From owner-proteins@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!rutgers!uwm.edu!cs.utexas.edu!news.sprintlink.net!uunet!in2.uu.net!mail.llu.edu!usenet From: bLangridge@ccmail.llu.edu (Bill Langridge) Newsgroups: bionet.molbio.proteins Subject: leptin Date: 2 Aug 1995 21:40:12 GMT Organization: Loma Linda University Lines: 11 Message-ID: <3vorbs$qpv@mail> NNTP-Posting-Host: 151.112.27.53 Mime-Version: 1.0 Content-Type: Text/Plain; charset=ISO-8859-1 X-Newsreader: WinVN 0.99.5 Can anyone tell me if they have heard of a protein called leptin? (may be a trade name) and can refer me to a publication? Leptin is supposedly involved in fat metabolism eg. oxidation of fatty acids!? and has been used in animal experiments in weight reduction (obesity). I believe there may have been a news segment on ABC network concerning lectin recently. Thanks for your help! Bill Langridge From owner-proteins@net.bio.net Tue Aug 01 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!ns1.faseb.org!darwin.sura.net!nih-csl!loglady.ninds.nih.gov!johnk From: johnk@spasm.niddk.nih.gov (John Kuszewski) Subject: Re: Prions Message-ID: <1995Aug2.205942.12648@alw.nih.gov> Sender: postman@alw.nih.gov (AMDS Postmaster) Nntp-Posting-Host: spasm.niddk.nih.gov Reply-To: johnk@spasm.niddk.nih.gov (John Kuszewski) Organization: National Insts. of Health References: <3vjshb$sbg@ns2.pb.net> Date: Wed, 2 Aug 1995 20:59:42 GMT Lines: 34 In article <3vjshb$sbg@ns2.pb.net>, smh@pb.net writes: |> Please refer me to a hypertext page where I can find info on problems associated with investigating prions, |> some experiments that have attempted to identify them, the diseased associated with them and how they |> are transmitted. You're not going to find anything significant on the web. Check the real scientific literature. For a painless starter, try the Scientific American article that appeared in the last year. That'll have some pointers into the professional literature. And please remember that we're not here to do your homework for you. --JK -- _____________ | ___/_ | |/ / -- /\ // /-- || || / /|| || || / / || || ||/ / || John Kuszewski || |/ /| || johnk@spasm.niddk.nih.gov || / /|| || \/ / / || \/ that's MISTER protein G to you! |/__/| | /_________| "Biophysics has driven me to an attitude of apocalyptic doom" --Frank Delaglio From owner-proteins@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!bcm!news.msfc.nasa.gov!newsfeed.internetmci.com!news.sprintlink.net!howland.reston.ans.net!vixen.cso.uiuc.edu!usenet From: David Clayton Newsgroups: bionet.molbio.proteins Subject: FLAG epitope tagging ??? Date: 2 Aug 1995 20:44:48 GMT Organization: University Of Illinois Lines: 10 Message-ID: <3voo40$e67@vixen.cso.uiuc.edu> NNTP-Posting-Host: claytond.life.uiuc.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) X-URL: news:bionet.molbio.proteins I want to stick an epitope on a recombinant protein to allow detection with a commercial antibody (ideally, via immunofluorescence against cultured cells and perhaps tissues). Questions: 1) is there a commercial source for the FLAG epitope and antibody? 2) can anyone offer advice regarding the utility of the FLAG epitope vs. other possibilities (e.g., His, myc, influenza)? Any advice appreciated! >David Clayton dclayton@uiuc.edu 217-244-4525 From owner-proteins@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!rutgers!uwm.edu!cs.utexas.edu!news.sprintlink.net!uunet!in1.uu.net!newsflash.concordia.ca!canopus.cc.umanitoba.ca!cc-hsc-k-box13.cc.umanitoba.ca!user From: egreif@ccu.umanitoba.ca (Elke) Newsgroups: bionet.molbio.proteins Subject: Drying 2D SDS PAGE? Followup-To: bionet.molbio.proteins Date: 2 Aug 1995 14:22:51 GMT Organization: University of Manitoba Lines: 33 Distribution: world Message-ID: NNTP-Posting-Host: cc-hsc-k-box13.cc.umanitoba.ca Hi there all you frustrated people doing 2D SDS PAGE. I have a problem. I have finally mastered the technique of actually running the IEF and second dimension gels up to the point of staining. The Coomassie has been giving me small problems that I can yet work that out, but the big Cahoona of a problem is the drying technique. I have tried drying the gels (12% acrylamide, 1mm thick) on a piece of regular filter paper on a Bio-Rad dryer at 80'C for 45 to 60 minutes, the results are always the same, CRACKING! I have adjusted the temps and the times but nothing worked. Then, I tried drying them between two sheets of clear cellophane (previously soaked with 10% glycerol, including the gel itself), stretching them between two frames that snap shut and leaving them at room