From owner-proteins@net.bio.net Sun Jan 01 22:00:00 1995 Path: biosci!FOX.NSTN.NS.CA!ewright From: ewright@FOX.NSTN.NS.CA ("Edwin Wright") Newsgroups: bionet.molbio.proteins Subject: Protein Classification Date: 2 Jan 1995 11:52:32 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 16 Sender: daemon@net.bio.net Distribution: world Message-ID: <57185.ewright@fox.nstn.ns.ca> NNTP-Posting-Host: net.bio.net Does anyone know of a comprehensive reference which lists all the major classifications of proteins with respect to superfamily, family, subfamily, etc.? Also, are any databases structured according to such classifications? Regards, ------- Edwin Wright 85 Spinnaker Drive, A-602 Halifax, Nova Scotia CANADA B3N 3E3 Telephone: (902) 477-5037 Email: ewright@fox.nstn.ns.ca =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= From owner-proteins@net.bio.net Sun Jan 01 22:00:00 1995 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!library.ucla.edu!agate!howland.reston.ans.net!usc!bloom-beacon.mit.edu!news.starnet.net!wupost!tulane!rs5.tcs.tulane.edu!dpn From: dpn@rs5.tcs.tulane.edu (David P Norwood) Newsgroups: bionet.molbio.proteins Subject: Re: ELISA slamming Date: 31 Dec 1994 22:12:10 GMT Organization: Computer Science Dept., Tulane Univ., New Orleans, LA Lines: 13 Message-ID: <3e4kvq$a0f@news.cs.tulane.edu> References: <259DED150FA@zool.umd.edu> <1994Dec30.140722.13400@es.dupont.com> NNTP-Posting-Host: rs5.tcs.tulane.edu X-Newsreader: TIN [version 1.2 PL2] kramerse@wmvx.lvs.dupont.com wrote: : In article <259DED150FA@zool.umd.edu>, GOODE@ZOOL.UMD.EDU ("Dennis Goode") writes: : Thanks in advance for any advice : Susan Kramer KODAK makes large (9") sheets of lens cleaning tissue that are designed to not-leave-lint. (I suppose other vendors make them too, we use Kodak...) Would these work? From owner-proteins@net.bio.net Sun Jan 01 22:00:00 1995 Path: biosci!agate!howland.reston.ans.net!newsserver.jvnc.net!synapse.bms.com!news-admin From: mamber@synapse.bms.com (mamber) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins,bionet.parasitology Subject: Re: Help with tubulin purification Date: 2 Jan 1995 16:03:01 GMT Organization: Bristol-Myers Squibb Lines: 32 Message-ID: <3e983l$6dr@synapse.bms.com> References: <3daqa8INNbij@newsman.csu.murdoch.edu.au> <3dcq27$omj@apollo.it.luc.edu> NNTP-Posting-Host: 140.176.141.202 Xref: biosci bionet.molbio.methds-reagnts:22490 bionet.molbio.proteins:3354 bionet.parasitology:421 In article <3dcq27$omj@apollo.it.luc.edu>, gnew@orion.it.luc.edu (Gerri Newfry) says: > >Megan Oxberry (oxberry@numbat.murdoch.edu.au) wrote: >: I am a PhD. student in Perth, Western Australia who is trying >: desperately to purify tubulin for use in an assay to measure tubulin >: polymerisation. So far I have tried using the polymerisation/depolymerisation method of >: Shelanski et al. (1973) and the homogenisation, centrifugation and >: dialysis method of Barrowman et al. (1984), both of which have been >: unsuccessful. >: Megan Oxberry oxberry@numbat.murdoch.edu.au (Long responses by gerri...gnew@orion.it.luc.edu and Ginnie Dress@biosci.arizona.edu) I have tried to rigorously follow the procedure of Williams and Lee, preparation of tubulin from brain, Methods in Enzymology 85:376-385, 1982. My only changes are that a) I pH the buffer at 6.6, and b) I store my 1XMTP in 3 M glycerol (PM3M buffer), and my 2XMTP in 0.4 M glycerol (PM0.4M buffer) at -80 C. What the respondents are saying about brain freshness is right on the money. When I obtain brains, I have the person removing the brain dip the brain into ice water first, then place in crushed ice. I have used goat and sheep brains as well as calf brains and the smaller animals' brains actually have given me better MTP yields, presumably because their smaller brains chill faster and thus retard tubulin degradation. But if I can't get the brains homogenized within 1.5-2 hr after slaughter, yield and quality really drops off. D. Murphy (Methods in Cell Biology 24:31-49, 1982) has graphed assembly vs time and states: 'Ideally one should use brain tissue obtained within 20 min of slaughter'. One other trick that I've read about (but don't remember where) may be to add a protease inhibitor like PMSF to the initial homogenate to minimize tubulin decomposition. Good luck, SWM mamber@synapse.bms.com From owner-proteins@net.bio.net Sun Jan 01 22:00:00 1995 Path: biosci!agate!howland.reston.ans.net!spool.mu.edu!torn!nermal.cs.uoguelph.ca!gadwall.cs.uoguelph.ca!dbwilson From: dbwilson@uoguelph.ca (Daniel B Wilson) Newsgroups: bionet.molbio.proteins Subject: AChE, BChE mAbs Date: 2 Jan 1995 20:50:18 GMT Organization: University of Guelph Lines: 5 Message-ID: <3e9oua$scr@nermal.cs.uoguelph.ca> NNTP-Posting-Host: gadwall.cs.uoguelph.ca X-Newsreader: TIN [version 1.2 PL2] I would much appreciate any information on where I might find monoclonal antibodies to acetylcholinesterase and/or butyrylcholinesterase. Thank you in advance for your assistance. DBW From owner-proteins@net.bio.net Sun Jan 01 22:00:00 1995 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!swrinde!gatech!newsxfer.itd.umich.edu!nntp.cs.ubc.ca!unixg.ubc.ca!worm.biochem.ubc.ca!user From: mleroux@unixg.ubc.ca (Michel Leroux) Newsgroups: bionet.molbio.proteins Subject: Re: Newsgroups concerned with proteins Date: 31 Dec 1994 22:48:32 GMT Organization: Dept. of Biochemistry, U.B.C Lines: 14 Distribution: world Message-ID: References: <9412291817.AA00636@wyatt.com> NNTP-Posting-Host: worm.biochem.ubc.ca In article <9412291817.AA00636@wyatt.com>, wyatt@wyatt.com wrote: > We would like to subscribe to newsgroup(s) that might be able to > answer questions on protein structure, size, molecular weights, > glycosylation, chromatographic separations, etc. Is this the right > place to start? > Many thanks! > Phil Wyatt > e-mail Yes it is! Another useful group, especially for asking technical questions such as chromatography, etc., is the bionet.molbio.methds-reagnts group. Welcome to the group. From owner-proteins@net.bio.net Mon Jan 02 22:00:00 1995 Path: biosci!bloom-beacon.mit.edu!news.starnet.net!wupost!waikato!comp.vuw.ac.nz!canterbury.ac.nz!chmeds.ac.nz!gjacobs From: gjacobs@chmeds.ac.nz Newsgroups: bionet.molbio.proteins Subject: Re: molten state of proteins? Message-ID: <1995Jan4.100347.812@chmeds.ac.nz> Date: 4 Jan 95 10:03:47 +1200 References: Lines: 14 In article , ubziobro@jetta (Rafal Ziobro) writes: > I am looking for information about molten state of proteins. > Please tell me where can i find it. Thanks a lot. > I tried to reply directly to you, but my mailer doesn't like that idea, so... Try reading Tom Crieghton's text "Proteins..." or (better) the book he editted on Protein Folding; follow up the refs. cited then flounder about the literature: you'll find more refs. that you care to read. Grant. From owner-proteins@net.bio.net Mon Jan 02 22:00:00 1995 Path: biosci!agate!ihnp4.ucsd.edu!munnari.oz.au!news.unimelb.EDU.AU!news From: david_small@muwayf.unimelb.edu.au (Your Name Here) Newsgroups: bionet.molbio.proteins Subject: Re: AChE, BChE mAbs Date: 3 Jan 1995 22:03:53 GMT Organization: The University of Melbounre Lines: 17 Message-ID: <3echk9$hgk@news.unimelb.EDU.AU> References: <3e9oua$scr@nermal.cs.uoguelph.ca> NNTP-Posting-Host: dsmallpc.path.unimelb.edu.au X-Newsreader: WinVN version 0.82 In article <3e9oua$scr@nermal.cs.uoguelph.ca>, dbwilson@uoguelph.ca (Daniel B Wilson) says: > >I would much appreciate any information on where I might find monoclonal >antibodies to acetylcholinesterase and/or butyrylcholinesterase. Thank >you in advance for your assistance. > >DBW A number of monoclonal antibodies to fetal bovine serum AChE have been made by B.P. Doctor at the Walter Reed Army Hospital in Washington. Jacques Grassi in France also has a number of monoclonals now. The best commercially available one to mammalian AchE is the Fambrough antibody, AE1, available from Chemicon. David Small NH&MRC Research Fellow Dept. of Pathology, University of Melbourne From owner-proteins@net.bio.net Mon Jan 02 22:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!agate!howland.reston.ans.net!Germany.EU.net!EU.net!Austria.EU.net!newsfeed.ACO.net!fuw.edu.pl!cyfronet!jetta!ubziobro From: ubziobro@jetta (Rafal Ziobro) Subject: molten state of proteins? Message-ID: Sender: news@cyf-kr.edu.pl (News Administrator) Nntp-Posting-Host: jetta.if.uj.edu.pl Organization: Academic Computer Centre, CYFRONET X-Newsreader: TIN [version 1.2 PL2] Date: Tue, 3 Jan 1995 16:32:47 GMT Lines: 3 I am looking for information about molten state of proteins. Please tell me where can i find it. Thanks a lot. From owner-proteins@net.bio.net Mon Jan 02 22:00:00 1995 Path: biosci!JASON.UTHCT.EDU!SHAUN From: SHAUN@JASON.UTHCT.EDU ("Shaun D. Black") Newsgroups: bionet.molbio.proteins Subject: RE: molten state of proteins? Date: 3 Jan 1995 15:55:59 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 35 Sender: daemon@net.bio.net Distribution: world Message-ID: <950103180255.2762@jason.uthct.edu> NNTP-Posting-Host: net.bio.net From: SMTP%"Postmaster@jason.uthct.edu" 3-JAN-1995 18:00:16.21 To: SHAUN CC: Subj: Undeliverable Mail Date: Tue, 3 Jan 1995 18:00:14 -0600 (CST) From: Postmaster@jason.uthct.edu Subject: Undeliverable Mail To: Bad address -- Error -- Nameserver error: Unknown host Start of returned message Date: Tue, 3 Jan 1995 18:00:13 -0600 (CST) From: "Shaun D. Black" To: ubziobro@jetta.uthct.edu Message-Id: <950103180013.2762@jason.uthct.edu> Subject: RE: molten state of proteins? Hi, Check out Creighton's book, "Protein Folding" (W.H. Freeman, 1992), especially chapter 6. There was also a good article in a recent Protein Science from Privalov's group, I believe. Hope this helps you. Best regards, =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= = Shaun D. Black, PhD | Internet address: shaun@jason.uthct.edu = = Dept. of Biochemistry | University of Texas Health Center, at Tyler = =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= End of returned message From owner-proteins@net.bio.net Tue Jan 03 22:00:00 1995 Path: biosci!newshost.lanl.gov!news.ttu.edu!seas.smu.edu!convex!convex!news.duke.edu!concert!bigblue.oit.unc.edu!joan From: joan@med.unc.edu (Joan Shields) Newsgroups: bionet.molbio.proteins Subject: Re: Westrn blot and quantification of protein. Date: 4 Jan 1995 15:54:36 GMT Organization: UNC-CH School of Medicine Lines: 26 Message-ID: <3eegbs$vm0@bigblue.oit.unc.edu> References: NNTP-Posting-Host: dallas.med.unc.edu Mary asks: We are currently using western blots to determine expression of a protein. Our antibody has not been impressive so far. It has been suggested that we use immunoprecipitation instead of the western technique. Can immunoprecipitation be used to compare amount of protein expression or is the western the better way to go? Thanks for your input. Mary ------ Immunoprecipatation alone isn't going to give you much, you still need some way to measure (or at least see) your protein expression. You could use immunoprecipitation and then run the sample on a gel and do a western. It could be that your antibody is reacting with something else along with what you're looking for. Immunoprecipitation might get rid of what may be interfering with the binding you want. The problem with doing the immunoprecipitation before the western is that you may loose some of what you're looking for (as will happen in any purification step which is pretty much what an immunoprecipitation step does). You could give it a try, probably wouldn't hurt. You might also want to look at your technique and solutions as the problem could be there. Good luck, joan From owner-proteins@net.bio.net Tue Jan 03 22:00:00 1995 Path: biosci!bloom-beacon.mit.edu!spool.mu.edu!howland.reston.ans.net!news.cac.psu.edu!news.pop.psu.edu!psuvax1!rutgers!utcsri!yonge.cs.toronto.edu!neuron.ai.toronto.edu!ai.toronto.edu!steeg Newsgroups: bionet.software,bionet.molbio.proteins,bionet.molec-model From: steeg@cs.toronto.edu ("Evan W. Steeg") Subject: Pictures/coordinates for states in protein folding Message-ID: <95Jan4.201837edt.280@neuron.ai.toronto.edu> Originator: root@yonge.cs.toronto.edu Nntp-Posting-Host: yonge.cs.toronto.edu Organization: CS Lab, University of Toronto Distribution: bionet Date: 5 Jan 95 01:19:19 GMT Lines: 37 Xref: biosci bionet.software:10540 bionet.molbio.proteins:3368 bionet.molec-model:220 A friend of mine would like some pictures of a protein in several different states of folding/unfolding. She'd really prefer Cytochrome C, but other proteins would suffice. Otherwise, her needs are very flexible: -- Intermediate states may be observed (NMR, etc.) or modelled (according to some accepted classical/qm simulation model). -- Pictures may be space-filling, or ribbon diagrams, or etc. -- Pictures in any unix-friendly format (gif, tiff, ppm, etc.) In addition to or in lieu of graphics, perhaps there is a database of partially-folded structures somewhere, akin to the brookhaven pdb of folded structure coordinate files? I'm curious about this myself. Last but not least, can anyone recommend offhand a particularly good paper on some theoretical or empirical aspects of folding that features a sequence of pictures as described above (especially of Cyt C!) ? Thanks for any pictures, pointers, or references! If I discover anything especially interesting, I'd be glad to summarize and repost. Regards, Evan Evan W. Steeg (416) 978-5182 steeg@ai.toronto.edu Dept of Computer Science steeg@t13.lanl.gov University of Toronto, Toronto, Canada M5S 1A4 FAX: (416) 978-1455 =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=- "We tend to scoff at the beliefs of the ancients. But we can't scoff at them personally, to their faces, and this is what annoys me." - Jack Handey =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=- From owner-proteins@net.bio.net Tue Jan 03 22:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!s.u-tokyo!news.tisn.ad.jp!gan!ksasaki From: ksasaki@ncc.go.jp Subject: Re: Address of Dr. Yonezawa and Sugiura at Kyoto, Japan To: bionet-molbio-proteins@gan.ncc.go.jp Message-ID: <9501050138.AA08181@gan.ncc.go.jp> Sender: ksasaki@gan.ncc.go.jp (Kazuki Sasaki) Organization: National Cancer Center JAPAN Date: Thu, 5 Jan 1995 01:38:17 GMT Return-Path: X-Mailer: Eudora-J(1.3.3J5) Lines: 23 >Does someone on the net know the address/e-mail/fax of Dr.Yonezawa and >Sugiara who work on FokI DNA-binding domain at Kyoto University, Japan. Dr. Yukio SUGIURA (Please note it is not SUGIARA) ^^ Professor, Chemical Institute, Kyoto Univ. snail-mail address: Gokashou Uji-shi, Kyoto, JAPAN Phone: +81-774-32-3111 ex. 2217 Regards, K.SASAKI, M.D. Growth Factor Division National Cancer Center Research Institute, Tokyo, JAPAN From owner-proteins@net.bio.net Tue Jan 03 22:00:00 1995 Path: biosci!bloom-beacon.mit.edu!gatech!howland.reston.ans.net!usc!usc!not-for-mail From: william@neuro.usc.edu (Fiberman) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: reprobe western blots Date: 4 Jan 1995 22:17:13 -0800 Organization: University of Southern California, Los Angeles, CA Lines: 11 Sender: william@neuro.usc.edu Message-ID: <3eg2t9$8hl@neuro.usc.edu> NNTP-Posting-Host: neuro.usc.edu Xref: biosci bionet.molbio.methds-reagnts:22572 bionet.molbio.proteins:3369 Hello, Netters, Can a western blot filter be "reprobed" like a Southern blot filter? If so, what