr03il.wh.bayer.com> NNTP-Posting-Host: net.bio.net Anti-HA (12CA5) can be purchased from Boehringer Mannheim cat# 1 583 816 (200ug) or 1 666 606 (5mg). Happy blotting/precipitating..... From owner-proteins@net.bio.net Thu Apr 10 23:00:00 1997 Path: biosci!rutgers.rutgers.edu!gatech!arclight.uoregon.edu!news.maxwell.syr.edu!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.nacamar.de!news-kar1.dfn.de!news-fra1.dfn.de!news-ge.switch.ch!01-newsfeed.univie.ac.at!03-newsfeed.univie.ac.at!news.univie.ac.at!news-admin@univie.ac.at From: a8803349@unet.univie.ac.at (Martin Offterdinger) Newsgroups: bionet.molbio.proteins Subject: beta casein antibody Date: Fri, 11 Apr 1997 13:18:12 GMT Organization: AKH Lines: 6 Message-ID: <334e39ab.13769642@news.univie.ac.at> NNTP-Posting-Host: grunt.imed1.akh-wien.ac.at X-Newsreader: Forte Free Agent 1.1/16.230 Hi I am looking for a commercially available antibody to human beta casein that can be used for immunostaing. Any hints will be appreciated. Thank you very much! Martin From owner-proteins@net.bio.net Thu Apr 10 23:00:00 1997 Path: biosci!agate!howland.erols.net!vixen.cso.uiuc.edu!uwm.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!daemon From: klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins Subject: Re: kinetic question Date: Fri, 11 Apr 1997 15:10:57 GMT Organization: UW-Madison Lines: 17 Distribution: world Message-ID: <5ilkdk$4kao@news.doit.wisc.edu> References: <334D5CF3.4F15@acpub.duke.edu> NNTP-Posting-Host: f182-120.net.wisc.edu X-Newsreader: News Xpress 2.01 X-No-Archive: Yes In article <334D5CF3.4F15@acpub.duke.edu>, TSL@ACPUB.DUKE.EDU ("T. S. Lai") wrote: #Hi there, # #I wonder if anyone has experience studying the kinetics of #metal-nucleotide complex. I don't but the actual problem is easy to solve: # I am working with an enzyme that can use GTP #as well as ATP as substrate for hydrolysis and I am trying to find out #what is the substrate of this enzyme. Is it the magnesium-nucleotide or #free nucleotdes ? Experimentally compare hydrolysis rates between Mg2+ and EDTA added. Most of the time metal cofactor is required. - Dima From owner-proteins@net.bio.net Thu Apr 10 23:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!europa.clark.net!cpk-news-hub1.bbnplanet.com!cpk-news-feed4.bbnplanet.com!news.bbnplanet.com!europa.chnt.gtegsc.com!boingo.amil.jhu.edu!blaze.cs.jhu.edu!news.jhu.edu!welchlink!stebby From: stebby@welchlink.welch.jhu.edu (Stephen C. Dahl) Newsgroups: bionet.molbio.proteins Subject: Re: Anti-HA (haemagglutinin epitope)? Date: 10 Apr 1997 17:41:24 GMT Organization: HCF - Johns Hopkins University, Baltimore, Maryland, USA Lines: 20 Message-ID: <5ij8o4$nl2@news.jhu.edu> References: NNTP-Posting-Host: 128.220.59.78 X-Newsreader: TIN [version 1.2 PL2] Fergus Doherty (Fergus.Doherty@nottingham.ac.uk) wrote: : Anyone know where to get an antihaemaglutinin antibody (MCYPYDVPDYASLG), : called 12CA5? Boehringer Mannheim--their catalog # 1-583-816 for 200 ug. Also Zymed which is handled by Cambridge BioScience in the UK (telephone # +44 1 223 316855 or on the web go to http//www.zymed.com Their product is catalog # 71-5500 and is more expensive than Boehringer here in the USA. I'm sure there are others, but these I'm certain about. I can't tell you anything about performance since I've never used either source. Regards, Steve Dahl From owner-proteins@net.bio.net Thu Apr 10 23:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!europa.clark.net!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!rill.news.pipex.net!pipex!warwick.netnews.ja.net!warwick!leicester!usenet From: "Dr E. Buxbaum" Newsgroups: bionet.molbio.proteins Subject: Re: kinetic question Date: 11 Apr 1997 10:41:58 GMT Organization: University of Leicester (PCFS User) Lines: 17 Message-ID: <5il4hm$1mb@falcon.le.ac.uk> References: <334D5CF3.4F15@acpub.duke.edu> NNTP-Posting-Host: pc47.msb.le.