temp overnight or in the fumehood with the fan on for a few hours. This was working alot better except the gels themselves don't last very long afterwards (maybe two weeks?) before they start cracking also. I have just heard of a technique where the gels are layed between two sheets of some sort and dried underneath a heating lamp? Apparently, you can store these gels indefinitely and are excellent to take a picture of. They are superb for handling. They dry as a hard plastic covering. Does anyone know of such a technique and could you please fill me in on it? This is a nightmare!!!!! I have good results but can't keep them for very long before they get damaged!!!! Tanks alot! Frustrated Winnipegger! Elke egreif@ccu.umanitoba.ca From owner-proteins@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!news.sprintlink.net!dispatch.news.demon.net!demon!uknet!bhamcs!news.ox.ac.uk!nmra.ocms!smb From: smb@bioch.ox.ac.uk (Simon Brocklehurst) Newsgroups: bionet.molbio.proteins Subject: Re: Modeling Software Date: 2 Aug 1995 11:17:38 GMT Organization: Oxford University Lines: 41 Message-ID: <3vnmsi$239@news.ox.ac.uk> References: <3ugpaf$hh6@newsbf02.news.aol.com> <3vl1dv$agf@sun4.bham.ac.uk> <1995Aug1.163717.20977@alw.nih.gov> NNTP-Posting-Host: nmra.ocms.ox.ac.uk In article <1995Aug1.163717.20977@alw.nih.gov>, johnk@spasm.niddk.nih.gov (John Kuszewski) writes: |> In article <3vl1dv$agf@sun4.bham.ac.uk>, s.m.williams.bcm@bham.ac.uk (Steve) writes: |> |> kballi@aol.com (KBalli) wrote: |> |> >Our lab is hoping to purchase some |> |> >software / hardware of our own soon. |> There are programs from Biosym (Discover), Tripos (whose name I can't |> recall), and Molecular Simulations (CHARMm, X-PLOR). They all do |> similar things. |> For non-profit making establishments, there is not really any advantage to be gained by paying big money to Software companies for this kind of stuff. You can get what you need for free (or virtually for free in some cases). X-PLOR (from Yale) is a great program to use for energy minimization/molecular dynamics simulations. You might also consider AMBER (from UCSF) if you're wanting to do a lot of molecular dynamics simulations. |> If your protein has fairly high homology to another protein whose |> structure is known, then there are different programs that can |> try to get an idea of what your protein's structure might look like. |> The best of these is Andrej Sali's MODELER (which is also sold by |> Molecular Simulations). MODELER is available free for academics I think(ftp from Harvard). Be aware that it uses a "restraint-based" method for building models. It's in no way clear that this is in fact the "best" way of going about building homology/comparative models. They do about as good a job as many other methods (I won't bore you with the details of pros and cons of various approaches), but the CPU time they require is MUCH greater than other approaches. -- Simon _____________________________________________________________________________ | | ,_ o Simon M. Brocklehurst, | / //\, Oxford Centre for Molecular Sciences, Department of Biochemistry, | \>> | University of Oxford, Oxford, UK. | \\, E-mail: smb@bioch.ox.ac.uk | WWW: http://www.ocms.ox.ac.uk/~smb/ |____________________________________________________________________________ From owner-proteins@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!news.cac.psu.edu!news.tc.cornell.edu!newsserver.sdsc.edu!nic-nac.CSU.net!charnel.ecst.csuchico.edu!csusac!csus.edu!news.ucdavis.edu!chip!szeliasf From: szeliasf@chip.ucdavis.edu (Elias Fernandez) Newsgroups: bionet.molbio.proteins Subject: omission mutagenesis Date: 2 Aug 1995 18:30:42 GMT Organization: University of California, Davis Lines: 16 Message-ID: <3vog8i$omr@mark.ucdavis.edu> NNTP-Posting-Host: chip.ucdavis.edu X-Newsreader: TIN [version 1.2 PL2] has anyone done a mutagenesis where a single residue was omitted in the middle of the protein? -- =============================================================================== Elias J. Fernandez | University of California | ## ## "It's not just an adventure; Section of Molecular and | (@ (@ it's a job!" Cellular Biology | \ Davis, CA 95616 | ##### | Tel. No. : (916)-752-3356 | ~\_====+ Fax No. : (916)-752-3085 e-mail : ejfernandez@ucdavis.edu ============================================================================== From owner-proteins@net.bio.net Tue Aug 01 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!ns1.faseb.org!darwin.sura.net!nih-csl!loglady.ninds.nih.gov!johnk From: johnk@spasm.niddk.nih.gov (John Kuszewski) Subject: Re: Modeling