is the best way to strip the original antibody? I am using a secondary antibody conjugated to alkaline phosphatase, and visualizing with addition of color substrates NBT and BCIP. I would appreciate your response. -fm From owner-proteins@net.bio.net Tue Jan 03 22:00:00 1995 Path: biosci!newshost.lanl.gov!news.ttu.edu!seas.smu.edu!convex!insosf1.infonet.net!solaris.cc.vt.edu!news.alpha.net!uwm.edu!vixen.cso.uiuc.edu!news.uoregon.edu!gaia.ucs.orst.edu!news.ohsu.edu!Rae.Nishi From: nishir@ohsu.edu (Rae Nishi) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: protein and RNA purification Date: 4 Jan 1995 17:52:17 GMT Organization: Oregon Health Sciences University, Portland OR Lines: 20 Sender: -Not-Authenticated-[3507] Message-ID: <3een8h$4ae@steele.ohsu.edu> References: <3daac4$1tu@neuro.usc.edu> NNTP-Posting-Host: 137.53.72.132 X-Posted-From: InterNews 1.0@steele.ohsu.edu. Xdisclaimer: No attempt was made to authenticate the sender's name. Xref: biosci bionet.molbio.methds-reagnts:22551 bionet.molbio.proteins:3365 In article <3daac4$1tu@neuro.usc.edu> william@neuro.usc.edu (Fiberman) writes: > Is there a protocol to get proteins and RNA from the same tissue in one > step? If you know of one, or can provide a reference, please let me know! > Thanks. > > -fm With the TriReagent (from Molecular Research Corp) or Trizol from GIBCO BRL you are supposed to be able to get RNA, DNA and protein in one extraction. We've only used in for isolating RNA but a colleague of mine who tried it for RNA and protein said that it worked fine. Rae Nishi Dept. Cell Biology & Anatomy Oregon Health Sciences University Portland Oregon 97201 **that's Orygun, NOT Ora-Gone** From owner-proteins@net.bio.net Tue Jan 03 22:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!CS.Arizona.EDU!uunet!news.iij.ad.jp!wnoc-tyo-news!news.u-tokyo.ac.jp!news.tisn.ad.jp!riksun!postman!macn405.riken.go.jp!user From: cgrunau@rkna50.riken.go.jp (Christoph Grunau) Subject: Test-please ignore Content-Type: text/plain; charset=ISO-8859-1 Message-ID: Followup-To: bionet.molbio.proteins Sender: news@postman.riken.go.jp (News Administrator) Nntp-Posting-Host: macn405 Content-Transfer-Encoding: 8bit Organization: RIKEN Mime-Version: 1.0 Date: Wed, 4 Jan 1995 00:16:08 GMT Lines: 1 only test - please ignore! From owner-proteins@net.bio.net Tue Jan 03 22:00:00 1995 Path: biosci!lhc!darwin.sura.net!newspump.wustl.edu!newsreader.wustl.edu!news.starnet.net!wupost!howland.reston.ans.net!news.sprintlink.net!EU.net!uknet!sunsite.doc.ic.ac.uk!daresbury!not-for-mail From: vachon@bio.grenet.fr (Gilles VACHON) Newsgroups: bionet.molbio.proteins Subject: Address of Dr. Yonezawa and Sugiara at Kyoto, Japan Date: 4 Jan 1995 16:02:27 -0000 Lines: 16 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <3eegqj$buh@mserv1.dl.ac.uk> Original-To: proteins@dl.ac.uk Posted-Date: Wed, 4 Jan 95 17:03:31 +0100 Hi all, Does someone on the net know the address/e-mail/fax of Dr.Yonezawa and Sugiara who work on FokI DNA-binding domain at Kyoto University, Japan. Thanks Dr. Gilles Vachon Laboratoire de Biologie Moleculaire Vegetale CERMO, 3eme etage Universite J. Fourier, BP 53X 38041 GRENOBLE CEDEX FRANCE Tel: (33) 76 51 48 92 fax: (33) 76 51 43 36 e-mail: vachon@bio.grenet.fr From owner-proteins@net.bio.net Tue Jan 03 22:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!agate!howland.reston.ans.net!swrinde!emory!darwin.sura.net!fconvx.ncifcrf.gov!reming From: reming@ncifcrf.gov (Mary Remington) Subject: Westrn blot and quantification of protein. Message-ID: Organization: Frederick Cancer Research and Development Center We are currently using western blots to determine expression of a protein. Our antibody has not been impressive so far. It has been suggested that we use immunoprecipitation instead of the western technique. Can immunoprecipitation be used to compare amount of protein expression or is the western the better way to go? Thanks for your input. Mary Date: Wed, 4 Jan 1995 13:07:10 GMT Lines: 1 From owner-proteins@net.bio.net Tue Jan 03 22:00:00 1995 Path: biosci!TIKAL.BIOSCI.ARIZONA.EDU!hallick_lab From: hallick_lab@TIKAL.BIOSCI.ARIZONA.EDU (Hallick Lab) Newsgroups: bionet.molbio.proteins Subject: coupling peptides Date: 4 Jan 1995 16:36:17 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 13 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net I have a question about coupling peptides to carrier proteins for use in immunizations. I am interested in using EDC to couple my peptides to BSA. I would like to know people's opinion of this method. Is it tricky? Is it a fairly successful method? I am worried about blocking free amino groups on the peptide. Is this necessary? (Each of my 10mer peptides has at least two internal lysines.) I would appreciate any hints or comments. Thanks, Kristin Jenkins Dept. of Molecular and Cellular Biology University of Arizona hallicklab@tikal.biosci.arizona.edu From owner-proteins@net.bio.net Wed Jan 04 22:00:00 1995 Path: biosci!JWCI.ORG!dale From: dale@JWCI.ORG ("Dale Yuzuki's Work Account") Newsgroups: bionet.molbio.proteins Subject: spermidine cell lysis Date: 5 Jan 1995 00:08:07 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 12 Sender: daemon@net.bio.net Distribution: world Message-ID: <9501041140.aa26055@jwi-adm.jwci.org> NNTP-Posting-Host: net.bio.net Being a newcomer to protein purification, I read about an enzymatic method of cell lysis of E.coli that uses sucrose and spermidine (Meth. Enzym. 182:149) but the scale is much too high for the amount of cells I have (pellet from ~2l). Is there a similar method available that is appropriately scaled down? Or should I use the above method at 1/200 volumes? Thanks in advance. - Dale Yuzuki M.A. M.Ed. - John Wayne Cancer Inst. - dale@jwci.org - __@ - 2200 Santa Monica Blvd - Santa Monica CA 90404 - (310) 449-5267 _ -\<'_ - I do not pray for success. I ask for faithfulness. Mother Theresa(_)-/-(_) From owner-proteins@net.bio.net Wed Jan 04 22:00:00 1995 Path: biosci!bloom-beacon.mit.edu!uhog.mit.edu!nntp.club.cc.cmu.edu!newsfeed.pitt.edu!uunet!fonorola!inforamp.net!woody16.inforamp.net!intermed From: intermed@inforamp.net (Ian Clarke) Newsgroups: bionet.molbio.proteins Subject: Attention Canadian Researchers Date: Thu, 5 Jan 1995 12:57:58 Organization: InfoRamp Inc. Lines: 19 Message-ID: NNTP-Posting-Host: woody16.inforamp.net X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] Inter Medico is the Canadian distributor for many of the world's leading molecular biology companies. Inter Medico represents Genzyme(cytokines, chemokines, cell adhesion molecules), Novagen (pET bacterial expression systems, mRNA purification systems), Amresco (fine chemicals, thermostable DNA polymerases, nucleic acid isolation kits), SLT (ELISA readers, fluorometers, microplate washers), BioDesign (a huge selection of biologically important antibodies), and The Binding Site ( clinically-relevant antibody-based diagnostic systems). As part of Inter Medico's continuing dedication to the Canadian research community, we have established a user-group to share the latest information about molecular biology products designed to save you time and money. There are valuable discounts available only to subscribers. There is no charge to join. If you are interested in joining, please send an e-mail message to Majordomo@inforamp.net. In the body of the letter, type Subscribe Research . Join today, and start enjoying the benefits immediately. From owner-proteins@net.bio.net Wed Jan 04 22:00:00 1995 Path: biosci!bloom-beacon.mit.edu!usc!usc!not-for-mail From: william@neuro.usc.edu (Fiberman) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: lab manual Date: 5 Jan 1995 01:50:14 -0800 Organization: University of Southern California, Los Angeles, CA Lines: 8 Sender: william@neuro.usc.edu Message-ID: <3egfcm$8ur@neuro.usc.edu> NNTP-Posting-Host: neuro.usc.edu Xref: biosci bionet.molbio.methds-reagnts:22576 bionet.molbio.proteins:3371 Hi, Netters, Can you recommend a good biochemistry laboratory manual, equivalent to the Sambrook and Maniatis manual for molecular biology? Thanks for any suggestions. -fm From owner-proteins@net.bio.net Wed Jan 04 22:00:00 1995 Path: biosci!agate!ihnp4.ucsd.edu!swrinde!howland.reston.ans.net!news.sprintlink.net!news.rain.org!usenet From: phil@wyatt.com (Philip J. Wyatt) Newsgroups: bionet.molbio.proteins Subject: Proteins and liquid chromatography Date: 5 Jan 1995 23:49:34 GMT Organization: RAIN Public Access Internet (805) 967-RAIN Lines: 39 Distribution: world Message-ID: <3ei0ie$d2k@news.rain.org> Reply-To: wyatt@wyatt.com NNTP-Posting-Host: port09.rain.org Keywords: separations, aggregation, liquid chromatography We are a small company that manufactures multi-angle laser light scattering detectors (MALLS) used in combination with chromatographically separated samples to determine their absolute molecular weights and sizes and the distributions of these quantities. During the past few years, a great number of our colleagues seem to be increasingly interested in proteins and their characterizations. (E. g. aggregation phenomena, self assembly, stability and just plain molecular weights of various preparation.) We get a lot of complaints, not so much about the instruments, but about the chromatography itself and what the separation process is doing to the samples. We're not experts in protein chemistry, but obviously feel we have a lot to learn both to help our customers and to interact better with the protein chemistry community. Right now we have the following four questions and would be most grateful for any comments or suggestions a protein or biochemist might have: Please be assured that this is NOT a commercial pitch, but it seems to touch on some pretty important measurements now going on in the protein community. 1) What do you think of the chromatographic separation process as a tool for characterizing protein samples (assuming that an absolute determination may result)? 2) If you use such techniques, what type do you use? (E. g. reverse phase, size exclusion, ion exchange, capillary electrophoresis) 3) Do you think the buffer and/or the mobile phase can (will) affect the composition of your sample? (E. g. might you detect aggregates that were not present in your sample? Could some of the sample stick to the columns during the separation process itself?) 4) If a column structure could be developed that would permit the use of any type of mobile phase without affecting the separation process itself (i. e. no sticking and no false aggregation), would that make such chromatographic techniques more attractive? (We do NOT manufacture columns!) Phil Wyatt wyatt@wyatt.com From owner-proteins@net.bio.net Wed Jan 04 22:00:00 1995 Path: biosci!rutgers!utcsri!yonge.cs.toronto.edu!neuron.ai.toronto.edu!ai.toronto.edu!steeg Newsgroups: bionet.software,bionet.molbio.proteins,bionet.molec-model From: steeg@cs.toronto.edu ("Evan W. Steeg") Subject: Re: Pictures/coordinates for states in protein folding Message-ID: <95Jan5.175343edt.850@neuron.ai.toronto.edu> Originator: root@yonge.cs.toronto.edu Nntp-Posting-Host: yonge.cs.toronto.edu Organization: CS Lab, University of Toronto References: <95Jan4.201837edt.280@neuron.ai.toronto.edu> Distribution: bionet Date: 5 Jan 95 22:54:35 GMT Lines: 53 Xref: biosci bionet.software:10554 bionet.molbio.proteins:3376 bionet.molec-model:222 If I may just follow up on my own posting of yesterday: In article <95Jan4.201837edt.280@neuron.ai.toronto.edu>, Evan W. Steeg wrote: > A friend of mine would like some pictures of a protein in several >different states of folding/unfolding. She'd really prefer Cytochrome C, >but other proteins would suffice. Otherwise, her needs are very >flexible: > > -- Intermediate states may be observed (NMR, etc.) or modelled (according > to some accepted classical/qm simulation model). > > -- Pictures may be space-filling, or ribbon diagrams, or etc. > > -- Pictures in any unix-friendly format (gif, tiff, ppm, etc.) > > In addition to or in lieu of graphics, perhaps there is a database >of partially-folded structures somewhere, akin to the brookhaven pdb >of folded structure coordinate files? I'm curious about this myself. > > Last but not least, can anyone recommend offhand a particularly good >paper on some theoretical or empirical aspects of folding that features >a sequence of pictures as described above (especially of Cyt C!) ? > 1. Lest there be any confusion... what I meant when I referred to various states of folding/unfolding was *well-characterized intermediates in the folding pathway*, and not "unfolded" or "random" configurations. Obviously, it makes no sense to have a database of "unfolded" protein coordinates. 2. I'm making this request for a friend who wants to do a research/reading project for a chemistry course. I talked her into being interested in the protein folding problem (oops) and she is already thinking of how she might give her presentation if she does follow through on this nontrivial topic. It would be nice if she could put up a couple of slides graphically illustrating the ("real" or hypothesized) folding of a particular protein. 3. It occurs to me that, besides my friend's immediate needs, it may be of interest to others (myself included) if there exists any database of either graphics (e.g., a WWW site) or databases of proposed or discovered intermediate structures. Thanks again for your attention. -- Evan Evan W. Steeg (416) 978-5182 steeg@ai.toronto.edu Dept of Computer Science steeg@t13.lanl.gov University of Toronto, Toronto, Canada M5S 1A4 FAX: (416) 978-1455 From owner-proteins@net.bio.net Wed Jan 04 22:00:00 1995 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!news.service.uci.edu!hammer.biochem.uci.edu!user From: anon Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: reprobe western blots Date: Thu, 05 Jan 1995 15:43:18 -0800 Organization: University of California, Irvine Lines: 25 Message-ID: References: <3eg2t9$8hl@neuro.usc.edu> NNTP-Posting-Host: hammer.biochem.uci.edu Xref: biosci bionet.molbio.methds-reagnts:22629 bionet.molbio.proteins:3375 In article <3eg2t9$8hl@neuro.usc.edu>, william@neuro.usc.edu (Fiberman) wrote: > Hello, Netters, > > Can a western blot filter be "reprobed" like a Southern blot filter? If > so, what is the best way to strip the original antibody? I am using a > secondary antibody conjugated to alkaline phosphatase, and visualizing > with addition of color substrates NBT and BCIP. I would appreciate your > response. > > -fm I think this method causes a precipitate to form, and you can't strip and reprobe when using this method. . . . . . . . . From owner-proteins@net.bio.net Wed Jan 04 22:00:00 1995 Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins,bionet.molbio.rapd,bionet.molbio.yeast,bionet.mycology,bionet.n2-fixation,bionet.neuroscience,bionet.photosynthesis,bionet.plants,bionet.population-bio,bionet.software,bionet.software.acedb,bionet.software.gcg,bionet.users.addresses,bionet.virology,bionet.women-in-bio,bionet.xtallography,biz.americast,biz.books.technical,biz.comp.hardware Path: biosci!bloom-beacon.mit.edu!gatech!newsfeed.pitt.edu!uunet!xnet!quake.xnet.com!research From: crta@xnet.com (Norman Fraley) Subject: New Research & Testing Association Formed Message-ID: Sender: news@amiserv.chi.il.us Nntp-Posting-Host: research.crta.org Organization: Contract Research & Testing Association X-Newsreader: News Xpress Version 1.0 Beta #2 Date: Thu, 5 Jan 1995 20:53:53 GMT Lines: 66 Xref: biosci bionet.molbio.methds-reagnts:22623 bionet.molbio.proteins:3374 bionet.molbio.rapd:925 bionet.molbio.yeast:2110 bionet.mycology:1351 bionet.neuroscience:5641 bionet.photosynthesis:527 bionet.plants:4862 bionet.population-bio:1001 bionet.software:10553 bionet.software.acedb:518 bionet.software.gcg:897 bionet.users.addresses:2135 bionet.virology:1302 bionet.women-in-bio:1694 bionet.xtallography:1410 biz.americast:1037 biz.books.technical:741 biz.comp.hardware:7143 As the primary resource of research information, the Internet was the primary choice for making all concerned individuals aware of the formation of the Contract Research & Testing Association. CRTA is an International Association designed to serve the needs of contract research, product and process development organizations and consultants throughout the world. Contract research organizations have specific public, governmental, and industry perception and promotion needs which are not addressed by existing scientific industry associations. CRTA operates as a non-profit, tax-exempt, corporation eligible for scientific research and public awareness charitable organization contributions as provided for in the IRC 501(c)(3) provisions. Being a scientific research and public awareness related organization, CRTA exists to benefit its members by providing: 1) An organization devoted to the promotion of Contract Research. 