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) TSL@ACPUB.DUKE.EDU ("T. S. Lai") wrote: I am working with an enzyme that can use GTP >as well as ATP as substrate for hydrolysis and I am trying to find out >what is the substrate of this enzyme. Is it the magnesium-nucleotide or >free nucleotdes ? If it is free nucleotide, then incubation in the absence of metall ions should result in hydrolysis. If, on the other hand, MgNTP complexes are used as substrate, then exchange stable analogues of this complex, like Cr(H2O)4NTP or Co(NH3)4NTP should work either as substrates or as competitive inhibitors of hydrolysis. These complexes were originally described by W.W. Cleland. Of course changing the Mg concentration should affect the reaction speed in this case. The same might happen if you increase [NTP] to >> [Mg], as the free NTP might act as a competitive inhibitor toward MgNTP From owner-proteins@net.bio.net Thu Apr 10 23:00:00 1997 Path: biosci!daresbury!uninett.no!due.unit.no!nntp.uio.no!news.maxwell.syr.edu!cpk-news-hub1.bbnplanet.com!su-news-hub1.bbnplanet.com!news.bbnplanet.com!xfer.kren.nm.kr!newsfeed.dacom.co.kr!usenet.dacom.co.kr!not-for-mail From: "Chang Zoon, Chun" Newsgroups: bionet.molbio.proteins Subject: Cellulase gene Date: Sat, 12 Apr 1997 14:53:26 +0900 Organization: Pukyong National University Lines: 6 Message-ID: <334F2353.1C105473@bora.dacom.co.kr> Reply-To: swchun@bora.dacom.co.kr NNTP-Posting-Host: pppps23.dacom.co.kr Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 X-Mailer: Mozilla 4.0b3 [en] (Win95; I) X-Priority: 3 (Normal) I am a graduate student at PKNU in korea. I am trying to clone a kind of cellulase gene from marine. I know the cellulase gene dosen't have conserved sequence, but I'd like to try hybridization. If anyone help me, let me know - if you could send to me any clone - let me know. From owner-proteins@net.bio.net Sat Apr 12 23:00:00 1997 Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!news.ecn.uoknor.edu!feed1.news.erols.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!rill.news.pipex.net!pipex!tank.news.pipex.net!pipex!idiscover.co.uk!orac.idiscover.net!194.216.165.19 From: "SpaceboY" Newsgroups: bionet.molbio.proteins Subject: Measure activity of pepsin Date: 13 Apr 97 20:00:43 GMT Organization: Internet Discovery Customer News Service Lines: 3 Message-ID: <01bc4844$a30ac7e0$13a5d8c2@nexus.idiscover.co.uk> NNTP-Posting-Host: orac-gw.idiscover.net X-Newsreader: Microsoft Internet News 4.70.1155 How could one measure the activity of pepsin experimentally? ... i.e. to measure the effect of ethanol on the activity of pepsin. From owner-proteins@net.bio.net Sat Apr 12 23:00:00 1997 Path: biosci!internet!biosci!not-for-mail From: biohelp (BIOSCI Administrator) Newsgroups: bionet.molbio.proteins Subject: BIOSCI/bionet miniFAQ & Fundraiser Date: 13 Apr 1997 02:00:13 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 239 Sender: daemon@net.bio.net Distribution: world Message-ID: <199704130900.CAA06787@net.bio.net> NNTP-Posting-Host: net.bio.net (LAST REVISION: 30-JUL-95) This BIOSCI "miniFAQ" is designed to answer the questions that come up the *most frequently*. The main BIOSCI FAQ (Frequently Asked Questions) is accessible on the World Wide Web at URL http://www.bio.net/. If you can not find an answer to your question in this or other documentation, the BIOSCI technical support staff answers e-mail queries sent to biosci-help@net.bio.net We can only answer questions about the use of the newsgroups and mailing lists. We unfortunately do not have the staff to do Internet information searches or answer scientific questions. Please post those to the appropriate BIOSCI/bionet newsgroups. Contents: -------- 0) BIOSCI NEEDS YOUR SUPPORT!! 1) Using the WWW to access the BIOSCI/bionet newsgroups. 2) What to do about "spams," i.e., junk mail, ads, etc. 3) Examples of subscribing and unsubscribing to the mailing lists. 4) The BIOSCI user address and research interest directory. 