Software Message-ID: <1995Aug2.210613.12760@alw.nih.gov> Sender: postman@alw.nih.gov (AMDS Postmaster) Nntp-Posting-Host: spasm.niddk.nih.gov Reply-To: johnk@spasm.niddk.nih.gov (John Kuszewski) Organization: National Insts. of Health References: <3ugpaf$hh6@newsbf02.news.aol.com> <3vl1dv$agf@sun4.bham.ac.uk> <1995Aug1.163717.20977@alw.nih.gov> <3vnmsi$239@news.ox.ac.uk> Date: Wed, 2 Aug 1995 21:06:13 GMT Lines: 43 In article <3vnmsi$239@news.ox.ac.uk>, smb@bioch.ox.ac.uk (Simon Brocklehurst) writes: |> For non-profit making establishments, there is not really any |> advantage to be gained by paying big money to Software companies for |> this kind of stuff. You can get what you need for free (or virtually for True; I should have pointed that out. X-plor is ~$250 for academic users (and ~$50000 for corporate users!). For academic licensce enquiries, send email to Axel Brunger at xplor@newton.biology.yale.edu |> free in some cases). X-PLOR (from Yale) is a great program to use for |> energy minimization/molecular dynamics simulations. You might also consider Yes. It's also great for NMR structure determination and refinement 8-) But X-PLOR is useless without experimental data. |> MODELER is available free for academics I think(ftp from Harvard). |> Be aware that it uses a "restraint-based" method for building models. |> It's in no way clear that this is in fact the "best" way of going about |> building homology/comparative models. They do about as good a job as many |> other methods (I won't bore you with the details of pros and cons of various |> approaches), but the CPU time they require is MUCH greater than other |> approaches. My impression is that it might require more CPU than other approaches, but it's not an unreasonable CPU cost. -- _____________ | ___/_ | |/ / -- /\ // /-- || || / /|| || || / / || || ||/ / || John Kuszewski || |/ /| || johnk@spasm.niddk.nih.gov || / /|| || \/ / / || \/ that's MISTER protein G to you! |/__/| | /_________| "Biophysics has driven me to an attitude of apocalyptic doom" --Frank Delaglio From owner-proteins@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!rutgers!gatech!howland.reston.ans.net!news.cac.psu.edu!news.math.psu.edu!psuvax1!gvls1!newsfeed.pitt.edu!dsinc!netnews.upenn.edu!mscf.med.upenn.edu!pathology From: pathology@mscf.med.upenn.edu Newsgroups: bionet.molbio.proteins Subject: Re: leptin Date: 3 Aug 95 01:17:13 GMT Organization: University of Pennsylvania, Med School Computer Facility Lines: 24 Distribution: world Message-ID: <1995Aug3.011713.1@mscf.med.upenn.edu> References: <3vorbs$qpv@mail> NNTP-Posting-Host: mscf.med.upenn.edu In article <3vorbs$qpv@mail>, bLangridge@ccmail.llu.edu (Bill Langridge) writes: > Can anyone tell me if they have heard of a protein called leptin? > (may be a trade name) and can refer me to a publication? Leptin is > supposedly involved in fat metabolism eg. oxidation of fatty acids!? > and has been used in animal experiments in weight reduction (obesity). > I believe there may have been a news segment on ABC network > concerning lectin recently. > Leptin is the name given to the protein product of the ob (obesity) gene. This gene product was expressed in E. coli and administered to obese mice with defective ob genes, resulting in substantial weight loss. The three latest studies on the ob gene appeared in Science, 7/28/95, Vol. 269, in articles by Halaas et al. pp 543-546; Campfield et al., 546-548; and Pelleymounter et al, 540-543. Take a look at the WWW page http://www.gene.com/ae/WN/SU/obgene.html for more information. Gregg Wells Department of Pathology University of Pennsylvania Philadelphia, PA email: pathology@a1.mscf.upenn.edu From owner-proteins@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!torn!news.bc.net!info.ucla.edu!library.ucla.edu!csulb.edu!paris.ics.uci.edu!news.service.uci.edu!rablab.biochem.uci.edu!user From: user@host.uci.edu (Enter Your Name Here) Newsgroups: bionet.molbio.proteins Subject: Re: Drying 2D SDS PAGE? Date: Thu, 03 Aug 1995 16:48:38 +1000 Organization: UC Irvine Lines: 18 Distribution: world Message-ID: References: NNTP-Posting-Host: rablab.biochem.uci.edu In article , egreif@ccu.umanitoba.ca (Elke) wrote: Drying 2D gels is as easy as drying 1D gels. Usually what I do after staining (Coomassie or Silver) is to equilibrate the gel in 40% methanol and 5% glycerol (500ml for a 15x15 cm gel) for at least two hours and dry the gel between two cellophan sheets maintained with clips on a plastic board for at least two days. Important: avoid air bubbles that get trapped around the edge of the gel. If