2) A unified voice on matters of common interest or concern. 3) Point of contact for media relations relative to contract research. 4) Business opportunity referrals as a research clearinghouse. 5) Professional networking opportunities for its members. 6) Periodic publishing of information beneficial to the membership. 7) Periodic dissemination of applicable research results to the public. 8) Governmental representation on issues affecting CRO's. 9) Public promotion of the strengths of its membership. 10) A directory of Contract Research Organizations and Consultants. CRTA will provide: 1) A forum for the exchange of information. 2) Formal recognition to the CRO's role in business. 3) Standards for the professionals so engaged. 4) Representation the profession in matters of common interest. 5) The development of techniques and methods to improve the practice and management of CROs. CRTA will also offer: 1) A monthly news publication. 2) Annual meetings 3) Active promotional media publicity programs. 4) A professional placement service 5) A Contract Research Service Directory. 6) Media topics and contacts directory If you have an interest in joining the Contract Research & Testing Association, please E-mail your reply to crta@xnet.com. Please include: 1) The word "membership" in your RE: or header information, 2) Your interest in the association / your area of work, 3) Your dues payment preference (check, money order, credit card, company check, wire xfer, etc.) DO NOT INCLUDE ANY CREDIT CARD INFORMATION! Only your preference for the manner of payment. 4) Most importantly, your email address, and additional contact information if you desire. We will then e-mail membership information and ALL FURTHER INFORMATION directly to you at your email location. Thank you for taking the time to read this announcement. If membership in this program this does not appeal to you, thank you for your patience and understanding. Sincerely, Membership Department Contract Research & Testing Association Best Regards, Norman Fraley CRTA@xnet.com Executive Director BBS:708-515-0494 Contract Research & Testing Association From owner-proteins@net.bio.net Wed Jan 04 22:00:00 1995 Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!library.ucla.edu!agate!howland.reston.ans.net!gatech!newsfeed.pitt.edu!dsinc!ub!galileo.cc.rochester.edu!uhura.cc.rochester.edu!ttha From: ttha@uhura.cc.rochester.edu (Tom Thatcher) Subject: Re: reprobe western blots Message-ID: <1995Jan6.025507.899@galileo.cc.rochester.edu> Sender: news@galileo.cc.rochester.edu Nntp-Posting-Host: uhura.cc.rochester.edu Organization: University of Rochester - Rochester, New York References: <3eg2t9$8hl@neuro.usc.edu> Date: Fri, 6 Jan 95 02:55:07 GMT Lines: 36 Xref: biosci bionet.molbio.methds-reagnts:22633 bionet.molbio.proteins:3379 In anon writes: >In article <3eg2t9$8hl@neuro.usc.edu>, william@neuro.usc.edu (Fiberman) wrote: >> Hello, Netters, >> >> Can a western blot filter be "reprobed" like a Southern blot filter? If >> so, what is the best way to strip the original antibody? I am using a >> secondary antibody conjugated to alkaline phosphatase, and visualizing >> with addition of color substrates NBT and BCIP. I would appreciate your >> response. >> >> -fm > I think this method causes a precipitate to form, and you can't strip >and reprobe when using this method. > You could reprobe with a second antibody (to a different protein) without stripping as long as you used primary and secondary antibodies with different specificities eg. Ab#1 from rabbit< Reply-To: wyatt@wyatt.com NNTP-Posting-Host: port30.rain.org Keywords: protein,HPLC,molecular weights We are a small company that manufactures multi-angle laser light scattering detectors (MALLS) used in combination with chromatographically separated samples to determine their absolute molecular weights and sizes and the distributions of these quantities. During the past few years, a great number of our colleagues seem to be increasingly interested in proteins and their characterizations. (E. g. aggregation phenomena, self assembly, stability and just plain molecular weights of various preparation.) We get a lot of complaints, not so much about the instruments, but about the chromatography itself and what the separation process is doing to the samples. We're not experts in protein chemistry, but obviously feel we have a lot to learn both to help our customers and to interact better with the protein chemistry community. Right now we have the following four questions and would be most grateful for any comments or suggestions a protein or biochemist might have: Please be assured that this is NOT a commercial pitch, but it seems to touch on some pretty important measurements now going on in the protein community. 1) What do you think of the chromatographic separation process as a tool for characterizing protein samples (assuming that an absolute determination may result)? 2) If you use such techniques, what type do you use? (E. g. reverse phase, size exclusion, ion exchange, capillary electrophoresis) 3) Do you think the buffer and/or the mobile phase can (will) affect the composition of your sample? (E. g. might you detect aggregates that were not present in your sample? Could some of the sample stick to the columns during the separation process itself?) 4) If a column structure could be developed that would permit the use of any type of mobile phase without affecting the separation process itself (i. e. no sticking and no false aggregation), would that make such chromatographic techniques more attractive? (We do NOT manufacture columns!) Phil Wyatt wyatt@wyatt.com From owner-proteins@net.bio.net Wed Jan 04 22:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!bloom-beacon.mit.edu!gatech!howland.reston.ans.net!pipex!news.oleane.net!oleane!jussieu.fr!univ-lyon1.fr!swidir.switch.ch!scsing.switch.ch!rzusuntk.unizh.ch!NewsWatcher!user From: maga@vetbio.unizh.ch (Giovanni Maga) Subject: Enzymology Message-ID: Followup-To: bionet.molbio.proteins Sender: newsadm@rzu-news.unizh.ch (CNEWS ADMINISTRATION) Organization: University of Zurich Irchel- Biochemistry Date: Thu, 5 Jan 1995 23:51:09 GMT Lines: 14 From: Giovanni Maga, Biochemistry, University of ZŸrich-Irchel ZH (CH) Dear Netters, my main scientific ineterest and work is devoted to enzyme kinetics. In the last years I have been working with enzymes involved in nucleotide biosynthesis in viruses, mammals and protozoa. Currently, I am working with mammalian DNA polymerases. Is anybody out there interested in corresponding (i.e. discussing problems, giving hints, helping to develope new approaches) about this fascinating topic? I have lots of questions and maybe some answers. If so, please E-mail at: maga@vetbio.unizh.ch Thanks again and Happy New Year!! From owner-proteins@net.bio.net Thu Jan 05 22:00:00 1995 Path: biosci!bloom-beacon.mit.edu!hookup!uwm.edu!psuvax1!news.pop.psu.edu!hudson.lm.com!netline-fddi.jpl.nasa.gov!nntp-server.caltech.edu!ingber From: ingber@alumni.caltech.edu (Lester Ingber) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins,bionet.molbio.rapd,bionet.molbio.yeast,bionet.mycology,bionet.n2-fixation,bionet.neuroscience,bionet.photosynthesis,bionet.plants,bionet.population-bio,bionet.software,bionet.software.acedb,bionet.software.gcg,bionet.users.addresses,bionet.virology,bionet.women-in-bio,bionet.xtallography,biz.americast,biz.books.technical,biz.comp.hardware Subject: Re: New Research & Testing Association Formed Date: 6 Jan 1995 14:04:51 GMT Organization: California Institute of Technology, Alumni Association Lines: 165 Message-ID: <3ejim3$eiq@gap.cco.caltech.edu> References: Reply-To: ingber@alumni.caltech.edu NNTP-Posting-Host: alumni.caltech.edu Xref: biosci bionet.molbio.methds-reagnts:22646 bionet.molbio.proteins:3383 bionet.molbio.rapd:927 bionet.molbio.yeast:2117 bionet.mycology:1356 bionet.neuroscience:5655 bionet.photosynthesis:528 bionet.plants:4871 bionet.population-bio:1002 bionet.software:10566 bionet.software.acedb:519 bionet.software.gcg:898 bionet.users.addresses:2136 bionet.virology:1305 bionet.women-in-bio:1699 bionet.xtallography:1412 biz.americast:1044 biz.books.technical:744 biz.comp.hardware:7159 Norman: I like the concept of what you are trying to form, and so I would like to explain why I will not join now, but might be interested at some future time. My criticism is meant to be constructive, and I hope it will be accepted in this context. Since I think your organization is a great idea, I'm making my response public so that other researchers who might be turned off by some of these problems in your presentation also will keep an open mind to joining your organization in the future. (You really did span quite few bulletin boards!) : From crta@xnet.com Thu Jan 5 21:47:00 1995 : Return-Path: : Date: Thu, 5 Jan 1995 23:46:55 -0600 : From: Norman Fraley : To: ingber@alumni.caltech.edu : Subject: Membership information for CRTA : : Dear Mr. Ingber : : WHAT IS CRTA? : : CRTA, formally known as the Contract Research and Testing Association, is : an : international scientific organization whose primary objective is to build : awareness of the role of contract research in industry and in society, : represent the industry in matters of common interest and to provide a forum : for the exchange of ideas and techniques in the fields of Testing and : Applied : Research. If you are appealing to a large audience over the InterNet, you should follow some commonly accepted guidelines, e.g., delivering text in a most readable format, e.g., limiting lines to 79 characters, so that lines do not spill over on most 80-character screens. This is the most vital context of my critique, that you will not come across as a knowledgeable and professional organization unless you present yourself as such. For example, I put your 424-line e-mail reply to my follow-up to this posting through ispell, and here is a partial list which also does not register in `webster`: copywrite lnternational nonanalytical nonmicrobiology proovided spectroscopists toxicologists : CRTA is known for the efforts it has made in helping to promote the new and : rapidly growing industry of contract research. This is accomplished by a : network of nearly 700 commercial, industry, government, and academic : laboratories and individual product and process development consultants. : These promotional activities and other CRTA programs offer you, the : technical : professional, avenues for professional growth and recognition that only an : organization of CRTA's caliber can provide ... plus access to a wellspring : of : analytical, research and technical information. I question whether you already have such a membership, or this is what you aspire to? This is a reasonable question. Many people, like myself, are more sympathetic to an honest statement from a new operation, than to misleading advertisements. I note a number of journals and publications you (intend to?) make available, and I expect they have a price. Your fees for members of $125/yr is fair, if all these services are available now. All this is fine, but those prices should be stated up front. : Email: crta@xnet.com : WWW: www.xnet.com/~crta I tried this site, http://www.xnet.com/~crta, and found a short notice that it is under construction. I think it better to first do your homework, before advertising such a site, especially since you really are charging commercial rates for a commercial venture, your non-profit status notwithstanding. : copywrite 1994 Contract Research & Testing Association. This one is a real killer, demonstrating that you are not sensitive to one of the main concerns of contract research, the copyright (not the "copywrite"). Lester In article , Norman Fraley wrote: :As the primary resource of research information, the Internet was the :primary choice for making all concerned individuals aware of the formation :of the Contract Research & Testing Association. : :CRTA is an International Association designed to serve the needs of contract :research, product and process development organizations and consultants :throughout the world. Contract research organizations have specific public, :governmental, and industry perception and promotion needs which are not addressed :by existing scientific industry associations. CRTA operates as a non-profit, :tax-exempt, corporation eligible for scientific research and public awareness :charitable organization contributions as provided for in the IRC 501(c)(3) provisions. : :Being a scientific research and public awareness related organization, CRTA :exists to benefit its members by providing: : : 1) An organization devoted to the promotion of Contract Research. : 2) A unified voice on matters of common interest or concern. : 3) Point of contact for media relations relative to contract research. : 4) Business opportunity referrals as a research clearinghouse. : 5) Professional networking opportunities for its members. : 6) Periodic publishing of information beneficial to the membership. : 7) Periodic dissemination of applicable research results to the public. : 8) Governmental representation on issues affecting CRO's. : 9) Public promotion of the strengths of its membership. : 10) A directory of Contract Research Organizations and Consultants. : :CRTA will provide: : 1) A forum for the exchange of information. : 2) Formal recognition to the CRO's role in business. : 3) Standards for the professionals so engaged. : 4) Representation the profession in matters of common interest. : 5) The development of techniques and methods to improve the practice and : management of CROs. : :CRTA will also offer: : 1) A monthly news publication. : 2) Annual meetings : 3) Active promotional media publicity programs. : 4) A professional placement service : 5) A Contract Research Service Directory. : 6) Media topics and