0) BIOSCI NEEDS YOUR SUPPORT!! ------------------------------ BIOSCI's government funding has been expended, and we are now operating solely from advertising revenue that we have raised from our Web site at http://www.bio.net/. We need just a few minutes of your time to help us serve you. You can do two important things which will take very little time for you individually and will immensely help us continue to help you. First, please use our WWW system at http://www.bio.net/ to access the archives. You can post or reply to messages via your Web browser as described in item #1 below. Your usage helps attract sponsors. If you contact any of our sponsors, please be sure to thank them for supporting BIOSCI. It is critical for them to get this feedback if they are to continue their sponsorship for the long term. Second, if you work for a company or organization that provides products or services of interest to the biology community, please pass this message on to your marketing or marketing communications department or other appropriate group. Please ask them to help support BIOSCI by sponsoring our Web site and explain the uses and benefits of the system to the biology community. If they are interested, they can then contact us for further information at our tech support address, biosci-help@net.bio.net. 1) Using the WWW to access the BIOSCI/bionet newsgroups. -------------------------------------------------------- As of 10 December 1995, all BIOSCI/bionet full newsgroups are accessible through the World Wide Web (WWW) at URL http://www.bio.net. One can read and reply publicly or privately to both recent postings and archived messages through one's Web browser if it is configured properly to send e-mail. Each newsgroup is equipped with its own WAIS index. The main BIOSCI home page also has access to the BIO-JOURNALS Table of Contents database WAIS index and the BIOSCI user address database described in another item further below. 2) What to do about "spams," i.e., junk mail, ads, etc. ------------------------------------------------------- BIOSCI is a set of parallel USENET newsgroups (the "bionet" groups), mailing lists, and a hypermail archive at URL http://www.bio.net/. The same postings are distributed on all media (except for a small number of mailing-list-only groups at net.bio.net). Unfortunately it is becoming a despicable practice on the Internet (by a few people out to make a fast buck) to do automated mass postings to thousands of newsgroups and mailing lists. These attempts to grab free advertising are refered to as "spams" in the usual, somewhat boneheaded, net terminology. USENET is more susceptible to this practice, and many spams originate on the USENET groups and then are passed on to the mailing lists. However, spammers also get lists of mailing addresses and hit these too, so neither medium is immune. What should you do personally if you get junk mail? --------------------------------------------------- Just delete it and move on without reading it further. Filing a protest is becoming increasingly useless because spammers are often disguising the addresses where the messages are sent from. Unless you really understand Internet mail systems, your attempt at protest by sending replies to the message will often end up being sent to the address of an innocent person that the spammer is victimizing. What can BIOSCI/bionet do to protect its newsgroups? ---------------------------------------------------- The only solution currently available is to moderate the newsgroup. If this newsgroup is already moderated, then you are in good shape. Moderation protects the USENET distribution from about 95% of the spams that are being sent to date and protects the mailing lists completely. Moderation means, however, that someone has to take the time to review each message before it goes out. We have set up software here that simply allows the moderator to forward to an address at net.bio.net messages that (s)he wishes to have distributed. This takes no more time than that needed to read the message and pass it on, say about 1 min. per message. Most newsgroups currently have a discussion leader who is responsible for their newsgroup. The discussions leaders and their e-mail addresses are listed