you follow this procedure, you should not get craking gels anymore and you will keep your gel ad vitam eternam. Good luck DT DMTHOMAS@uci.edu From owner-proteins@net.bio.net Wed Aug 02 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!ns1.faseb.org!darwin.sura.net!nih-csl!NewsWatcher!user From: newitt@nih.gov (John A. Newitt) Subject: Re: Antigenicity Epitope Prediction Message-ID: Sender: postman@alw.nih.gov (AMDS Postmaster) Nntp-Posting-Host: 128.231.113.35 Organization: National Institutes of Health References: <3vr45r$r3h@saffron.hnrc.tufts.edu> Date: Thu, 3 Aug 1995 21:37:03 GMT Lines: 18 In article <3vr45r$r3h@saffron.hnrc.tufts.edu>, Jim Sadowski wrote: > Does anybody know where I can find a program to determine antigenic > epitopes from known protein amino acid sequences? We're trying to > design peptides for immunization protocols to develop antibodies in > rabbits. Thank you. > The GCG suite contains a program called PEPTIDESTRUCTURE that will calculate an antigenic index according to the Jameson-Wolf method (CABIOS 4:181-186, 1988). The antigenic index results can then be plotted using another GCG program called PLOTSTRUCTURE. Regards, John A. Newitt, Ph.D. | National Institutes of Health | FAX: 301-402-0387 Bethesda, Maryland USA | From owner-proteins@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!news.sprintlink.net!uunet!in2.uu.net!d2.tufts.edu!saffron.hnrc.tufts.edu!usenet From: Jim Sadowski Newsgroups: bionet.molbio.proteins Subject: Antigenicity Epitope Prediction Date: 3 Aug 1995 18:22:51 GMT Organization: USDA Human Nutrition Research Center on Aging at Tufts University Lines: 11 Message-ID: <3vr45r$r3h@saffron.hnrc.tufts.edu> NNTP-Posting-Host: vitk-quadra-720.hnrc.tufts.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) X-URL: news:bionet.molbio.proteins Does anybody know where I can find a program to determine antigenic epitopes from known protein amino acid sequences? We're trying to design peptides for immunization protocols to develop antibodies in rabbits. Thank you. Jim Sadowski, Ph.D. Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University Sadowski_VK@Mint.HNRC.Tufts.edu From owner-proteins@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!botany.uq.edu.au!J.Marcus From: J.Marcus@botany.uq.edu.au ("Marcus, Dr J.") Newsgroups: bionet.molbio.proteins Subject: Reduction/Pyridethylation/HPLC Purification of proteins(?) Date: 3 Aug 1995 16:58:00 -0700 Organization: Dept of Botany, Univ of Qld Lines: 39 Sender: daemon@net.bio.net Distribution: world Message-ID: <1F7AF6B2027@botany.uq.edu.au> NNTP-Posting-Host: net.bio.net Dear Netters, I am attempting to reduce and alkylate a protein that I have. My problem comes when I want to repurify by RP-HPLC; I get all kinds of spurious peaks with tremendous absorbance at 280 and 254 making the identification of my protein very difficult. My questions are these: 1) Is it common to get many large peaks of material from such an reaction (even my mock reaction with no protein added)? 2) How critical is it to run the reaction under nitrogen or argon? Will I get polymerization and other reactions going on in the presence of oxygen? 3) How do you typically separate your reduced/alkylated protein from the reagents? a) gel filtration (desalting column or sizing column?) b) RP HPLC c) other Any tidbits of advice will be much appreciated. Regards, John _________________________________________________________ John Marcus Marcus@tpp.uq.edu.au (Dr J.Marcus) Cooperative Research Centre for Tropical Plant Pathology 5th Level John Hines Building University of Queensland St. Lucia, QLD 4072 AUSTRALIA Fax: 61-7-365-4771 Phone: 61-7-365-4764 From owner-proteins@net.bio.net Wed Aug 02 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!ns1.faseb.org!darwin.sura.net!nih-csl!loglady.ninds.nih.gov!johnk From: johnk@spasm.niddk.nih.gov (John Kuszewski) Subject: Re: Modeling Software Message-ID: <1995Aug3.181013.709@alw.nih.gov> Sender: postman@alw.nih.gov (AMDS Postmaster) Nntp-Posting-Host: spasm.niddk.nih.gov Reply-To: johnk@spasm.niddk.nih.gov (John Kuszewski) Organization: National Insts. of Health References: <3ugpaf$hh6@newsbf02.news.aol.com> <3vl1dv$agf@sun4.bham.ac.uk> <1995Aug1.163717.20977@alw.nih.gov> <3vnmsi$239@news.ox.ac.uk> <1995Aug2.210613.12760@alw.nih.gov> <3vqjbl$6te@news.ox.ac.uk> Date: Thu, 3 Aug 1995 18:10:13 GMT Lines: 52 In article <3vqjbl$6te@news.ox.ac.uk>, smb@bioch.ox.ac.uk (Simon Brocklehurst) writes: |> In article <1995Aug2.210613.12760@alw.nih.gov>, johnk@spasm.niddk.nih.gov (John Kuszewski) writes: |> |> In article <3vnmsi$239@news.ox.ac.uk>, smb@bioch.ox.ac.uk (Simon Brocklehurst) writes: |> |> |> |> But X-PLOR is useless without experimental data. |> |> |> Why do you think that? Because xplor doesn't have any energy terms for modeling data like MODELER's. It doesn't even have a solvation energy term. MD simulations of normal force fields (CHARMm, AMBER, etc) produce garbage for anything more complicated than ideal gasses, unless you add in energy terms that force the structure to look like experimental terms (eg., the NOE, chemical shift, and xray terms in xplor, many of which are my code). |> |> |> |> My impression is that it might require more CPU than other approaches, |> |> but it's not an unreasonable CPU cost. |> |> |> Whether it's unreasonable or not will depend on: |> |> o the size of the protein(s) you want to model (at best, |> cpu-time requirements for restraint-based methods scale linearly with the |> number of residues in the system - and some are much worse than this). |> |> o the _number_ of models you want to build (e.g. to evaluate different |> (but similar) alignments etc) - fragment-assembly-based methods can work orders |> of magnitude faster than restraint-based methods. And of course, how fast your computer is. Suffice it to say that MODELER impresses me immensely, and it's worth buying a (bunch of) fast computers to run it if you really need good models. --JK -- _____________ | ___/_ | |/ / -- /\ // /-- || || / /|| || || / / || || ||/ / || John Kuszewski || |/ /| || johnk@spasm.niddk.nih.gov || / /|| || \/ / / || \/ that's MISTER protein G to you! |/__/| | /_________| "Biophysics has driven me to an attitude of apocalyptic doom" --Frank Delaglio From owner-proteins@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!xlink.net!rz.uni-karlsruhe.de!news.uni-stuttgart.de!uni-regensburg.de!lrz-muenchen.de!ipp-garching.mpg.de!assel.biochem.mpg.de!jiang From: jiang@assel.biochem.mpg.de (Weiping Jiang) Newsgroups: bionet.molbio.proteins Subject: replacement of plasmid by another Date: 3 Aug 1995 10:07:17 GMT Organization: Rechenzentrum der Max-Planck-Gesellschaft in Garching Lines: 10 Message-ID: <3vq74l$17ue@sat.ipp-garching.mpg.de> NNTP-Posting-Host: assel.biochem.mpg.de X-Newsreader: TIN [version 1.2 PL2] Hi, netters, I transfered a plasmid into E coli, and got a supressor mutation on E. coli genome. Now I would like to transfer another plasmid into the mutated strain above, but the plasmid which is already in interupts observation. My question is: Is it possible to replace one plasmid by another one? Offered messages relevant to this from anyone are welcome. Thanks in advance. Weiping From owner-proteins@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!daresbury!nntp-trd.UNINETT.no!Norway.EU.net!EU.net!howland.reston.ans.net!tank.news.pipex.net!pipex!warwick!bham!bhamcs!news.ox.ac.uk!nmra.ocms!smb From: smb@bioch.ox.ac.uk (Simon Brocklehurst) Newsgroups: bionet.molbio.proteins Subject: Re: Modeling Software Date: 3 Aug 1995 13:35:49 GMT Organization: Oxford University Lines: 33 Message-ID: <3vqjbl$6te@news.ox.ac.uk> References: <3ugpaf$hh6@newsbf02.news.aol.com> <3vl1dv$agf@sun4.bham.ac.uk> <1995Aug1.163717.20977@alw.nih.gov> <3vnmsi$239@news.ox.ac.uk> <1995Aug2.210613.12760@alw.nih.gov> NNTP-Posting-Host: nmra.ocms.ox.ac.uk In article <1995Aug2.210613.12760@alw.nih.gov>, johnk@spasm.niddk.nih.gov (John Kuszewski) writes: |> In article <3vnmsi$239@news.ox.ac.uk>, smb@bioch.ox.ac.uk (Simon Brocklehurst) writes: |> |> But X-PLOR is useless without experimental data. |> Why do you think that? |> |> It's in no way clear that this is in fact the "best" way of going about |> |> building homology/comparative models. They do about as good a job as many |> |> other methods (I won't bore you with the details of pros and cons of various |> |> approaches), but the CPU time they require is MUCH greater than other |> |> approaches. |> |> My impression is that it might require more CPU than other approaches, |> but it's not an unreasonable CPU cost. |> Whether it's unreasonable or not will depend on: o the size of the protein(s) you want to model (at best, cpu-time requirements for restraint-based methods scale linearly with the number of residues in the system - and some are much worse than this). o the _number_ of models you want to build (e.g. to evaluate different (but similar) alignments etc) - fragment-assembly-based methods can work orders of magnitude faster than restraint-based methods. _____________________________________________________________________________ | | ,_ o Simon M. Brocklehurst, | / //\, Oxford Centre