contacts directory : :If you have an interest in joining the Contract Research & Testing Association, :please E-mail your reply to crta@xnet.com. Please include: : :1) The word "membership" in your RE: or header information, :2) Your interest in the association / your area of work, :3) Your dues payment preference (check, money order, credit card, company check, wire xfer, etc.) : DO NOT INCLUDE ANY CREDIT CARD INFORMATION! Only your preference for the manner of payment. :4) Most importantly, your email address, and additional contact information if you desire. : :We will then e-mail membership information and ALL FURTHER INFORMATION :directly to you at your email location. Thank you for taking the time :to read this announcement. If membership in this program this does not :appeal to you, thank you for your patience and understanding. : :Sincerely, :Membership Department :Contract Research & Testing Association : : :Best Regards, : :Norman Fraley CRTA@xnet.com :Executive Director BBS:708-515-0494 :Contract Research & Testing Association -- /* Prof. Lester Ingber * * Lester Ingber Research E-Mail: ingber@alumni.caltech.edu * * P.O. Box 857 WWW: http://alumni.caltech.edu/~ingber/ * * McLean, VA 22101 Archive: ftp.alumni.caltech.edu:/pub/ingber/ */ From owner-proteins@net.bio.net Thu Jan 05 22:00:00 1995 Path: biosci!daresbury!trane.uninett.no!sunic!uunet!cs.utexas.edu!howland.reston.ans.net!news.sprintlink.net!news.world.net!serval.net.wsu.edu!netnews.nwnet.net!news.u.washington.edu!root From: Bassuk@U.Washington.Edu (Jim Bassuk) Newsgroups: bionet.molbio.proteins Subject: Re: Westrn blot and quantification of protein. Date: 6 Jan 1995 07:39:47 GMT Organization: University of Washington Lines: 23 Message-ID: <3eis43$o29@nntp1.u.washington.edu> References: NNTP-Posting-Host: stein2.u.washington.edu X-Newsreader: WinVN 0.92.5 In article , reming@ncifcrf.gov (Mary Remington) says: > > We are currently using western blots to determine expression of a protein. > Our antibody has not been impressive so far. It has been suggested that we > use immunoprecipitation instead of the western technique. Can immunoprecipitation > be used to compare amount of protein expression or is the western the > better way to go? Thanks for your input. Mary Mary, Both techniques have their pros and cons. To quantitate a radioactive signal in a western, you must work within the linear range of your system, i.e., run several different amounts of your purified protein on your gel, do western, and scan the autoradiogram with a densitometer or similar device. If the area of integration is linearly proportional to your mass, then you can use the western to do what you want. In immunoprecipitation is used, then you must be very careful to process all samples equally. You can easily loose protein during the washing stages. Otherwise, this technique is another reliable method. Hope this helps. Jim Bassuk BioStructure Univ. Washington From owner-proteins@net.bio.net Thu Jan 05 22:00:00 1995 Path: biosci!CS.Arizona.EDU!uunet!cs.utexas.edu!swrinde!gatech!newsxfer.itd.umich.edu!caen!msunews!harbinger.cc.monash.edu.au!lugb!ucsvc.ucs.unimelb.edu.au!wehi.edu.au!wehi.edu.au!davis Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: reprobe western blots Message-ID: <1995Jan6.082920.1@wehi.edu.au> From: davis@wehi.edu.au Date: 6 Jan 95 08:29:20 +1000 References: <3eg2t9$8hl@neuro.usc.edu> Organization: Walter & Eliza Hall Institute NNTP-Posting-Host: wehit.wehi.edu.au Lines: 17 Xref: biosci bionet.molbio.methds-reagnts:22636 bionet.molbio.proteins:3380 In article <3eg2t9$8hl@neuro.usc.edu>, william@neuro.usc.edu (Fiberman) writes: > > Hello, Netters, > > Can a western blot filter be "reprobed" like a Southern blot filter? Yes, it can. Use the following stripping buffer: 100 mM 2-ME, 2% SDS, 62.5 mM Tris.HCl pH 6.7. Submerge membrane in buffer at 50 deg C for 30 minutes. Wash twice for 10 minutes in your usual wash buffer. Reblock. Reprobe. I have used this several times and it works. Ian Davis davis@licre.ludwig.edu.au Ludwig Institute for Cancer Research Melbourne Tumour Biology Branch Melbourne, Australia. From owner-proteins@net.bio.net Thu Jan 05 22:00:00 1995 Path: biosci!daresbury!trane.uninett.no!sunic!uunet!cs.utexas.edu!howland.reston.ans.net!news.sprintlink.net!news.world.net!serval.net.wsu.edu!netnews.nwnet.net!news.u.washington.edu!root From: Bassuk@U.Washington.Edu (Jim Bassuk) Newsgroups: bionet.molbio.proteins Subject: Digital chromatograms: parts list? Date: 6 Jan 1995 07:55:26 GMT Organization: University of Washington Lines: 21 Message-ID: <3eit1e$o29@nntp1.u.washington.edu> NNTP-Posting-Host: stein2.u.washington.edu X-Newsreader: WinVN 0.92.5 In standard chromatography of proteins, we monitor the absorbance of the column eluate with a Pharmacia UV-1 flow cell coupled to a chart recorder. This analog signal from the UV-1 is 10 mV. To design a system to convert the signal to a digital format, I'll need a A/D converter, some sort of board for a yet-to-be purchased 486 PC, and some sort of software to acquire, process, and store the data. We'll likely couple two UV-1 units in tandem (214 and 280 nm).....this is 2 channels.....and then a simple closure switch to indicate the advancement of the fraction collector....this is another channel... a total of 3 channels..... No need to buy a big, expensive HPLC software program, because I won't be controlling any pumps, etc... Comments on boards, A/D units, and software sources and experiences are respectfully requested. Thanks in advance, Jim Bassuk BioStructure Univ Washington From owner-proteins@net.bio.net Thu Jan 05 22:00:00 1995 Path: biosci!agate!howland.reston.ans.net!news.sprintlink.net!news.rain.org!usenet From: phil@wyatt.com (Philip J. Wyatt) Newsgroups: bionet.molbio.proteins Subject: Re: Proteins and liquid chromatography Date: 6 Jan 1995 18:49:41 GMT Organization: RAIN Public Access Internet (805) 967-RAIN Lines: 146 Distribution: world Message-ID: <3ek3c5$nsl@news.rain.org> References: <3ejjqo$75s@vixen.cso.uiuc.edu> Reply-To: wyatt@wyatt.com NNTP-Posting-Host: port17.rain.org In article <3ejjqo$75s@vixen.cso.uiuc.edu> elarson@ux1.cso.uiuc.edu (larson eric) writes: > phil@wyatt.com (Philip J. Wyatt) writes: > > >We are a small company that manufactures multi-angle laser light > >scattering detectors (MALLS) used in combination with chromatographically > >separated samples to determine their absolute molecular weights and sizes > >and the distributions of these quantities. > > > 1) What do you think of the chromatographic separation process as > >a tool for characterizing protein samples (assuming that an absolute > >determination may result)? > >=============== > > The only technique that seems to yield definitive results is size-exclusion > gel filtration. By definitive I mean results you can talk about results as > reasonably solid evidence that there is an aggregation difference. Even in > the absence of exact stoichiometries (i.e. eluted mol. wt./subunit mol. > wt.) stating that there is a difference is reasonable. > > I have one protein under study that seems to elute from ion exchange resins > as a split peak. Subject the eluted samples to a molecular weight sizing > gel and both elute at the same point. Re-elute these samples from the mol. > wt. sizing gel on ion exchange and both now elute at the same salt > concentration. I believe the separation afforded by the first ion exchange > column is real, but cannot definitively determine "why" the protein split > on the column. I suspect an aggregation phenomenon -- something an on-line > mol. wt. detector could determine (we looked at these several years ago and > they couldn't correctly assess the mol. wt. of a test protein we had -- > might be time to see if the years have been good to the technique -- hmmm, > send me some literature. :-) > > > 2) If you use such techniques, what type do you use? (E. g. > >reverse phase, size exclusion, ion exchange, capillary electrophoresis) > > Of your list, size exclusion and ion exchange are the most popular (at > least with our lab.) Reverse phase and capillary electrophoresis > techniques almost always use conditions that are far too harsh for most > proteins. It would be foolish to try and assess aggregation state of a > protein after it had been subjected to trifluoroacetic acid, phosphoric > acid, organic solvents, etc. Reverse phase and capillary seem suited to > purifying a polypeptide chain (as opposed to purifying a native protein.) > > > 3) Do you think the buffer and/or the mobile phase can (will) > >affect the composition of your sample? (E. g. might you detect > >aggregates that were not present in your sample? Could some of the sample > >stick to the columns during the separation process itself?) > > I would say yes. With two proteins we currently work with, aggregation, > whether part of the native "operational mode" of the protein, or caused by > buffer conditions is observed (one proteins aggregation state depends on > the presence or absence of one substrate.) > > > 4) If a column structure could be developed that would permit > >the use of any type of mobile phase without affecting the separation > >process itself (i. e. no sticking and no false aggregation), would that > >make such chromatographic techniques more attractive? (We do NOT > >manufacture columns!) > > This would be a neat trick if you could do it. Your description pretty > much describes the goal of a mol. wt. sizing column. A priori, this would > not seem to apply to the ion exchange, reverse phase, affinity > chromatography, capillary, etc.. > > It sounds like your company is focussing on devices for on-line > identification of mol.wt. from chromatographic resin (be they ion exchange, > mol. wt. sizing, or whatever.) This is useful. However, many of the > aforementioned chromatographic techniques are being used in the hopes that > differential aggregation (in our case it would protein-protein interactions > between a regulatory protein and its substrate) can be analyzed. Of just > as much utility would be direct measurement of the mol. wt. distribution > within a single (stirred?) vessel versus time (i.e. add substrate protein, > check mol. wt., add regulatory protein, check mol. wt.) Evidence of > changes, even if not definitive in terms of mol. wt., would be very useful. > > -- > Eric Larson | University of Illinois at Urbana-Champaign > USDA/Agronomy | 190 PABL; 1201 W. Gregory; Urbana, IL 61801 > elarson@ux1.cso.uiuc.edu | Voice 217.244.3079 Fax 217.244.4419 > Fidonet: 1:233/4.1 | My opinions are my own, but correct :-) Let me comment on your insightful remarks referring to my questions 1 through 4. 1) Regarding the split peak you observe on elution from ion exchange, putting in line a light scattering detector like our miniDAWN would show immediately the molecular weights and (if the protein is large enough) the rms radius of each slice and, of course, each peak without any need for calibration. (Dr. Charles Zukoski of the Dept. of Chemical Engineering has a large 18 detector DAWN system that will work equally well. You might want to give him a call and discuss this with him at 333 7379.) We've seen similar differences between GPC and reverse phase separations, yet whenever we make the light scattering measurements, the results are clarified. Nevertheless, we may see the SAME sample separating differently with different injections on the SAME GPC column. We often attribute this to sample attaching to the column and eluting with a later sample, often with some weird aggregation (based on LS MW determination) effects! 2) There is another method that appears quite promising that we shall be working with during this next few months. At present it is not user friendly, was developed by another company, and adds another $20,000 to an already expensive LS detector, but we hope to have better news to report soon. We'll keep you posted. 3) If the separation technique mentioned briefly in 2), above, were to obviate these concerns, would anyone want to spend this additional amount of money to achieve it? 4) See above regarding the separation techniques. As for the ablity to monitor MW and size with time, a brute force demonstration of this (using LS) was presented by Wilson and Benight in J. Bio. Chem. vol 265, p 7351 (1990). With our newer instruments, this type of measurement is very easily done. Thanks so much for your comments. If you need any more information from us, please don't hesitate to ask. We'll keep you informed as to the new separation experiments. Phil Wyatt From owner-proteins@net.bio.net Thu Jan 05 22:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!bloom-beacon.mit.edu!gatech!howland.reston.ans.net!torn!nntp.cs.ubc.ca!news.UVic.CA!spruce.pfc.forestry.ca!PFC.Forestry.CA!AEKRAMODDOUL From: aekramoddoul@PFC.Forestry.CA (Ekramoddoullah, Abul Kalam M.) Subject: Re: Westrn blot and quantification of protein. Message-ID: <1995Jan5.215513.578@spruce.pfc.forestry.ca> Sender: news@spruce.pfc.forestry.ca Nntp-Posting-Host: pfc.pfc.forestry.ca Reply-To: aekramoddoul@PFC.Forestry.CA Organization: Forestry Canada (Pacific Forestry Centre) References: Date: Thu, 5 Jan 1995 21:55:13 GMT Lines: 10 In article , reming@ncifcrf.gov (Mary Remington) writes: > If you have an access to a densitometer and software for digitization, Western blot is the ideal. We use regularly Western-immunoblot for quantitation. If you like you can send some blots and we will quantify the protein in question. Good Luck. Abul EKRAMODDOULLAH, ABUL KALAM M. Title: Research Scientist Forestry Canada Phone: (604) 363-0600 Victoria, B.C. Internet: AEKRAMODDOUL@A1.PFC.Forestry.CA From owner-proteins@net.bio.net Thu Jan 05 22:00:00 1995 Path: biosci!agate!howland.reston.ans.net!vixen.cso.uiuc.edu!ux1.cso.uiuc.edu!elarson From: elarson@ux1.cso.uiuc.edu (larson eric) Newsgroups: bionet.molbio.proteins Subject: Re: Proteins and liquid chromatography Date: 6 Jan 1995 14:24:24 GMT Organization: University of Illinois at Urbana Lines: 78 Message-ID: <3ejjqo$75s@vixen.cso.uiuc.edu> References: <3ei0ie$d2k@news.rain.org> NNTP-Posting-Host: ux1.cso.uiuc.edu Keywords: separations, aggregation, liquid chromatography phil@wyatt.com (Philip J. Wyatt) writes: >We are a small company that manufactures multi-angle laser light >scattering detectors (MALLS) used in combination with chromatographically >separated samples to determine their absolute molecular weights and sizes >and the distributions of these quantities. > 1) What do you think of the chromatographic separation process as >a tool for characterizing protein samples (assuming that an absolute >determination may result)? >=============== The only technique that seems to yield definitive results is size-exclusion gel filtration. By definitive I mean results you can talk about results as reasonably solid evidence that there is an aggregation difference. Even in the absence of exact stoichiometries (i.e. eluted mol. wt./subunit mol. wt.) stating that there is a difference is reasonable. I have one protein under study that seems to elute from ion exchange resins as a split peak. Subject the eluted samples to a molecular weight sizing gel and both elute at the same point. Re-elute these samples from the mol. wt. sizing gel on ion exchange and both now elute at the same salt concentration. I believe the separation afforded