in the BIOSCI Information Sheet which is available on the Web at http://www.bio.net/. If a newsgroup is being hit with too many junk postings, please contact the discussion leader for that group and see if there is interest in moderating the group. Please do not assume that by simply posting a complaint to the newsgroup itself, anyone on the BIOSCI staff will act on your complaint. With close to 100 newsgroups to run, the BIOSCI staff has to rely on the discussion leaders of each newsgroup to report problems directly to us at biosci-help@net.bio.net. We will moderate any of our newsgroups if the discussion leader tells us that the readership of the group wishes to do so and if a moderator is willing to do the work. For most BIOSCI/bionet groups, this entails only a few minutes of work each day. Moderating a newsgroup will resolve probably 95% of the junk postings on the USENET distribution. Unfortunately there are easy ways for determined spammers to override the moderation mechanism on USENET, but we can protect our e-mail subscribers from unwanted postings if the newsgroup is moderated. You can also access our newsgroups over the WWW at URL http://www.bio.net. While this Web interface will not stop spammers from trying to post to the groups, this will give you yet another way, besides using USENET news, to keep the junk out of your personal mail files. For those of you with local USENET news systems, the Web interface will also give you faster access to new newsgroups and recent postings. 3) Examples of subscribing and unsubscribing to the mailing lists. ------------------------------------------------------------------ PLEASE NOTE: The BIOSCI management does NOT act on subscription/unsubscription requests that are posted improperly to the newsgroups and mailing lists. People who do this only bother everyone on the lists to no avail. Please be sure to follow the proper procedures below. Gory details are in the BIOSCI Information sheets on the Web at http://www.bio.net. Below we give an example utilizing the METHODS-AND-REAGENTS list at both of our two BIOSCI sites: Users in the Americas and Pacific Rim countries who use the BIOSCI ------------------------------------------------------------------ node at computer net.bio.net: ---------------------------- A) Determine the "listname" which is the <=8 character mail address ^^^^^^^^^^^^^ for the group. These can be found in the BIOSCI Info. Sheet. For the METHODS-AND-REAGENTS group the mailing address is methods@net.bio.net. The listname is the portion of the address to the left of the @ sign, i.e., "methods". The listname is used with the "subscribe" and "unsubscribe" commands illustrated below. B) Mail all commands in the body of a mail message addressed to biosci-server@net.bio.net. Do NOT send commands to the newsgroup posting addresses! Leave the Subject: line blank, any text on it will be ignored. C) In the body of your message put one or more of the following commands with an "end" command on the last line, e.g., subscribe methods unsubscribe methods end Do NOT put your e-mail address or other text on these lines. The server only allows you to cancel your subscription if the address on your mail header matches the address on our mailing list. Please ask for help at biosci-help@net.bio.net if your address has changed, e.g., if you know you are on the list but the server tells you that you are not a member. Users in Europe, Africa, and Central Asia who use the BIOSCI node at -------------------------------------------------------------------- computer daresbury.ac.uk (also known as dl.ac.uk): ------------------------------------------------- To subscribe and unsubscribe to/from the BIOSCI lists, you need to specify the full USENET newsgroup name with "bionet-news." prepended. The USENET newsgroup names are listed in the BIOSCI Information sheet on the Web at http://www.bio.net/. For the METHODS-AND-REAGENTS list the USENET newsgroup name is bionet.molbio.methds-reagnts, thus the appropriate commands are sub