for Molecular Sciences, Department of Biochemistry, | \>> | University of Oxford, Oxford, UK. | \\, E-mail: smb@bioch.ox.ac.uk | WWW: http://www.ocms.ox.ac.uk/~smb/ |____________________________________________________________________________ From owner-proteins@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!bloom-beacon.mit.edu!gatech!newsfeed.internetmci.com!news.dacom.co.kr!news.kreonet.re.kr!worak.kaist.ac.kr!usenet From: Hak Sung Kim Newsgroups: bionet.molbio.proteins Subject: Biological methyl transfer Date: 3 Aug 1995 09:29:35 GMT Organization: KAIST(Korea Advanced Institute of science and Technology) Lines: 11 Message-ID: <3vq4u0$2ln@worak.kaist.ac.kr> NNTP-Posting-Host: hskim.kaist.ac.kr Hello bionetters : I would like to know the biological methyl transfer catalyzed by enzymes. I think that reaction involving methyl moiety is hard to occur in the chemical point of view. I know some cases of biological methyl transfer.. (in the process of chemotaxis, synthesis of methione etc. ). Any information regarding the enzyme involved, mechanism, or references are much appreciated. Personal contacts at jylee@chiak.kaist.ac.kr or hskim@sorak.kaist.ac.kr would be especially welcomed so that I can have easy access to the information. Thank you J.Y.Lee Ph.D. From owner-proteins@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!daresbury!nntp-trd.UNINETT.no!nntp.uio.no!tolindba From: tolindba@bioslave.uio.no (Toril Lindback) Newsgroups: bionet.molbio.proteins Subject: rbs Date: 3 Aug 1995 09:17:55 GMT Organization: University of Oslo Lines: 2 Message-ID: <3vq483$ea@hermod.uio.no> NNTP-Posting-Host: bioslave.uio.no X-Newsreader: TIN [version 1.2 PL2] Can anyone tell me how to calculate the value of an bacterial ribosome binding site (RBS) in kcal/mol? I will be happy for any reply. From owner-proteins@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!newsjunkie.ans.net!hermes.louisville.edu!news From: Elias Klein Newsgroups: bionet.molbio.proteins Subject: Iminodiacetic acid affinity matrix Date: 3 Aug 1995 18:34:01 GMT Organization: University of Louisville, Louisville KY USA Lines: 1 Message-ID: <3vr4qp$rnp@hermes.louisville.edu> NNTP-Posting-Host: eklein1.kdp-baptist.louisville.edu This is my third try to send a message on this board; hope it makes it. I'm trying to make affinity membranes using nylon as the starting material to bind histidine containing proteins. We start by reacting glutaraldehyde with the primary amine end groups of the nylon. When reduced, this gives a good yield of terminal aldehyde groups. Then these are reacted with iminodiacetic acid and reduced again, but we are getting very poor yields of the hoped for diaectic acid derivative. If you have any expeience with this reaction I would love to hear from you. Especially on how to measure yield (we are trying acid base titrations) , how much Cu or Ni you can bind per ml of bed volume, etc. My E-mail address is e0klei01@ulkyvm.louisville.edu From owner-proteins@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!agate!news.mindlink.net!vanbc.wimsey.com!unixg.ubc.ca!howie From: howie@unixg.ubc.ca (Howie Damude) Newsgroups: bionet.molbio.proteins Subject: Re: Drying 2D SDS PAGE? Date: 4 Aug 1995 04:40:16 GMT Organization: University of British Columbia, Vancouver, B.C., Canada Lines: 24 Distribution: world Message-ID: <3vs8bg$p5r@nnrp.ucs.ubc.ca> References: NNTP-Posting-Host: interchg.ubc.ca X-Newsreader: TIN [version 1.2 PL2] The procedure used below is exactly what I do except I take the gel directly out of commassie destain and put it between the 2 cellophane sheets. Definitely, no air bubbles. Put the gel in the fumehood to get it to dry faster. Enter Your Name Here (user@host.uci.edu) wrote: : In article , : egreif@ccu.umanitoba.ca (Elke) wrote: : Drying 2D gels is as easy as drying 1D gels. Usually what I do after : staining (Coomassie or Silver) is to equilibrate the gel in 40% methanol : and 5% glycerol (500ml for a 15x15 cm gel) for at least two hours and dry : the gel between two cellophan sheets maintained with clips on a plastic : board for at least two days. : Important: avoid air bubbles that get trapped around the edge of the gel. : If you follow this procedure, you should not get craking gels anymore and : you will keep your gel ad vitam eternam. : Good luck : DT DMTHOMAS@uci.edu From owner-proteins@net.bio.net Wed Aug 02 23:00:00 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: HPLC Troubleshooting question Date: Thu, 3 Aug 1995 16:40:43 Organization: Muscle Biology Group, University of Arizona Lines: 47 Message-ID: References: <11EF24B23F5@botany.uq.edu.au> <3v5l1q$6v2@fu-berlin.de> <3viqua$cva@amcnix.amc.uva.nl> NNTP-Posting-Host: val.agshantz.arizona.edu X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] In article <3viqua$cva@amcnix.amc.uva.nl> seppen@amc.uva.nl writes: >From: seppen@amc.uva.nl >Subject: Re: HPLC Troubleshooting question >Date: Mon, 31 Jul 95 16:56:07 -100 >In Article <3v5l1q$6v2@fu-berlin.de> >strecker@chemie.fu-berlin.de (Andreas Strecker) writes: >>In article <11EF24B23F5@botany.uq.edu.au>, >> J.Marcus@botany.uq.edu.au ("Marcus, Dr J.") wrote: >>>Dear Netters, >>> I have been struggling with our Waters HPLC >>>for over a month now and would appreciate ANY clues >>>that might help sort out the problem. If there is any >>>interest, I will post a summary of responses. >>> The problem manifests itself as an extremely wavy >>>baseline which coincides with the pump cycle of pump B >>>(6000A Series pump with dual piston design). After >>>changing the pump seals and check valves, the problem >>>remains. Every indication now seems to point to a bubble in >>>one of the pump heads; but, try as I may, I have not been >>>able to get rid of it. I have tried pumping methanol; I >>>have used the solvent draw-off valve to pressurize the >>>intake line while the pump is going at 10ml/min; I have >>>tried tapping the pump head. >> >> >>>John Marcus >> I have another possibility that I haven't seen mentioned. I have a Waters 625 pump. I have had many problems with air bubbles. They are coming from the mixer. It doesn't necessarily happen only when mixing solvents. On the 625 pump, the tubing going into the check valves is white (semi-clear) plastic. I can see the bubbles going from the mixer to the check valves. Waters has replaced the mixer once a couple of years ago and it seemed to work for a while. But the problem is back. I haven't quite decided what to do about it. It is no longer under warranty and we don't have a service contract for it. I'm debating whether or not the problem is bad enough to warrant spending the money for a service call. Other than this annoyance I have been really please with the system. Good luck with your problem. Valery F. Thompson Sr. Research Specialist Muscle Biology Group University of Arizona valeryt@ag.arizona.edu From owner-proteins@net.bio.net Thu Aug 03 23:00:00 1995 Path: biosci!daresbury!nntp-trd.UNINETT.no!Norway.EU.net!nac.no!nntp.uio.no!tolindba From: tolindba@bioslave.uio.no (Toril Lindback) Newsgroups: bionet.molbio.proteins Subject: RBS Date: 4 Aug 1995 09:38:58 GMT Organization: University of Oslo Lines: 6 Message-ID: <3vspri$999@hermod.uio.no> NNTP-Posting-Host: bioslave.uio.no X-Newsreader: TIN [version 1.2 PL2] Can anyone tell me how to calculate the deltaG value of a bacterial ribosome binding site? I will be happy for any suggestions. Regards Toril Lindback From owner-proteins@net.bio.net Thu Aug 03 23:00:00 1995 Path: biosci!daresbury!nntp-trd.UNINETT.no!Norway.EU.net!nac.no!nntp.uio.no!tolindba From: tolindba@bioslave.uio.no (Toril Lindback) Newsgroups: bionet.molbio.proteins Subject: ribosome binding site Date: 4 Aug 1995 10:36:20 GMT Organization: University of Oslo Lines: 6 Message-ID: <3vst74$9pa@hermod.uio.no> NNTP-Posting-Host: bioslave.uio.no X-Newsreader: TIN [version 1.2 PL2] Can anyone tell me how to calculate the delta G value of a bacterial ribosome binding site. I will be happy for any suggestions. Regards Toril Lindback From owner-proteins@net.bio.net Thu Aug 03 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!tank.news.pipex.net!pipex!oleane!jussieu.fr!univ-lyon1.fr!swidir.switch.ch!scsing.switch.ch!news.belwue.de!news.uni-freiburg.de!sun1.tumorbio.uni-freiburg.de!said From: said@sun1.tumorbio.uni-freiburg.de (Said Hashemolhossein) Newsgroups: bionet.molbio.proteins Subject: How to detect GFP via FACS? Date: 24 Jul 1995 11:14:41 GMT Organization: Rechenzentrum der Universitaet Freiburg, Germany Lines: 31 Message-ID: <3uvvb1$rie@n.ruf.uni-freiburg.de> NNTP-Posting-Host: sun1.tumorbio.uni-freiburg.de X-Newsreader: TIN [version 1.2 PL2] Hi, I try to detect GFP after transfection in NIH3T3 or 293 without success. Transfection is for sure not the problem ( I am able to transfect with luciferase + detect in luminometer ). I want to detect the GFP containing cells via FACS (Becton Dickinson , FL1H / FL2A) , but cannot see anything. Even under the microscope it is not possible to detect positive cells. Does anyone can help???.... thanks a lot .... said ------------------------------------------------------------------------------- Dr. Said Hashemolhosseini phone (761)2061531 Tumor Biology Center fax (761)2061505 Dep Exp Cancer Research email said@sun1.tumorbio.uni-freiburg.de Breisacherstr. 