by the first ion exchange column is real, but cannot definitively determine "why" the protein split on the column. I suspect an aggregation phenomenon -- something an on-line mol. wt. detector could determine (we looked at these several years ago and they couldn't correctly assess the mol. wt. of a test protein we had -- might be time to see if the years have been good to the technique -- hmmm, send me some literature. :-) > 2) If you use such techniques, what type do you use? (E. g. >reverse phase, size exclusion, ion exchange, capillary electrophoresis) Of your list, size exclusion and ion exchange are the most popular (at least with our lab.) Reverse phase and capillary electrophoresis techniques almost always use conditions that are far too harsh for most proteins. It would be foolish to try and assess aggregation state of a protein after it had been subjected to trifluoroacetic acid, phosphoric acid, organic solvents, etc. Reverse phase and capillary seem suited to purifying a polypeptide chain (as opposed to purifying a native protein.) > 3) Do you think the buffer and/or the mobile phase can (will) >affect the composition of your sample? (E. g. might you detect >aggregates that were not present in your sample? Could some of the sample >stick to the columns during the separation process itself?) I would say yes. With two proteins we currently work with, aggregation, whether part of the native "operational mode" of the protein, or caused by buffer conditions is observed (one proteins aggregation state depends on the presence or absence of one substrate.) > 4) If a column structure could be developed that would permit >the use of any type of mobile phase without affecting the separation >process itself (i. e. no sticking and no false aggregation), would that >make such chromatographic techniques more attractive? (We do NOT >manufacture columns!) This would be a neat trick if you could do it. Your description pretty much describes the goal of a mol. wt. sizing column. A priori, this would not seem to apply to the ion exchange, reverse phase, affinity chromatography, capillary, etc.. It sounds like your company is focussing on devices for on-line identification of mol.wt. from chromatographic resin (be they ion exchange, mol. wt. sizing, or whatever.) This is useful. However, many of the aforementioned chromatographic techniques are being used in the hopes that differential aggregation (in our case it would protein-protein interactions between a regulatory protein and its substrate) can be analyzed. Of just as much utility would be direct measurement of the mol. wt. distribution within a single (stirred?) vessel versus time (i.e. add substrate protein, check mol. wt., add regulatory protein, check mol. wt.) Evidence of changes, even if not definitive in terms of mol. wt., would be very useful. -- Eric Larson | University of Illinois at Urbana-Champaign USDA/Agronomy | 190 PABL; 1201 W. Gregory; Urbana, IL 61801 elarson@ux1.cso.uiuc.edu | Voice 217.244.3079 Fax 217.244.4419 Fidonet: 1:233/4.1 | My opinions are my own, but correct :-) From owner-proteins@net.bio.net Thu Jan 05 22:00:00 1995 Path: biosci!agate!sunsite.doc.ic.ac.uk!daresbury!bioftp.unibas.ch!rc1.vub.ac.be!usenet From: scorteel@resulb.ulb.ac.be (Stephane Corteel) Newsgroups: bionet.molbio.proteins Subject: Microwaves action on milk given to infants Date: 7 Jan 1995 00:29:39 GMT Organization: ULB Lines: 12 Message-ID: <3ekn9k$ctt@rc1.vub.ac.be> NNTP-Posting-Host: slip_pc28.ulb.ac.be. X-Newsreader: WinVN 0.90.4 As a young father (my baby now is 7 weeks old) I read a lot... I've read (and some people also told me) that warwing up a feeding bottle using a microwave oven is not recommended because the microwaves cause molecular changes in some proteins of the milk. But, at this time, nobody could confirm that or give me an more scientific explanation. If someone could provide me a answer that an engineer like me can accept, it would be very nice. Thank you very much for any information From owner-proteins@net.bio.net Thu Jan 05 22:00:00 1995 Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Path: biosci!ns1.faseb.org!darwin.sura.net!gatekeeper.es.dupont.com!esds01.es.dupont.com!KRAMERSE@ldoc03.ldc.dupont.com From: kramerse@wmvx.lvs.dupont.com Subject: Re: reprobe western blots Message-ID: <1995Jan6.165540.12347@es.dupont.com> Sender: news@es.dupont.com (USENET News System) Nntp-Posting-Host: ldoc03.lvs.dupont.com Reply-To: kramerse@wmvx.lvs.dupont.com Organization: DuPont (Opinions are those of the writer only) References: <3eg2t9$8hl@neuro.usc.edu> ,<1995Jan6.025507.899@galileo.cc.rochester.edu> Date: Fri, 6 Jan 1995 16:55:40 GMT Lines: 30 Xref: biosci bionet.molbio.methds-reagnts:22670 bionet.molbio.proteins:3388 In article1995Jan6.025507.: >In >In article <3eg2t9$8hl@neuro.usc.edu>, william@neuro.usc.edu (Fiberman) wrote: > >>> Hello, Netters, >>> >>> Can a western blot filter be "reprobed" like a Southern blot filter? If >>> so, what is the best way to strip the original antibody? I am using a >>> secondary antibody conjugated to alkaline phosphatase, and visualizing >>> with addition of color substrates NBT and BCIP. I would appreciate your >>> response. >>> >>> -fm > > Stripping of antibodies from membranes has been described for 125-I labeled proteins and the extension of theis technique to chemiluminescence substrates is feasible. Reference: Kaufman,et al., Analytical Biochem., 161:89-95,(1987) Removing chromogenic substrates is difficult, I don't reccommend it. > >-- >Tom Thatcher | You c >University of Rochester Cancer Center | and h From owner-proteins@net.bio.net Fri Jan 06 22:00:00 1995 Path: biosci!bloom-beacon.mit.edu!gatech!swrinde!howland.reston.ans.net!darwin.sura.net!blaze.cs.jhu.edu!jhunix1.hcf.jhu.edu!welchlink.welch.jhu.edu!ntheo From: ntheo@welchlink.welch.jhu.edu (NICHOLAS THEODORAKIS ) Newsgroups: bionet.molbio.proteins Subject: Re: Microwaves action on milk given to infants Date: 8 Jan 1995 04:37:49 GMT Organization: Johns Hopkins University, Welch Medical Library Lines: 29 Message-ID: <3enq6t$656@jhunix1.hcf.jhu.edu> References: <3ekn9k$ctt@rc1.vub.ac.be> NNTP-Posting-Host: 128.220.59.78 In article <3ekn9k$ctt@rc1.vub.ac.be>, Stephane Corteel wrote: >As a young father (my baby now is 7 weeks old) I read a lot... >I've read (and some people also told me) that warwing up a feeding >bottle using a microwave oven is not recommended because the microwaves >cause molecular changes in some proteins of the milk. > >But, at this time, nobody could confirm that or give me an more >scientific explanation. > >If someone could provide me a answer that an engineer like me can >accept, it would be very nice. > >Thank you very much for any information As a new father of twin girls (now 3 mo. old), I heard a different, and more believable reason not to microwave milk or formula (and this reason would probably appeal to an engineer). Microwave ovens often warm things up unevenly, and thus might leave "hot spots" in the milk that could could burn, even if the outside of the container feels the right temperature -- ++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ Nick Theodorakis ntheo@welchlink.welch.jhu.edu Johns Hopkins Medical School, Baltimore, MD From owner-proteins@net.bio.net Sat Jan 07 22:00:00 1995 Path: biosci!bloom-beacon.mit.edu!uhog.mit.edu!news.mtholyoke.edu!news.amherst.edu!not-for-mail From: rmsmith@unix.amherst.edu Newsgroups: bionet.molbio.proteins Subject: Re: coupling peptides Date: 8 Jan 1995 09:51:39 -0500 Organization: Amherst College, Amherst MA, USA Lines: 40 Distribution: world Message-ID: <3eou5r$r2p@amhux3.amherst.edu> References: NNTP-Posting-Host: amhux3.amherst.edu X-Newsreader: TIN [version 1.2 PL0] Hallick Lab (hallick_lab@TIKAL.BIOSCI.ARIZONA.EDU) wrote: : I have a question about coupling peptides to carrier proteins for use in : immunizations. I am interested in using EDC to couple my peptides to BSA. I : would like to know people's opinion of this method. Is it tricky? Is it a : fairly successful method? I am worried about blocking free amino groups on : the peptide. Is this necessary? (Each of my 10mer peptides has at least two : internal lysines.) I would appreciate any hints or comments. : Thanks, : Kristin Jenkins : Dept. of Molecular and Cellular Biology : University of Arizona : hallicklab@tikal.biosci.arizona.edu We do this kind of thing in our lab all the time. While I have never done this I believe the method we use involves activating the epsilon amino groups on protein lysines with Trauts reagent to yield more reactive thiol groups. We then use a heterobifunctional crosslinker (a maleomidocaproate or something like that) to react first with the peptide. Your unprotected peptide lysines would be a problem here in our scheme but you might be able to find a way around this such as to couple via the C-terminus. The Trauts reagent strategy along with a plethora of crosslinking reagents can be found in the Pierce catalog (with a few references). If you want procedures or any other information feel free to email me. Gook Luck. Rob. ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Robert M Smith rmsmith@amhux3.amherst.edu Department of Chemistry, a Program in Molecular and Cellular Biology Amherst College, n University of Massachusetts, Amherst, MA 01002 d Amherst, MA 01003 Phone: (413) 542-2558 FAX: (413) 542-2735 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ From owner-proteins@net.bio.net Sat Jan 07 22:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!agate!howland.reston.ans.net!pipex!uunet!sangam!iitb!powai!e9403001 From: e9403001@powai.cc.iitb.ernet.in (m madhusoodanan) Subject: article in protein science Message-ID: Date: Sat, 7 Jan 1995 10:38:48 GMT Organization: Computer Centre, Indian Institute of Technology, Bombay X-Newsreader: TIN [version 1.2 PL2] Lines: 16 dear netters, i came across an interesting article in one of the recent issues of the journal Protien Science. unfortunately i do not have access to that journal. is it possible for me to obtain atleast the absract of the article. could someone be kind enough to send me the abstract. i shall give the particulars of the article on hearing from you. thanks in advance sincerely, ramkrishna Ramkrishna research scholar dept. of Chemistry I.I.T., Bombay india From owner-proteins@net.bio.net Sat Jan 07 22:00:00 1995 Path: biosci!agate!howland.reston.ans.net!Germany.EU.net!netmbx.de!zib-berlin.de!informatik.tu-muenchen.de!lrz-muenchen.de!ipp-garching.mpg.de!alf.biochem.mpg.de!krasel From: krasel@alf.biochem.mpg.de (Cornelius Krasel) Newsgroups: bionet.molbio.proteins Subject: Re: article in protein science Date: 8 Jan 1995 13:40:33 GMT Organization: Rechenzentrum der Max-Planck-Gesellschaft in Garching Lines: 21 Message-ID: <3eoq0h$i39@sat.ipp-garching.mpg.de> References: NNTP-Posting-Host: alf.biochem.mpg.de X-Newsreader: TIN [version 1.2 PL2] m madhusoodanan (e9403001@powai.cc.iitb.ernet.in) wrote: > i came across an interesting article in one of the recent issues > of the journal Protien Science. unfortunately i do not have access > to that journal. is it possible for me to obtain atleast the absract > of the article. could someone be kind enough to send me the abstract. The contents and abstracts of Protein Science are posted to the newsgroup bionet.journals.contents which you can search by gophering to net.bio.net. Protein Science has also a WWW server at the URL http://www.prosci.uci.edu/ You can find there a lot of additional useful data. Hope that helps, --Cornelius. -- /* Cornelius Krasel, Abt. Lohse, Genzentrum, D-82152 Martinsried, Germany */ /* email: krasel@alf.biochem.mpg.de fax: +49 89 8578 3795 */ /* "Science is the game you play with God to find out what His rules are." */ From owner-proteins@net.bio.net Sun Jan 08 22:00:00 1995 Path: biosci!FOX.NSTN.NS.CA!ewright From: ewright@FOX.NSTN.NS.CA ("Edwin Wright") Newsgroups: bionet.molbio.proteins Subject: Neutral Alleles Date: 9 Jan 1995 16:32:56 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 16 Sender: daemon@net.bio.net Distribution: world Message-ID: <74035.ewright@fox.nstn.ns.ca> NNTP-Posting-Host: net.bio.net Does anyone know of a program that (a) searches a database for all neutral alleles of a sequence, and (b) predicts all possible neutral alleles of a sequence? Regards, ------- Edwin Wright 85 Spinnaker Drive, A-602 Halifax, Nova Scotia CANADA B3N 3E3 Telephone: (902) 477-5037 Email: ewright@fox.nstn.ns.ca =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= From owner-proteins@net.bio.net Sun Jan 08 22:00:00 1995 Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Path: biosci!bcm!cs.utexas.edu!swrinde!sgiblab!darwin.sura.net!fconvx.ncifcrf.gov!fcsparc6!pnh From: pnh@fcsparc6.ncifcrf.gov (Paul N Hengen) Subject: Re: tris-glycine vs tricine Message-ID: Summary: advantages Sender: Paul N. Hengen Nntp-Posting-Host: fcsparc6.ncifcrf.gov Organization: Frederick Cancer Research and Development Center References: <3es5r2$mm3@neuro.usc.edu> Distribution: bionet Date: Mon, 9 Jan 1995 23:51:30 GMT Lines: 29 Xref: biosci bionet.molbio.methds-reagnts:22728 bionet.molbio.proteins:3397 In article <3es5r2$mm3@neuro.usc.edu> william@neuro.usc.edu (Fiberman) writes: | Does anyone know what are the advantages/disadvantages of tris-glycine | vs. tricine gels for electrophoresis of proteins? The use of tricine allows resolution of proteins in the < 5,000 Dalton range better than glycine. Also, the proteins may be recovered for microsequencing without modification problems. @article{Schagger1987, author = "H. {Sch\"{a}gger} and G. von Jagow", title = "Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 {kDa}", journal = "Anal. Biochem.", volume = "166", pages = "368-379", year = "1987"} ******************************************************************************* * Paul N. Hengen, Ph.D. /--------------------------/* * National Cancer Institute |Internet: pnh@ncifcrf.gov |* * Laboratory of Mathematical Biology | Phone: (301) 846-5581 |* * Frederick Cancer Research and Development Center| FAX: (301) 846-5598 |* * Frederick, Maryland 21702-1201 USA /--------------------------/* * - - - Methods FAQ list -> http://www.ncifcrf.gov:2001/~pnh/info.html - - - * * - - - Anonymous FTP from ftp.ncifcrf.gov as file pub/methods/FAQlist - - - * ******************************************************************************* From owner-proteins@net.bio.net Sun Jan 08 22:00:00 1995 Path: biosci!rutgers!gatech!newsxfer.itd.umich.edu!jobone!lynx.unm.edu!dns1.NMSU.Edu!jubongia From: jubongia@nmsu.edu (Juliet K. Bongianni) Newsgroups: bionet.molbio.proteins Subject: chicken antibodies Date: 9 Jan 1995 17:35:17 GMT Organization: New Mexico State University, Las Cruces, NM Lines: 8 Message-ID: <3ers4l$ftl@dns1.NMSU.Edu> NNTP-Posting-Host: verdi.nmsu.edu X-Newsreader: TIN [version 1.2 PL2] Does anybody know, or has anybody heard of using chickens to produce primary antibodies against an antigen? Apparently the chicken can be injected with the protein of choice, and then the antibodies can be isolated from the egg white?? If anyone knows about this method, or has used it, would you please comment on it and the efficiency of this technique. Thank you. julie From owner-proteins@net.bio.net Sun Jan 08 22:00:00 1995 Path: biosci!ns1.faseb.org!darwin.sura.net!nntp.st.usm.edu!wave.st.usm.edu!rbateman From: rbateman@wave.st.usm.edu (Dr. Robert Bateman) Newsgroups: bionet.molbio.proteins Subject: Re: Microwaves action on milk given to infants Date: 9 Jan 1995 22:43:35 GMT Organization: University of Southern Mississippi Lines: 34 Message-ID: <3ese6n$kmg@server.st.usm.edu> References: <3ekn9k$ctt@rc1.vub.ac.be> <3enq6t$656@jhunix1.hcf.jhu.edu> NNTP-Posting-Host: wave.st.usm.edu X-Newsreader: TIN [version 1.2 PL1] NICHOLAS THEODORAKIS (ntheo@welchlink.welch.jhu.edu) wrote: : In article <3ekn9k$ctt@rc1.vub.ac.be>, : Stephane Corteel wrote: : >As a young father (my baby now is 7 weeks old) I read a lot... : >I've read (and some people also told me) that warwing up a feeding : >bottle using a microwave oven is not recommended because the microwaves : >cause molecular changes in some proteins of the milk. : > : >But, at this time, nobody could confirm that or give me an more : >scientific explanation. : > : >If someone could provide me a answer that an engineer like me can : >accept, it would be very nice. : > : >Thank you very much for any information : As a new father of twin girls (now 3 mo. old), I heard a different, and : more believable reason not to microwave milk or formula (and this reason : would probably appeal to an engineer). Microwave ovens often warm things up : unevenly, and thus might leave "hot spots" in the milk that could could : burn, even if the outside of the container feels the right temperature I microwaved milk for each of my sons and it worked fine. Just use medium power for 1 minute for each 6 oz milk and swirl the bottle a time or two to equilibrate the temperature. Bob Bateman : -- : ++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ : Nick Theodorakis : ntheo@welchlink.welch.jhu.edu : Johns Hopkins Medical School, Baltimore, MD From owner-proteins@net.bio.net Sun Jan 08 22:00:00 1995 Path: biosci!bloom-beacon.mit.edu!gatech!swrinde!howland.reston.ans.net!usc!usc!not-for-mail From: william@neuro.usc.edu (Fiberman) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: tris-glycine vs tricine Date: 9 Jan 1995 12:20:50 -0800 Organization: University of Southern California, Los Angeles, CA Lines: 7 Sender: william@neuro.usc.edu Message-ID: <3es5r2$mm3@neuro.usc.edu> NNTP-Posting-Host: neuro.usc.edu Xref: biosci bionet.molbio.methds-reagnts:22716 bionet.molbio.proteins:3394 Hello, netters, Does anyone know what are the advantages/disadvantages of tris-glycine vs. tricine gels for electrophoresis of proteins? -fm From owner-proteins@net.bio.net Sun Jan 08 22:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!news2.near.net!news.delphi.com!usenet From: Marla Brunker Newsgroups: bionet.molbio.proteins Subject: Re: Microwaves action on milk given to infants Date: Mon, 9 Jan 95 21:01:24 -0500 Organization: Delphi (info@delphi.com email, 800-695-4005 voice) Lines: 14 Message-ID: References: <3ekn9k$ctt@rc1.vub.ac.be> <3enq6t$656@jhunix1.hcf.jhu.edu> <3ese6n$kmg@server.st.usm.edu> NNTP-Posting-Host: bos1b.delphi.com X-To: Dr. Robert Bateman Dr. Robert Bateman writes: >: As a new father of twin girls (now 3 mo. old), I heard a different, and >: more believable reason not to microwave milk or formula (and this reason >: would probably appeal to an engineer). Microwave ovens often warm things up >: unevenly, and thus might leave "hot spots" in the milk that could could >: burn, even if the outside of the container feels the right temperature I heard this one myself when my son was still on bottles (he's 7 now), and thought it was one of the dumber things they tell new mothers...and you do hear dumb things as a new mother. It's a LIQUID, for Pete's sake...if you jostle it at all, the heat equilibrates throughout the bottle. Nuke away. From owner-proteins@net.bio.net Sun Jan 08 22:00:00 1995 Path: biosci!FOX.NSTN.NS.CA!ewright From: ewright@FOX.NSTN.NS.CA ("Edwin Wright") Newsgroups: bionet.molbio.proteins Subject: RE: Protein Sequence Insertions Date: 9 Jan 1995 16:29:07 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 33 Sender: daemon@net.bio.net Distribution: world Message-ID: <73805.ewright@fox.nstn.ns.ca> NNTP-Posting-Host: net.bio.net Does anyone know the extent to which protein sequence insertions have more than one level? For example, given the hypothetical sequence XXXXXXXXXX[YYYYY]XXXXXXXXXX where [YYYYY] represents an insertion, is it possible for the insertion itself to have an insertion in another sequence, and so on, i.e. XXXXXXXXXX[YY[ZZZ]YYY]XXXXXXXXXX where [ZZZ] represents an insertion in the above [YYYYY] insertion? Regards, ------- Edwin Wright Spinnaker Drive, A-602 Halifax, Nova Scotia CANADA B3N 3E3 Telephone: (902) 477-5037 Email: ewright@fox.nstn.ns.ca =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= Edwin Wright 85 Spinnaker Drive, A-602 Halifax, Nova Scotia CANADA B3N 3E3 Telephone: (902) 477-5037 Email: ewright@fox.nstn.ns.ca =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= From owner-proteins@net.bio.net Sun Jan 08 22:00:00 1995 Path: biosci!agate!howland.reston.ans.net!gatech!rutgers!dziuxsolim.rutgers.edu!er6.rutgers.edu!not-for-mail From: alkaiser@er6.rutgers.edu (Alan Kaiser) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: tris-glycine vs tricine Date: 9 Jan 1995 18:55:27 -0500 Organization: Rutgers University Lines: 26 Distribution: world Message-ID: <3esidf$r77@er6.rutgers.edu> References: <3es5r2$mm3@neuro.usc.edu> NNTP-Posting-Host: er6.rutgers.edu Xref: biosci bionet.molbio.methds-reagnts:22733 bionet.molbio.proteins:3401 In <3es5r2$mm3@neuro.usc.edu> william@neuro.usc.edu (Fiberman) writes: >Hello, netters, >Does anyone know what are the advantages/disadvantages of tris-glycine >vs. tricine gels for electrophoresis of proteins? >-fm Tricine gels are used to resolve low MW proteins and peptides. According to something I read from Bio-Rad, the glycine front runs at a position that could interfer with the running of low MW peptides. I think that it formed micells or something like that. With the tricine you don't get this problem. For a good protocol look up the low MW standards from Sigma. These standards are designed to be used with tricine gels and the protocol comes with them (or you could get it separetly from tech service). I use this protocol to resolve a peptide with a MW of 2500 and it works just fine. -- Alan Kaiser alkaiser@eden.rutgers.edu Graduate Program in Microbiology and Molecular Genetics Rutgers University New Brunswick,NJ From owner-proteins@net.bio.net Sun Jan 08 22:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!agate!sunsite.doc.ic.ac.uk!daresbury!trane.uninett.no!sunic!ki.se!micgt.imm.ki.se!user From: Ralf.Morgenstern@imm.ki.se (Ralf Morgenstern) Subject: Re: chicken antibodies Sender: news@ki.se (News admin) Message-ID: Date: Tue, 10 Jan 1995 07:06:01 GMT References: <3ers4l$ftl@dns1.NMSU.Edu> Nntp-Posting-Host: micgt.imm.ki.se Organization: IMM, Karolinska Institutet Followup-To: bionet.molbio.proteins Lines: 24 In article <3ers4l$ftl@dns1.NMSU.Edu>, jubongia@nmsu.edu (Juliet K. Bongianni) wrote: > > Does anybody know, or has anybody heard of using chickens to > produce primary antibodies against an antigen? Apparently the chicken can > be injected with the protein of choice, and then the antibodies can be > isolated from the egg white?? If anyone knows about this method, or has > used it, would you please comment on it and the efficiency of this technique. > Thank you. > julie Dear Julie, I have used this technique which is efficient and convenient. There are also commercial sites that can do this for you (at least in sweden and most likely elsewhere). Immunization is by standard methods (we injected our protein into the breast muscle as with conventional rabbit protocols and Freunds adjuvant). The IgG is isolated from egg YOLK. A good reference is Jensenius et. al. (1981) Journal of Immunological Methods, 46, 63-68. Good luck, -- Ralf Morgenstern, IMM, Karolinska Institutet, Box 210, S-17177, SWEDEN Tlf. +46-8-7287574, Fax, +46-8-334467, E-mail: ralf.morgenstern@imm.ki.se From owner-proteins@net.bio.net Mon Jan 09 22:00:00 1995 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!agate!dog.ee.lbl.gov!news.cs.utah.edu!cs.utexas.edu!howland.reston.ans.net!spool.mu.edu!uwm.edu!msunews!harbinger.cc.monash.edu.au!bunyip.cc.uq.oz.au!news From: J.Marcus@tpp.uq.oz.au (John Marcus) Newsgroups: bionet.molbio.proteins Subject: Re: TCA-precipitation? Date: 11 Jan 1995 03:24:21 GMT Organization: CRC for Tropical Plant Pathology Lines: 29 Message-ID: <3evj15$3an@dingo.cc.uq.oz.au> References: <3euc3i$3ib@sunserver.lrz-muenchen.de> NNTP-Posting-Host: biglab1.tpp.uq.oz.au X-Newsreader: WinVN 0.92.6+ In article <3euc3i$3ib@sunserver.lrz-muenchen.de>, u7q11aa@sunmail.lrz-muenchen.de (Peter Alliger) says: > >I am looking for good method to precipitate a highly purified protein >before >analysing it on a SDS-PAGE. >The fraction has about 1M KCl and should be concentrated from 500 ul to >about 20. > >Thanks for suggestions > >PIT > Have you thought about using a Centricon or Microcon centrifugal filtration device. Essentially, you put your protein sample into the microcon (~500ul capacity) and then spin in a microcentrifuge for 1 min to 30 min (depending on the molecular weight cutoff of the filter that you are using at the time). I think Amicon may have molecular weight cuttoffs as low as 1000Da. When you are done spinning you should be left with about 10ul volume if you use a microcon; although, this holdup volume is not very predictable. Alternatively, you could desalt your sample with the microcon; recover it and then proceed to dry your sample with a stream of nitrogen or else a speedi-vac hooked up to a vacuum pump. I hope that is helpful. Regards, John Marcus From owner-proteins@net.bio.net Mon Jan 09 22:00:00 1995 Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!spool.mu.edu!torn!utnut!utgpu!japhale From: japhale@gpu.utcc.utoronto.ca (J. Aphale) Subject: Re: tris-glycine vs tricine Message-ID: Organization: UTCC Public Access References: <3es5r2$mm3@neuro.usc.edu> Date: Tue, 10 Jan 1995 18:08:09 GMT Lines: 5 Xref: biosci bionet.molbio.methds-reagnts:22763 bionet.molbio.proteins:3409 Yes. Tricine gels are MUCH better at resolving protein (peptides) in the 1 kD to 10 kD range! Cheers. Jayant japhale@utcc.utoronto.ca From owner-proteins@net.bio.net Mon Jan 09 22:00:00 1995 Path: biosci!mani.unet.umn.edu!npv From: npv@mani.unet.umn.edu (Nora Plesofsky-Vig) Newsgroups: bionet.molbio.proteins Subject: re:chicken antibodies Date: 10 Jan 1995 11:30:08 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 23 Sender: daemon@net.bio.net Distribution: world Message-ID: <9501101715.AA00652@mani.cbs.umn.edu> Reply-To: nora@molbio.cbs.umn.edu NNTP-Posting-Host: net.bio.net I made antibodies in chickens and have isolated the IgGs from egg yolks, but I was not entirely pleased with the results. I had high background reactivity of the pre-immune yolk IgGs from the three chickens I used, and it was therefore absolutely essential to include a control reaction with the pre-immune IgGs in all experiments. However, this problem may depend on the conditions under which the soon to be immunized chickens are kept, and an affinity isolation of the specific IgGs should get around the problem. Also, if you are considering immunoprecipitation with the antibodies, you should be aware that chicken IgGs do not react with protein A or protein G, which therefore cannot be used to amplify the immunoprecipitation (unless you use an anti-chicken IgG antibody from mammals first, as I did). However, there are now other tools on the market that may get around this problem. Promega (Madison, Wisc) offers an immobilized anti-chicken IgY (IgG) for immunoprecipitation, which may be useful. Good luck. Nora Plesofsky-Vig nora@molbio.cbs.umn.edu From owner-proteins@net.bio.net Mon Jan 09 22:00:00 1995 Path: biosci!bloom-beacon.mit.edu!news2.near.net!howland.reston.ans.net!vixen.cso.uiuc.edu!ux1.cso.uiuc.edu!elarson From: elarson@ux1.cso.uiuc.edu (larson eric) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: tris-glycine vs tricine Date: 10 Jan 1995 15:21:51 GMT Organization: University of Illinois at Urbana Lines: 34 Distribution: bionet Message-ID: <3eu8mf$4r7@vixen.cso.uiuc.edu> References: <3es5r2$mm3@neuro.usc.edu> NNTP-Posting-Host: ux1.cso.uiuc.edu Xref: biosci bionet.molbio.methds-reagnts:22749 bionet.molbio.proteins:3406 pnh@fcsparc6.ncifcrf.gov (Paul N Hengen) writes: >| Does anyone know what are the advantages/disadvantages of tris-glycine >| vs. tricine gels for electrophoresis of proteins? >The use of tricine allows resolution of proteins in the < 5,000 Dalton >range better than glycine. Also, the proteins may be recovered for >microsequencing without modification problems. >author = "H. {Sch\"{a}gger} > and G. von Jagow", >title = "Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis >for the separation of proteins in the range from 1 to 100 {kDa}", >journal = "Anal. Biochem.", >volume = "166", >pages = "368-379", >year = "1987"} ========= Okajima, T., T. Tanage, and T. Yasuda (1993) Anal. Biochem 211: 293-300 Has a procedure adapted from Laemmli that allows for small mol. wt. peptide separation. The technique works (though I had to change to 16% gels from the 20% the authors used.) Basically, it's a Laemmli gel with 2X higher concentrations of buffer in stacking and running gel. This makes the procedure "simple" in that two sets of buffer stocks don't need to be around. I haven't tried the gels with normal proteins (because the % acrylamide is too high.) -- Eric Larson | University of Illinois at Urbana-Champaign USDA/Agronomy | 190 PABL; 1201 W. Gregory; Urbana, IL 61801 elarson@ux1.cso.uiuc.edu | Voice 217.244.3079 Fax 217.244.4419 Fidonet: 1:233/4.1 | My opinions are my own, but correct :-) From owner-proteins@net.bio.net Mon Jan 09 22:00:00 1995 Path: biosci!agate!howland.reston.ans.net!pipex!sunsite.doc.ic.ac.uk!daresbury!bioftp.unibas.ch!rc1.vub.ac.be!ensor!dimitri From: dimitri@ensor.ulb.ac.be (Dimitri Gilis) Newsgroups: bionet.molbio.proteins Subject: fractal and proteins Date: 10 Jan 1995 10:50:25 GMT Organization: UCMB - ULB Lines: 16 Sender: dimitri@ensor (Dimitri Gilis) Distribution: world Message-ID: <3etoph$7it@rc1.vub.ac.be> NNTP-Posting-Host: ucmbsgi6.ulb.ac.be. Hello, Does somebody have informations, references.... about application of fractal dimension to polymers and/or proteins? -- ================================================================= Dimitri Gilis Unite de Conformation des Macromolecules Biologiques ULB (Belgium) tel: +32 2 648 52 00 fax: +32 2 648 89 54 e-mail : dimitri@ucmb.ulb.ac.be ================================================================= From owner-proteins@net.bio.net Mon Jan 09 22:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!bloom-beacon.mit.edu!uhog.mit.edu!europa.eng.gtefsd.com!newsxfer.itd.umich.edu!gatech!swrinde!pipex!news.oleane.net!oleane!jussieu.fr!univ-lyon1.fr!swidir.switch.ch!scsing.switch.ch!rzusuntk.unizh.ch!NewsWatcher!user From: maga@vetbio.unizh.ch (Giovanni Maga) Subject: Re: chicken antibodies Message-ID: Followup-To: bionet.molbio.proteins Sender: newsadm@rzu-news.unizh.ch (CNEWS ADMINISTRATION) Organization: University of Zurich Irchel- Biochemistry References: <3ers4l$ftl@dns1.NMSU.Edu> Date: Tue, 10 Jan 1995 14:52:49 GMT Lines: 28 In article <3ers4l$ftl@dns1.NMSU.Edu>, jubongia@nmsu.edu (Juliet K. Bongianni) wrote: > > Does anybody know, or has anybody heard of using chickens to > produce primary antibodies against an antigen? Apparently the chicken can > be injected with the protein of choice, and then the antibodies can be > isolated from the egg white?? If anyone knows about this method, or has > used it, would you please comment on it and the efficiency of this technique. > Thank you. > julie Dear Julie, we are using chikens and they work fine (all they have to do is to lay eggs, indeed). The tecnique is very fast and simple. You can inject with your antigen according to your protocol of choice and then recover your Abs from the egg's YOLK (yellow part, not white) simply by PEG 6000 extractions. Here it is a reference of something done by our lab fey years ago. If you have problems in getting the paper or if you need some hints drop me an e-mail. Gassmann, M., Thoemmes, P., Weiser, T. and Huebscher, U. FASEB Journal, 4, 2528-2532 (1990). Have fun with your hens (and remember, if you want to make a cake take only pre-immune eggs). G. Maga, PhD Biochemistry University of Zuerich (CH) e-mail: maga@vetbio.unizh.ch From owner-proteins@net.bio.net Mon Jan 09 22:00:00 1995 Path: biosci!agate!howland.reston.ans.net!ee.und.ac.za!nntp.und.ac.za!agpc049.cc.unp.ac.za!Morty From: Morty@bchm.unp.ac.za (Rory.Morty) Newsgroups: bionet.molbio.proteins Subject: Stability of Okadaic Acid & Calyculin A Date: Tue, 10 Jan 1995 08:23:19 GMT Organization: University of Natal , Pietermaritzburg, South Africa Lines: 9 Message-ID: NNTP-Posting-Host: agpc049.cc.unp.ac.za Does anybody know how stable the protein phosphatase inhibitors okadaic acid, calyculin A and tautomycin are, once made up in solution. How long do they retain their inhibitory activity, how to store them, etc? Many thank's in advance Rory Morty morty@biochem.unp.ac.za From owner-proteins@net.bio.net Mon Jan 09 22:00:00 1995 Path: biosci!agate!spool.mu.edu!bloom-beacon.mit.edu!gatech!swrinde!pipex!news.oleane.net!oleane!jussieu.fr!univ-lyon1.fr!swidir.switch.ch!scsing.switch.ch!news.dfn.de!zeus.rbi.informatik.uni-frankfurt.de!terra.wiwi.uni-frankfurt.de!news.th-darmstadt.de!zib-berlin.de!informatik.tu-muenchen.de!lrz-muenchen.de!usenet From: u7q11aa@sunmail.lrz-muenchen.de (Peter Alliger) Newsgroups: bionet.molbio.proteins Subject: TCA-precipitation? Date: 10 Jan 1995 16:20:02 GMT Organization: Institute for Immunology, Munich Lines: 21 Sender: u7q11aa@sunserver-et.lrz-muenchen.de Distribution: world Message-ID: <3euc3i$3ib@sunserver.lrz-muenchen.de> NNTP-Posting-Host: kine.ifi.med.uni-muenchen.de X-Posted-From: InterNews 1.0.5@kine.ifi.med.uni-muenchen.de X-Authenticated: u7q11aa on POP host sunserver-et.lrz-muenchen.de I am looking for good method to precipitate a highly purified protein before analysing it on a SDS-PAGE. The fraction has about 1M KCl and should be concentrated from 500 ul to about 20. Thanks for suggestions PIT =============================================================== Peter Alliger, Ph.D. Institute for Immunology home address: University of Munich Goethestr. 31 Maximilian-Wetzger-Str.7 D-80336 Munich, Germany D-80636 Munich, Germany Phone:(49-89)5996-673 (49-89)183426 Fax: (49-89)5160-2236 ---------------------------------------------------------------- From owner-proteins@net.bio.net Mon Jan 09 22:00:00 1995 Path: biosci!ROCKVAX.ROCKEFELLER.EDU!cheethj From: cheethj@ROCKVAX.ROCKEFELLER.EDU (Jim Cheetham) Newsgroups: bionet.molbio.proteins Subject: Re: Stability of Okadaic Acid & Calyculin A Date: 10 Jan 1995 07:41:55 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 26 Sender: daemon@net.bio.net Distribution: world Message-ID: <199501101541.KAA00623@rockvax.rockefeller.edu> NNTP-Posting-Host: net.bio.net >Does anybody know how stable the protein phosphatase inhibitors okadaic >acid, calyculin A and tautomycin are, once made up in solution. How long do >they retain their inhibitory activity, how to store them, etc? > >Many thank's in advance > > >Rory Morty >morty@biochem.unp.ac.za While we have not measured this parameter extensively, I would set an upper limit for stabilility of these phosphatase inhibitors in aqueous solution of 24 hours. It is probably better to use these compounds as soon as possible after making up the solution. Jim James J. Cheetham, Ph.D. Laboratory of Molecular and Cellular Neuroscience The Rockefeller University 1230 York Ave New York, NY 10021 USA cheethj@Rockvax.Rockefeller.EDU From owner-proteins@net.bio.net Mon Jan 09 22:00:00 1995 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!scripps.edu!usenet From: mp@scripps.edu (Mike Pique) Newsgroups: bionet.molbio.proteins Subject: Re: fractal and proteins Date: 11 Jan 1995 04:32:28 GMT Organization: The Scripps Research Institute, La Jolla, CA USA Lines: 16 Message-ID: <3evn0s$of4@riscsm.scripps.edu> References: <3etoph$7it@rc1.vub.ac.be> Reply-To: mp@scripps.edu NNTP-Posting-Host: riscfs2.scripps.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit Summary: See JMB (1992) 228, 13-22 Kuhn et al. Keywords: fractal, protein, surface See our article in Journal of Molecular Biology, 5 November 1992, 228, 13-22, "The interdependence of protein surface topography and bound water molecules revealed by surface accessibility and fractal density measures", by Leslie Kuhn, Michael Siani, Michael Pique, Cindy Fisher, Elizabeth Getzoff, and John Tainer, and the references therein. All the software of ours mentioned there is now available for free, sources included, from the Computational Center for Macromolecular Structure, ccms-help@sdsc.edu. - Mike Pique From owner-proteins@net.bio.net Mon Jan 09 22:00:00 1995 Path: biosci!agate!library.ucla.edu!europa.eng.gtefsd.com!gatech!howland.reston.ans.net!news.moneng.mei.com!uwm.edu!lll-winken.llnl.gov!enews.sgi.com!decwrl!tribune.usask.ca!quartz.ucs.ualberta.ca!unixg.ubc.ca!port48.annex4.net.ubc.ca!user From: lomas@unixg.ubc.ca (Cyprien Lomas) Newsgroups: bionet.molbio.proteins Subject: s-s bonds in cytoplasm? Date: Tue, 10 Jan 1995 21:34:40 -0800 Organization: The University of British Columbia Lines: 3 Message-ID: NNTP-Posting-Host: port48.annex4.net.ubc.ca Can anyone give me a reference for disulphide bonds found in the cytoplasmic domain of a membrane protein? I thought that they only existed in the ER lumenal part of proteins. From owner-proteins@net.bio.net Mon Jan 09 22:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!agate!sunsite.doc.ic.ac.uk!uknet!festival!leeds.ac.uk!news From: mphcw@gps.leeds.ac.uk (C Webster) Subject: Crossed Immunoelectrophoresis Originator: c.webster@leeds.ac.uk Message-ID: <1995Jan10.135459.13249@leeds.ac.uk> Sender: news@leeds.ac.uk Organization: University of Leeds, England Date: Tue, 10 Jan 1995 13:54:58 +0000 (GMT) Lines: 19 Does anyone have experience of using crossed immunoelectrophoresis for the study of glycoforms of protein ? I am currently working on the microheterogenity of acid glycoprotein using concanvalin A in the first dimension but having problems in getting the method to work. I think my problem is the first dimension i.e. with the con A. Does anyone have a method for using con A in CIE with regard to buffers etc. I would appreciate any help on this matter. TIA -- Mr Craig Webster, Department of Chemical Pathology and Immunology The Old Medical School, Thoresby Place, Leeds, LS2 9JT Tel:- 0532 923285 email:- craigw@pathology.leeds.ac.uk Fax:- 0532 335672 mphcw@gps.leeds.ac.uk From owner-proteins@net.bio.net Tue Jan 10 22:00:00 1995 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!library.ucla.edu!agate!howland.reston.ans.net!vixen.cso.uiuc.edu!ux1.cso.uiuc.edu!elarson From: elarson@ux1.cso.uiuc.edu (larson eric) Newsgroups: bionet.molbio.proteins Subject: Re: TCA-precipitation? Date: 11 Jan 1995 14:17:32 GMT Organization: University of Illinois at Urbana Lines: 30 Message-ID: <3f0p9s$p2g@vixen.cso.uiuc.edu> References: <3euc3i$3ib@sunserver.lrz-muenchen.de> <3evj15$3an@dingo.cc.uq.oz.au> NNTP-Posting-Host: ux1.cso.uiuc.edu J.Marcus@tpp.uq.oz.au (John Marcus) writes: >>I am looking for good method to precipitate a highly purified protein >>before >>analysing it on a SDS-PAGE. >>The fraction has about 1M KCl and should be concentrated from 500 ul to >>about 20. >> >>Thanks for suggestions >> >>PIT >> >Have you thought about using a Centricon or Microcon centrifugal >filtration device. Essentially, you put your protein sample into >the microcon (~500ul capacity) and then spin in a microcentrifuge >for 1 min to 30 min (depending on the molecular weight cutoff of >the filter that you are using at the time). I think Amicon may >have molecular weight cuttoffs as low as 1000Da. When you are done >spinning you should be left with about 10ul volume if you use a >microcon; although, this holdup volume is not very predictable. ============ An excellent suggestion, especially since the KCl will have to be removed or reduced to prevent precipitation of the SDS. -- Eric Larson | University of Illinois at Urbana-Champaign USDA/Agronomy | 190 PABL; 1201 W. Gregory; Urbana, IL 61801 elarson@ux1.cso.uiuc.edu | Voice 217.244.3079 Fax 217.244.4419 Fidonet: 1:233/4.1 | My opinions are my own, but correct :-) From owner-proteins@net.bio.net Tue Jan 10 22:00:00 1995 Path: biosci!CS.Arizona.EDU!news.Arizona.EDU!val.AgShantz.Arizona.EDU!valeryt From: valeryt@ag.arizona.edu (Valery F. Thompson) Newsgroups: bionet.molbio.proteins Subject: re: chicken antibodies Date: Wed, 11 Jan 1995 16:37:17 Organization: Muscle Biology Group, University of Arizona Lines: 6 Message-ID: NNTP-Posting-Host: val.agshantz.arizona.edu X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] This doesn't apply to a lot of people, but my experience with chicken antibodies is that chickens don't lay many eggs during the summer in Arizona. It's just too hot for them. Ours were housed in a facility that used only evaporative cooling which doesn't work too well during the humid months of July and August. It's just something to consider if you happen to be in an area where weather conditions are similar to Tucson. From owner-proteins@net.bio.net Tue Jan 10 22:00:00 1995 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!scripps.edu!NewsWatcher!user From: Bibbs@scripps.edu (Lisa Bibbs) Newsgroups: bionet.molbio.proteins Subject: Heat Shock Proteins Date: 11 Jan 1995 21:24:38 GMT Organization: The Scripps Research Institute, La Jolla, CA Lines: 10 Message-ID: NNTP-Posting-Host: pbfnuts.scripps.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit I would like to hear from people who have had experiences with heat shock protein. As I understand it, it binds to Ig heavy chains. That would seem to make it light up on westerns. Does anyone have info for me? Thanks in advance. Lisa Bibbs Bibbs@scripps.edu -- These opinions are mine, not my employers. From owner-proteins@net.bio.net Tue Jan 10 22:00:00 1995 Path: biosci!ARSERRC.GOV!CTHOMPSON From: CTHOMPSON@ARSERRC.GOV Newsgroups: bionet.molbio.proteins Subject: FTIR (Collagen) Date: 11 Jan 1995 12:58:51 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 11 Sender: daemon@net.bio.net Distribution: world Message-ID: <01HLQ4VRSUB6000RHO@arserrc.gov> NNTP-Posting-Host: net.bio.net Hello, I am working with Type I and Type III collagen and gelable products from them. I was wondering if anyone could help me with a peak that shows up in the FTIR scan between 1030 and 1050 1/cm for the gelable byproducts but not does not show up for the original collagen I and III samples. Thanks in advance, An Inorganic Chemist Craig Thompson CTHOMPSON@ARSERRC.GOV From owner-proteins@net.bio.net Tue Jan 10 22:00:00 1995 Path: biosci!agate!sunsite.doc.ic.ac.uk!daresbury!bioftp.unibas.ch!rc1.vub.ac.be!ensor!dimitri From: dimitri@ensor.ulb.ac.be (Dimitri Gilis) Newsgroups: bionet.molbio.proteins Subject: Re: Combinatorial help Date: 12 Jan 1995 07:03:46 GMT Organization: UCMB - ULB Lines: 16 Sender: dimitri@ensor (Dimitri Gilis) Distribution: world Message-ID: <3f2k8i$agd@rc1.vub.ac.be> NNTP-Posting-Host: ucmbsgi6.ulb.ac.be. Maybe it is the fact that one amino acid is coded by three DNA bases. Furthermore, DNA code is degenerated (more than one DNA base triplet corresponds to one amino acid). Hope this help... -- ================================================================= Dimitri Gilis Unite de Conformation des Macromolecules Biologiques ULB (Belgium) tel: +32 2 648 52 00 fax: +32 2 648 89 54 e-mail : dimitri@ucmb.ulb.ac.be ================================================================= From owner-proteins@net.bio.net Tue Jan 10 22:00:00 1995 Path: biosci!agate!news.duke.edu!convex!cs.utexas.edu!howland.reston.ans.net!spool.mu.edu!uwm.edu!lll-winken.llnl.gov!enews.sgi.com!decwrl!tribune.usask.ca!canopus.cc.umanitoba.ca!zahradka2.sbrc.umanitoba.ca!davidw From: davidw@sbrc.umanitoba.ca (David Wilson) Newsgroups: bionet.molbio.proteins Subject: Re: milk microwave Date: Wed, 11 Jan 1995 22:52:44 Organization: St. Boniface Hospital Research Centre Lines: 4 Message-ID: NNTP-Posting-Host: zahradka2.sbrc.umanitoba.ca X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] I would suggest caution, none of the respondents to your query have given you a sound scientific answer. Old and new Fathers/wives tails are the reason the question was asked. Keep reading the mail, I won't tell you my stories since they have little scientific merit either. From owner-proteins@net.bio.net Tue Jan 10 22:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!agate!garnet.berkeley.edu!owens From: owens@garnet.berkeley.edu () Newsgroups: bionet.molbio.proteins Subject: xl amino acid data? Date: 12 Jan 1995 00:08:52 GMT Organization: University of California, Berkeley Lines: 13 Message-ID: <3f1ruk$asq@agate.berkeley.edu> NNTP-Posting-Host: garnet.berkeley.edu I just now realized that this question isn't about proteins per se, but here goes anyway: does anyone know of references/data about crystalline amino acids? specifically, their cell parameters and/or crystal types? thanks in advance for any help you can give me! christine owens dept. of geology uc berkeley From owner-proteins@net.bio.net Tue Jan 10 22:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!rutgers!gatech!newsfeed.pitt.edu!uunet!emba-news.uvm.edu!hender From: hender@med.uvm.edu (Shaw Henderson) Subject: bionet.molbio.proteins Message-ID: <1995Jan11.162407.6071@emba.uvm.edu> Sender: news@emba.uvm.edu Organization: EMBA Computer Facility, University of Vermont X-Newsreader: TIN [version 1.2 PL1] Date: Wed, 11 Jan 1995 16:24:07 GMT Lines: 2 test From owner-proteins@net.bio.net Tue Jan 10 22:00:00 1995 Path: biosci!daresbury!sunsite.doc.ic.ac.uk!yama.mcc.ac.uk!io.salford.ac.uk!usenet From: R.Lauder@lancaster.ac.uk (Bob Lauder) Newsgroups: bionet.molbio.proteins Subject: Collagens - esp FACIT Date: 11 Jan 1995 18:23:12 GMT Organization: Lancaster University Lines: 22 Message-ID: <3f17mg$kir@io.salford.ac.uk> NNTP-Posting-Host: bsa046000001.lancs.ac.uk X-Newsreader: WinVN 0.92.6 Hi, I'd like to make contact with anybody reading this who works in the collagen field. My main area of interest is connective tissue proteoglycans, and I'm hoping to do some work with collagens. So, I'd very much like to pose a few questions concerning the FACIT collagens. If anybody has experience of isolating collagen types IX, XII or XIV I'd be grateful if you could give me some idea of abundance in various tissues. Also how did you perform the isolation? Is anybody doing any work examining the interaction of these FACIT collagens with other ECM components? Finaly, if anybody is willing to give me some antibodies to type XIV I'd be delighted. (Unlikley but I thought I'd ask anyway). Thanks in advance Bob. ================================================================ Bob Lauder R.Lauder@lancaster.ac.uk Postdoctoral Research Asscociate Tel +(44)(0)1524 65201 Division of Biological Sciences Fax +(44)(0)1524 843854 Lancaster University, Lancaster, UK From owner-proteins@net.bio.net Wed Jan 11 22:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!CS.Arizona.EDU!news.Arizona.EDU!hamblin.math.byu.edu!sol.ctr.columbia.edu!news.moneng.mei.com!uwm.edu!vixen.cso.uiuc.edu!howland.reston.ans.net!gatech!newsxfer.itd.umich.edu!nntp.cs.ubc.ca!news.UVic.CA!spruce.pfc.forestry.ca!PFC.Forestry.CA!AEKRAMODDOUL From: aekramoddoul@PFC.Forestry.CA (Ekramoddoullah, Abul Kalam M.) Subject: Re: TCA-precipitation? Message-ID: <1995Jan12.001034.13211@spruce.pfc.forestry.ca> Sender: news@spruce.pfc.forestry.ca Nntp-Posting-Host: pfc.pfc.forestry.ca Reply-To: aekramoddoul@PFC.Forestry.CA Organization: Forestry Canada (Pacific Forestry Centre) References: <3euc3i$3ib@sunserver.lrz-muenchen.de> Date: Thu, 12 Jan 1995 00:10:34 GMT Lines: 28 In article <3euc3i$3ib@sunserver.lrz-muenchen.de>, u7q11aa@sunmail.lrz-muenchen.de (Peter Alliger) writes: >I am looking for good method to precipitate a highly purified protein >before >analysing it on a SDS-PAGE. >The fraction has about 1M KCl and should be concentrated from 500 ul to >about 20. > >Thanks for suggestions > >PIT > >=============================================================== >Peter Alliger, Ph.D. >Institute for Immunology home address: >University of Munich >Goethestr. 