bionet-news.bionet.molbio.methds-reagnts unsub bionet-news.bionet.molbio.methds-reagnts These commands are included in a message addressed to mxt@dl.ac.uk, NOT to the newsgroup mailing addresses. As usual, include the text in the body of the message as text on the Subject: line is ignored. To unsubscribe from all the lists at the UK node, use unsub bionet-news Please note that if the address in the list is different than the one in your mail message header, you will not be able to unsubscribe by this method. If you have problems, please mail biosci@daresbury.ac.uk. 4) The BIOSCI user address and research interest directory. ----------------------------------------------------------- Please take this opportunity to add your name, address, and research interest information to the BIOSCI User Address Database if you have not already done so. You can fill out the address form directly through our Web page at URL http://www.bio.net/adrform.html. The address database is reindexed nightly for WWW access (the URL is http://www.bio.net/). If you are not directly on the Internet but can reach it by e-mail, please use our waismail server to access the user directory. waismail use is described above. You can also request a user address form by e-mail from biosci-help@net.bio.net. Please check your database entry from time-to-time to see if your address information is still up-to-date. Because of our limited personnel resources, we ask that you resubmit a *complete* form to revise your entry; we only replace complete entries and do not have resources to edit old forms. Sincerely, Dave Kristofferson BIOSCI/bionet Manager biosci-help@net.bio.net From owner-proteins@net.bio.net Sat Apr 12 23:00:00 1997 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!ais.net!uunet!in1.uu.net!208.206.176.15!dimensional.com!newsfeed2!news.ycc.yale.edu!news From: Elias Fernandez Newsgroups: bionet.molbio.proteins Subject: Coating culture plates with Heparin Date: Wed, 09 Apr 1997 16:13:54 -0400 Organization: Yale University Lines: 6 Message-ID: <334BF882.33E3@pharm.med.yale.edu> Reply-To: elias@pharm.med.yale.edu NNTP-Posting-Host: jervaf.med.yale.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (Win95; I) Has anyone devised a way to immobilize heparin on microtitre/tissue culture plates? Elias From owner-proteins@net.bio.net Sun Apr 13 23:00:00 1997 Path: biosci!news.Stanford.EDU!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.nacamar.de!news-kar1.dfn.de!news-fra1.dfn.de!news-ge.switch.ch!news-zh.switch.ch!rzunews.unizh.ch!newsadm From: Rolf Kocherhans Newsgroups: bionet.molbio.proteins Subject: Free practical programs for molecular biologists !!! Date: Mon, 14 Apr 1997 09:39:51 +0100 Organization: University of Zurich Lines: 49 Message-ID: <3351ED56.2EFB@vetvir.unizh.ch> Reply-To: rolfk@vetvir.unizh.ch NNTP-Posting-Host: 130.60.22.38 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Macintosh; I; PPC) I wrote a few practical and simple to use programs which facilitate your daily work in the lab such as predicting the size of DNA fragments after digestion (graphical) with restriction enzymes. I would like to make these programs accessible to a broad user group by the Internet. All programs have been tested on MacOS and Windows95. My programs are accessible over the WWW and made functional using Netscape 2.x or Internet Explorer 2.x or higher in association with a free plugin which you have to download and install first. This is what you do: - Download the Roadster plugin http://www.unizh.ch/vetvir/plugin.html) install it on your computer. - Then connect to: http://www.unizh.ch/vetvir/programs.html That's it !! These are my programs which make your live as a molecular biologist easier ! Find here a few more examples or my programs: a. Digest Preview: Enter the size(s) of your DNA fragment(s) and see their migration pattern in a virtual gel in comparison to a 1 kb ladder. b. Adapter Design: Helps to create in frame adapters in order to link incompatible DNA ends together. c. Dilution Calculator: Does all the calculations when you have to make up solutions There are many other programs such as Oligo Tm, Compatible ends etc. Please have a look, comments are welcome! Have fun Rolf Kocherhans mailto:rolfk@vetvir.unizh.ch From owner-proteins@net.bio.net Sun Apr 13 23:00:00 1997 Path: biosci!bcm.tmc.edu!cs.utexas.edu!uwm.edu!newsfeeds.sol.net!hammer.uoregon.edu!news.uoregon.edu!marlin.ucsf.edu!overload.lbl.gov!news From: trey simmons Newsgroups: bionet.molbio.proteins,bionet.molecules.peptides,sci.chem.analytical,bionet.biophysics,bionet.molbio.methds-reagnts Subject: Re: Ion Exchange Chro/HPLC with 6M Guanidine or Some Detergent? Date: Mon, 14 Apr 1997 14:00:30 -0700 Organization: lawrence berkeley Lines: 55 Message-ID: <33529AEE.318A@csg.lbl.gov> References: <5frrbl$e16$1@snunews.snu.ac.kr> Reply-To: trey@csg.lbl.gov NNTP-Posting-Host: xlab1.als.lbl.gov Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.0b2 (WinNT; I) To: newera@plaza.snu.ac.kr X-Priority: 3 (Normal) Xref: biosci bionet.molbio.proteins:10475 bionet.molecules.peptides:745 sci.chem.analytical:8345 bionet.biophysics:2816 bionet.molbio.methds-reagnts:56688 newera@plaza.snu.ac.kr wrote: Do not 6M ganidine HCl, 8M urea or some zwitter-ionic detergent make any difference to cation exchange chromatography or affinity chromatography? My protein tends to be aggregated during some purification steps such as dialysis or concentration with a Centriprep. In the midst of misfortune, my protein has no cysteines and seems to be easily renatured; so I plan to add 6M guanidine HCl/8M urea and 0.5~2% or more zwitter-ionic detergents such as CHAPS, sarkosyl to my current buffer system(20mM Na Phosphate, pH 6.8). I am deeply concerned that they severely interfere with my protein's binding ability to BioRex70 cation exchange resin, BlueSepharose affinity chromatography resin, to which my protein seems to quite tightly bind since it begins to elute at around 0.5M NaCl, and C18 reverse phase HPLC. Thank you very much. Key word: 6M Guanidine HCl/8M Urea CHAPS/Sarkosyl Cation Exchange Chromatography Affinity Chromatography Reverse Phase HPLC -- email : newera@plaza.snu.ac.kr address : Lee, Ji Hyun Laboratory of Physical Pharmacy(Prof. Lee, Bong Jin) Seoul National University College of Pharmacy Shinlim-Dong, Kwanak-Gu Seoul 151-742, Korea. Just saw your post, so you may have an answer already, but just in case: Ion exchange chromatography works very well in 8M urea, as long as the urea is first deionized. We pass a 10M urea solution over a standard mixed bed ion-exchange resin before making up buffers for chromatography, and check for complete deionization using a conductivity meter. Although it is possible to store deionized urea solutions at 4 degrees for short periods, I don't recommend it, because urea breaks down, especially at alkaline pH, into reactive species which modify amine groups on proteins, and raise the ionic strength of the solution besides. It's a good idea to make up urea buffers fresh every day if you're going to use them for ion exchange chromatography. Good luck. Trey Simmons Macromolecular Crystallography Facility Lawrence Berkeley Laboratory trey@csg.lbl.gov From owner-proteins@net.bio.net Sun Apr 13 23:00:00 1997 Path: biosci!agate!nature.berkeley.edu!lhom From: lhom@nature.berkeley.edu (Louis Hom) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Denaturing proteins for IP Date: 15 Apr 1997 05:42:05 GMT Organization: University of California, Berkeley Lines: 9 Message-ID: <5iv4fe$3p$1@agate.berkeley.edu> NNTP-Posting-Host: nature.berkeley.edu Xref: biosci bionet.molbio.methds-reagnts:56704 bionet.molbio.proteins:10478 I need to IP my S35-labeled protein out of my cell lysates (there's an interfering protein of similar molecular weight), but unfortunately my antibody only binds the denatured form. Any suggestions on how I might go about this? -- _______________________________________________________________________________ Lou Hom >K '93 lhom@nature.berkeley.edu http://www.ocf.berkeley.edu/~lhom From owner-proteins@net.bio.net Sun