31 Maximilian-Wetzger-Str.7 >D-80336 Munich, Germany D-80636 Munich, Germany > >Phone:(49-89)5996-673 (49-89)183426 >Fax: (49-89)5160-2236 >---------------------------------------------------------------- > Why not prcipitate with 8X volume of cold acetone (at -20C) for a 1 h and redissolve the ppt in sample buffer required for SDS-PAGE. Good Luck. Abul EKRAMODDOULLAH, ABUL KALAM M. Title: Research Scientist Forestry Canada Phone: (604) 363-0600 Victoria, B.C. Internet: AEKRAMODDOUL@A1.PFC.Forestry.CA From owner-proteins@net.bio.net Wed Jan 11 22:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!pipex!news.oleane.net!oleane!jussieu.fr!univ-lyon1.fr!swidir.switch.ch!scsing.switch.ch!rzusuntk.unizh.ch!NewsWatcher!user From: maga@vetbio.unizh.ch (Giovanni Maga) Subject: Hill plot Message-ID: Followup-To: bionet.molbio.proteins Sender: newsadm@rzu-news.unizh.ch (CNEWS ADMINISTRATION) Organization: University of Zurich Irchel- Biochemistry Date: Thu, 12 Jan 1995 22:01:38 GMT Lines: 15 From G. Maga, Biochemistry University Zuerich Zuerich (CH) e-mail: maga@vetbio.unizh.ch I have a question about the use of Hill plot as a test for cooperativity. Given that you collect your experimental data (in my case initial reaction velocities at different substrate concentrations), if you obtain a non Mich.-Menten curve, but a sigmoidal one, can you use Hill plot as a test for real cooperativity? Or it will give you a h coeff. different from 1 even in those cases in which the non-linearity has nothing to do with cooperativity (for example if your enzyme has alternative pathways for binding two molecules of substrate with no irreversible steps in between)? And if so, how can I convincingly prove (or disprove) cooperativity? Thanks in advance. From owner-proteins@net.bio.net Wed Jan 11 22:00:00 1995 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!agate!library.ucla.edu!europa.eng.gtefsd.com!newsxfer.itd.umich.edu!news.itd.umich.edu!usenet From: Rick Neubig Newsgroups: bionet.molbio.proteins Subject: Re: Hill plot Date: 12 Jan 1995 18:14:30 GMT Organization: University of Michigan Lines: 18 Message-ID: <3f3ri6$d4n@lastactionhero.rs.itd.umich.edu> References: NNTP-Posting-Host: warbler.med.umich.edu maga@vetbio.unizh.ch (Giovanni Maga) wrote: stuff deleted > Can you use Hill plot as a test > for real cooperativity? Or it will give you a h coeff. different from 1 > even in those cases in which the non-linearity has nothing to do with > cooperativity (for example if your enzyme has alternative pathways for > binding two molecules of substrate with no irreversible steps in between)? > And if so, how can I convincingly prove (or disprove) cooperativity? You are absolutely right that a sigmoidal plot will give a Hill slope greater than 1 whether you have positive cooperativity or some other mechanism or artifact causing the sigmoid nature. A bit more information about the experimental design might help. Rick Neubig http://www.umich.edu/~rneubig From owner-proteins@net.bio.net Wed Jan 11 22:00:00 1995 Path: biosci!DXI.NIH.GOV!lmarekov From: lmarekov@DXI.NIH.GOV Newsgroups: bionet.molbio.proteins Subject: Derivatizad PVDF membranes Date: 12 Jan 1995 13:42:25 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 9 Sender: daemon@net.bio.net Distribution: world Message-ID: <9501122142.AA26479@dxi.nih.gov> NNTP-Posting-Host: net.bio.net Does anyone know the chemistry for derivatizing PVDF membranes to Arylamino-PVDF for sequencing purposes? Millipore used to sell them as Sequelon-AA, but they have been out of stock for more than two months. Lyuben Marekov NIH/NIAMS, Bethesda MD 20892 lmarekov@dxi.nih.gov From owner-proteins@net.bio.net Wed Jan 11 22:00:00 1995 Path: biosci!bcm!cs.utexas.edu!sdd.hp.com!news.cs.indiana.edu!purdue!news.bu.edu!rmandel From: rmandel@bu.edu (Richard Mandel) Newsgroups: bionet.molbio.proteins Subject: Immunofluores and cell permeability Date: 12 Jan 1995 20:36:27 GMT Organization: Boston University Lines: 38 Message-ID: <3f43sb$32b@news.bu.edu> NNTP-Posting-Host: acs.bu.edu X-Newsreader: TIN [version 1.2 PL0] [ Article crossposted from bionet.molbio.methds-reagnts ] [ Author was ] [ Posted on 12 Jan 1995 20:33:20 GMT ] We are trying to detect a protein on the cell surface while the protein is mostly found intracellularly. We are fixing with 4% paraformaldehyde but find that there is some permeabilization of the cells. Has anyone found washing and fixing conditions that cause very minimal damage to the cell membrane? Does glutaraldehyde or formaldehyde leave a more intact cell membrane than paraformaldehyde? Should washing be done with PBS or BSS? The cells are Chinese hamster ovary cells attached to a slide. What is the minimum % of paraformaldehyde which can be used in fixation and will this improve the situation? Thanks for your input. E-mail or post here. -- Ciao, Rich --------------------------------------------------------------------------- Richard Mandel | E-mail address: rmandel@acs.bu.edu Boston Univ. School of Medicine | Phone No. 617-638-4512 Boston, MA 02118 | Fax No. 617-638-4085 ----------------------------------------------------------------------------- -- Ciao, Rich --------------------------------------------------------------------------- Richard Mandel | E-mail address: rmandel@acs.bu.edu Boston Univ. School of Medicine | Phone No. 617-638-4512 Boston, MA 02118 | Fax No. 617-638-4085 ----------------------------------------------------------------------------- From owner-proteins@net.bio.net Wed Jan 11 22:00:00 1995 Path: biosci!JASON.UTHCT.EDU!SHAUN From: SHAUN@JASON.UTHCT.EDU ("Shaun D. Black") Newsgroups: bionet.molbio.proteins Subject: RE: s-s bonds in cytoplasm? Date: 12 Jan 1995 06:43:42 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 11 Sender: daemon@net.bio.net Distribution: world Message-ID: <950112085033.489a@jason.uthct.edu> As far as I know, disulfides are functions of secreted, extracellular proteins. (or, of course, within the ER during sorting). However, some cytoplasmic proteins do have disulfides, but such are not aimed at structural rigidity. Rather, these are used in redox reactions. Two examples that I know of are the active sites of thioredoxin and glutathione reductase. I hope this helps. Bye for now. -Shaun =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= = Shaun D. Black, PhD | Internet address: shaun@jason.uthct.edu = = Dept. of Biochemistry | University of Texas Health Center, at Tyler = =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= From owner-proteins@net.bio.net Wed Jan 11 22:00:00 1995 Path: biosci!JASON.UTHCT.EDU!SHAUN From: SHAUN@JASON.UTHCT.EDU ("Shaun D. Black") Newsgroups: bionet.molbio.proteins Subject: RE: xl amino acid parameters Date: 12 Jan 1995 06:32:39 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 17 Sender: daemon@net.bio.net Distribution: world Message-ID: <950112083930.489a@jason.uthct.edu> Hi all, Christine Owens asked about crystal parameters for amino acids. To the best of my knowledge, the most complete info available in that regard is in the Cambridge Structural Database (CSD). It is not a public database (i.e., by subscription only), but most campuses (and probably UC Berkeley) will have at least one subscription; check with your chemistry department, for example. But, if you'd like info on the CSD, let me know and I'll post more; also, there is a World Wide Web amino acid server that has a reasonable collection of data that I'd be glad to post the reference to if there is any interest. Bye for now. -Shaun =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= = Shaun D. Black, PhD | Internet address: shaun@jason.uthct.edu = = Dept. of Biochemistry | University of Texas Health Center, at Tyler = =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= From owner-proteins@net.bio.net Wed Jan 11 22:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!gatech!newsfeed.pitt.edu!uunet!world!news.mtholyoke.edu!news.umass.edu!gauss.ummed.edu!umassmed.UMMED.EDU!achern From: achern@umassmed.UMMED.EDU (Andrew Cherniack) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Ras antibodies Date: 12 Jan 1995 20:48:23 GMT Organization: University of Massachusetts Medical Center, Worcester Lines: 19 Distribution: world Message-ID: <3f44in$vk@gauss.ummed.edu> NNTP-Posting-Host: hcx9.ummed.edu Xref: biosci bionet.molbio.methds-reagnts:22862 bionet.molbio.proteins:3435 We are looking for antibodies that can distinguish between the various Ras forms (H-, K-, N- and R-Ras). Does anybody know who might have such antibodies ? Thanks Jes K. Klarlund jklarlun@umassmed.ummed.edu Program in Molecular Medicine U Mass Medical Center Worcester MA 01605 From owner-proteins@net.bio.net Wed Jan 11 22:00:00 1995 Path: biosci!CMSA.BERKELEY.EDU!RWG%SHCC.BITNET From: RWG%SHCC.BITNET@CMSA.BERKELEY.EDU Newsgroups: bionet.molbio.proteins Subject: TWO HYBRID SYSTEM Date: 12 Jan 1995 16:24:37 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 11 Sender: daemon@net.bio.net Distribution: world Message-ID: <01HLRKBI779S8WW37K@SHCC.BITNET> NNTP-Posting-Host: net.bio.net Is anyone using a yeast two-hybrid interaction system (Clontech or other) to study the interactions of extracellular matrix proteins or protein domains? If so I would like to hear from you about your experiences. We have been trying without much success and I was wondering whether there is some specific reason why matrix proteins are just not good candidates for this analytical system. Robert Glanville Shriners Hospital Portland, OR From owner-proteins@net.bio.net Wed Jan 11 22:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!europa.eng.gtefsd.com!news.umbc.edu!haven.umd.edu!purdue!lerc.nasa.gov!magnus.acs.ohio-state.edu!mwadhwa From: mwadhwa@magnus.acs.ohio-state.edu (Manpreet S Wadhwa) Newsgroups: bionet.molbio.proteins Subject: peptide helicity at different pH values Date: 13 Jan 1995 03:40:59 GMT Organization: The Ohio State University Lines: 15 Message-ID: <3f4sob$2cp@charm.magnus.acs.ohio-state.edu> NNTP-Posting-Host: top.magnus.acs.ohio-state.edu hi, i remember from my proteins class that the propensity of a peptide or protein to form a helical secondary structure can be calculated by a method of averaging out empirical values assigned to amino acid residues (work done by chou and fasman?). i have recently become interested at computing the helicity of short (20-40 mer) peptides, however at different pH values. i think the values assigned to acidic and basic residuess would vary with pH, i'm not sure about neutral residues. does anyone know of any references which deal with this question? also, does this kind of computation of helicity give fairly reliable estimates? thanks, /manpreet. From owner-proteins@net.bio.net Wed Jan 11 22:00:00 1995 Path: biosci!UMASSMED.UMMED.EDU!jklarlun From: jklarlun@UMASSMED.UMMED.EDU (Jes Klarlund) Newsgroups: bionet.molbio.proteins Subject: Ras Antibodies Date: 12 Jan 1995 07:55:20 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 25 Sender: daemon@net.bio.net Distribution: world Message-ID: <9501121555.AA20716@umassmed.UMMED.EDU> -- Login name: jklarlun In real life: Jes Klarlund Office: Mol Med, 856-6858 Directory: /resh/jklarlun Shell: /bin/sh No Plan. E-MAIL ADDRESS: jklarlun@umassmed.ummed.edu We are looking for antibodies that can distinguish between the various Ras forms (H-, K-, N- and R-Ras). Does anybody know who might have such antibodies ? Thanks Jes K. Klarlund jklarlun@umassmed.ummed.edu Program in Molecular Medicine U Mass Medical Center Worcester MA 01605 From owner-proteins@net.bio.net Wed Jan 11 22:00:00 1995 Path: biosci!MANI.CBS.UMN.EDU!npv From: npv@MANI.CBS.UMN.EDU (Nora Plesofsky-Vig) Newsgroups: bionet.molbio.proteins Subject: re:heat shock proteins Date: 12 Jan 1995 15:53:39 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 9 Sender: daemon@net.bio.net Distribution: world Message-ID: <9501122351.AA01499@mani.cbs.umn.edu> Reply-To: nora@molbio.cbs.umn.edu NNTP-Posting-Host: net.bio.net Lisa: My understanding is that the ER-localized hsp70, also called BIP, binds to the unassembled Ig heavy chains. It should not bind to them when they are assembled with the light chains and, therefore, BIP would not light up on a typical Western. Nora Plesofsky-Vig nora@molbio.cbs.umn.edu From owner-proteins@net.bio.net Thu Jan 12 22:00:00 1995 Path: biosci!JASON.UTHCT.EDU!SHAUN From: SHAUN@JASON.UTHCT.EDU ("Shaun D. Black") Newsgroups: bionet.molbio.proteins Subject: Re: Hill plot Date: 13 Jan 1995 10:45:56 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 11 Sender: daemon@net.bio.net Distribution: world Message-ID: <950113125223.534a@jason.uthct.edu> NNTP-Posting-Host: net.bio.net Hi All, I find this thread of real interest. Will any of the experts 'cooperate' with me to answer a question? My question is: What else could cause a sigmoidal binding plot besides cooperativity? Thanks. Best to all, Shaun =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= = Shaun D. Black, PhD | Internet address: shaun@jason.uthct.edu = = Dept. of Biochemistry | University of Texas Health Center, at Tyler = =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= From owner-proteins@net.bio.net Thu Jan 12 22:00:00 1995 Path: biosci!agate!news.duke.edu!godot.cc.duq.edu!hudson.lm.com!news.pop.psu.edu!psuvax1!news.ecn.bgu.edu!usenet.ins.cwru.edu!po.CWRU.Edu!pxb33 From: pxb33@po.CWRU.Edu (Parkash Badola) Newsgroups: bionet.molbio.proteins Subject: Protein elution from Ni-NTA resin Date: 13 Jan 1995 16:40:35 GMT Organization: Case Western Reserve Uni