Apr 13 23:00:00 1997 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news.apfel.de!univ-lyon1.fr!jussieu.fr!saphir.jouy.inra.fr!NewsWatcher!user From: granier@versailles.inra.fr (Fabienne Granier) Newsgroups: bionet.molbio.proteins Subject: far western Date: 14 Apr 1997 15:01:19 GMT Organization: INRA Lines: 4 Message-ID: NNTP-Posting-Host: saphir.jouy.inra.fr do you have any protocol allowing the detection of interactions between proteins by far western? thanks for answer Fabienne Granier From owner-proteins@net.bio.net Sun Apr 13 23:00:00 1997 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!europa.clark.net!news.msfc.nasa.gov!bcm.tmc.edu!mtuvin From: mtuvin@bcm.tmc.edu (Michael J. Tuvim) Newsgroups: bionet.immunology,bionet.molbio.methds-reagnts,bionet.molbio.proteins,sci.med.immunology Subject: Mast Cells Granules Antibody!! Date: 14 Apr 1997 19:09:21 GMT Organization: Baylor College of Medicine, Houston, Tx Lines: 14 Sender: Michael J. Tuvim Distribution: world Message-ID: <5itvd1$k2s@gazette.bcm.tmc.edu> NNTP-Posting-Host: condor.mbcr.bcm.tmc.edu Summary: Need specific monoclonal AB to Mast Cells Granules Keywords: Mast Cells, Granules, Exocytosis, Antibody Xref: biosci bionet.immunology:11473 bionet.molbio.methds-reagnts:56686 bionet.molbio.proteins:10474 sci.med.immunology:9441 Hi! Does anyone know of a Mast Cells Granules specific Antibody? To some granule marker like protease. Should be monoclonal since I need to colocalize a protein to the granules and my AB is polyclonal. Preferably for Rat Peritoneal Mast Cells, but mouse will also do. Please dubb your responce also to my address: mtuvin@bcm.tmc.edu I appreciate your help. Michael Tuvim From owner-proteins@net.bio.net Sun Apr 13 23:00:00 1997 Path: biosci!ihnp4.ucsd.edu!gondor!newshub.sdsu.edu!news.sgi.com!su-news-hub1.bbnplanet.com!cam-news-hub1.bbnplanet.com!news.bbnplanet.com!uunet!in3.uu.net!194.158.97.27!grolier!pressimage!world-net!not-for-mail From: David Rouquie Newsgroups: bionet.molbio.proteins Subject: electrotransfert of LMW proteins Date: 14 Apr 1997 18:33:50 GMT Organization: sunflower.ensam.inra.fr via ( ( EchO! ) ) Lines: 20 Message-ID: <5ittaf$6le$1@storm.worldnet.net> NNTP-Posting-Host: 195.3.8.50 Hi all Does anyone have experience with the electroblotting of low molecular weight proteins (< 20 kD)? After immunodetection with transfert standard procedures (semidry or wet tranfert in Tris/Glycine/MetOH buffer onto nitrocellulose or PVDF membranes) bands of interest are very weak. I wonder if the tranfert works well and if someone knows an optimized procedure for this kind of proteins. Thank you for your help. --- David Rouquie Lab de Bioch et Physio Molec des Plantes INRA MONTPELLIER FRANCE rouquie@ensam.inra.fr Cet article a ete poste via ( (EchO!) ) - http://echo.fr/News From owner-proteins@net.bio.net Sun Apr 13 23:00:00 1997 Path: biosci!CC.USU.EDU!arsphys From: arsphys@CC.USU.EDU (Phil Harrison) Newsgroups: bionet.molbio.proteins Subject: Enzyme kinetics software Date: 14 Apr 1997 13:13:32 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 16 Sender: daemon@net.bio.net Distribution: world Message-ID: <1.5.4.32.19970414205953.006ae7d0@cc.usu.edu> NNTP-Posting-Host: net.bio.net I am looking for a program that will plot enzyme kinetics. One of the enzymes I need to plot data for (a sucrose-sucrose fructosyl transferase) does not become saturated with substrate (sucrose), even at 1M sucrose. So I need to plot some non-standard kinetic data. I also want to look at possible temperature effects (Arrhenius plots?). If anyone can recommend a program for DOS or Windows 95 that fits this description, I would appreciate hearing from you. Phil Harrison USDA-Agricultural Research Service Utah State University, UMC 6300 Logan, UT 84322-6300 e-mail: arsphys@cc.usu.edu From owner-proteins@net.bio.net Mon Apr 14 23:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!europa.clark.net!worldnet.att.net!howland.erols.net!panix!news.panix.com!not-for-mail From: iayork@panix.com (Ian A. York) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: Denaturing proteins for IP Date: 15 Apr 1997 10:46:02 -0400 Organization: Panix Lines: 20 Message-ID: <5j04ba$u9@panix.com> References: <5iv4fe$3p$1@agate.berkeley.edu> NNTP-Posting-Host: panix.com X-Newsposter: trn 4.0-test55 (26 Feb 97) Xref: biosci bionet.molbio.methds-reagnts:56719 bionet.molbio.proteins:10486 In article <5iv4fe$3p$1@agate.berkeley.edu>, Louis Hom wrote: >I need to IP my S35-labeled protein out of my cell lysates (there's an >interfering protein of similar molecular weight), but unfortunately my >antibody only binds the denatured form. Any suggestions on how I might go >about this? The most common approach is to denature with SDS and then dilute the SDS to a concentration that won't affect the antibody. One other appraoch that worked nicely for me is to simply heat your lysate--I usually heated to 75oC for 30 minutes. Some proteins will precipitate with this treatment, so it may not work for your protein, but it's simple and fast. Don't forget to spin out the sample after the heating to get rid of those proteins that have precipitated. Ian -- Ian York (iayork@panix.com) "-but as he was a York, I am rather inclined to suppose him a very respectable Man." -Jane Austen, The History of England From owner-proteins@net.bio.net Mon Apr 14 23:00:00 1997 Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!news.ecn.uoknor.edu!feed1.news.erols.com!worldnet.att.net!newsadm From: Mark Dalton Newsgroups: bionet.molbio.proteins Subject: Re: far western Date: Tue, 15 Apr 1997 20:25:08 -0500 Organization: AT&T Lines: 15 Message-ID: <33542A74.26A9@worldnet.att.net> References: Reply-To: mdalton@worldnet.att.net NNTP-Posting-Host: 207.146.217.189 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.02E (Macintosh; U; PPC) Fabienne Granier wrote: > > do you have any protocol allowing the detection of interactions between > proteins by far western? > thanks for answer > Fabienne Granier The few times our lab has done this we do it just like a western blot except first probe with a fusion protein solution at least 5ug/ml for several hours. followed by the normal western blot protocol. anti-fusion protein 2hr. wash 3x with TBST. secondary anti primary HRP. wash 3x with TBST. Then develop by ECL. To reduce background each wash should be at minimum 15 minutes. Good Luck Mark From owner-proteins@net.bio.net Mon Apr 14 23:00:00 1997 Path: biosci!daresbury!uninett.no!news-feed.inet.tele.dk!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news-peer.sprintlink.net!news.sprintlink.net!sprint!news-peer.gsl.net!news-hk.gsl.net!news.gsl.net!newsgate.cuhk.edu.hk!newsfeeder.ust.hk!news.ust.hk!shellily From: shellily@uxmail.ust.hk (CHAN LAI YEE) Newsgroups: bionet.molbio.proteins Subject: Help!! Q. about Protein Refolding !!! Date: 15 Apr 1997 12:56:14 GMT Organization: Hong Kong University of Science and Technology Lines: 26 Message-ID: <5ivtte$362@ustsu10.ust.hk> NNTP-Posting-Host: uststf1.ust.hk X-Newsreader: TIN [version 1.2 PL2] Dear all, I have expressed a protein with 13kDa and it was found to be trapped in inclusion bodies. So I have denatured the inclusion bodies by using 6M Urea and then purified by Nickle Column. Could anybody enlighten me the way to remove the Urea from the protein?? Cos' I have tried to achieve this by dialysis against PBS pH7.3 but unfortunately ppt was formed. By the way, what are the factors that will affect the solubility of a protein??? How to decide a suitable dialysis buffer( what kind, pH and concentration )??? Thank you for your kindest help! Regards, Shelley Chan shellily@uxmail.ust.hk From owner-proteins@net.bio.net Mon Apr 14 23:00:00 1997 Path: biosci!daresbury!not-for-mail From: Cormac Shaw Newsgroups: bionet.molbio.proteins Subject: Re: Enzyme kinetics software Date: 15 Apr 1997 13:03:41 +0100 Organization: University Collge Dublin Lines: 34 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <5ivqqt$5o8@mserv1.dl.ac.uk> Reply-To: cormac.shaw@ucd.ie Original-To: arsphys@CC.USU.EDU (Phil Harrison), PROTEIN