117 D-79106 Freiburg, Germany ------------------------------------------------------------------------------- From owner-proteins@net.bio.net Thu Aug 03 23:00:00 1995 Path: biosci!agate!howland.reston.ans.net!newsfeed.internetmci.com!news.sprintlink.net!dispatch.news.demon.net!demon!tank.news.pipex.net!pipex!warwick!bham!usenet From: r.jefferis@bham.ac.uk (Roy Jefferis) Newsgroups: bionet.molbio.proteins Subject: Re: Fragment B of staphylococcal protein A Date: 4 Aug 1995 20:09:58 GMT Organization: University of Birmingham Lines: 22 Message-ID: <3vtuqm$gm6@sun4.bham.ac.uk> References: <3vn3h6$4kd@hpg30a.csc.cuhk.hk> NNTP-Posting-Host: med340.bham.ac.uk X-Newsreader: WinVN 0.92.1 In article <3vn3h6$4kd@hpg30a.csc.cuhk.hk>, b535762@mailserv.cuhk.hk (Chung Yuk Ka) says: > >Hi all, > >I am looking for the 3d structure of the Fragment B of >staphylococcal protein A in PDB but I can't find it. >I have check from the web site www.pdb.bnl.gov that it >should have the id 1ESF but I can't found it in the >ftp site. I would like to know if I got the wrong id >or not. Anyone who has this information please post >the relevant information here or email to >yukkachung@cuhk.hk. > >Thank you very much. > >Chung Yuk Ka >(Johnnie Walker) >Statistics Dept., The Chinese University of Hong Kong The only study that I am awarte of is the X-ray crystal structure determined as a complex with IgG-Fc. The paper is Deisenhofer J. (1981) Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of Protein A. Biochemistry 20:2361-2370. From owner-proteins@net.bio.net Thu Aug 03 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!swrinde!tank.news.pipex.net!pipex!uknet!bhamcs!news.ox.ac.uk!nmra.ocms!smb From: smb@bioch.ox.ac.uk (Simon Brocklehurst) Newsgroups: bionet.molbio.proteins Subject: Re: Modeling Software Date: 4 Aug 1995 13:32:41 GMT Organization: Oxford University Lines: 49 Message-ID: <3vt7hp$5v8@news.ox.ac.uk> References: <3ugpaf$hh6@newsbf02.news.aol.com> <3vl1dv$agf@sun4.bham.ac.uk> <1995Aug1.163717.20977@alw.nih.gov> <3vnmsi$239@news.ox.ac.uk> <1995Aug2.210613.12760@alw.nih.gov> <3vqjbl$6te@news.ox.ac.uk> <1995Aug3.181013.709@alw.nih.gov> NNTP-Posting-Host: nmra.ocms.ox.ac.uk In article <1995Aug3.181013.709@alw.nih.gov>, johnk@spasm.niddk.nih.gov (John Kuszewski) writes: |> In article <3vqjbl$6te@news.ox.ac.uk>, smb@bioch.ox.ac.uk (Simon Brocklehurst) writes: |> |> In article <1995Aug2.210613.12760@alw.nih.gov>, johnk@spasm.niddk.nih.gov (John Kuszewski) writes: |> |> |> In article <3vnmsi$239@news.ox.ac.uk>, smb@bioch.ox.ac.uk (Simon Brocklehurst) writes: |> |> |> |> |> |> But X-PLOR is useless without experimental data. |> |> |> |> |> Why do you think that? |> |> Because xplor doesn't have any energy terms for modeling data like |> MODELER's. It doesn't even have a solvation energy term. |> MD simulations of normal force fields (CHARMm, AMBER, etc) |> produce garbage for anything more complicated than ideal gasses, So you think that not only X-PLOR is useless, but so is CHARMm, AMBER etc when it comes to anything non-experimental. Well at least that dismisses everyone equally. To reject the results from all simulations of proteins (OBVIOUSLY including solvent - which CAN be done easily enough) by describing them as garbage is not credible. |> unless |> you add in energy terms that force the structure to look like |> experimental terms (eg., the NOE, chemical shift, and xray terms in |> xplor, I think there's some serious typing errors in that bit! What are you saying, that you can't get good time-averaged models of proteins "de novo" without experimental data? Of course that's true. Unless you want to tell us all about some new method that's effectively solved the folding problem that is ;-) |> And of course, how fast your computer is. Suffice it to say that MODELER |> impresses me immensely, and it's worth buying a (bunch of) fast computers |> to run it if you really need good models. |> Why do you think the models produced by MODELER are of higher quality than models produced by other homology/comparative modelling programs? As far as I've seen, it does about as well (or as badly - depending on how you look at these things!) as many other approaches. -- Simon _____________________________________________________________________________ | | ,_ o Simon M. Brocklehurst, | / //\, Oxford Centre for Molecular Sciences, Department of Biochemistry, | \>> | University of Oxford, Oxford, UK. | \\, E-mail: smb@bioch.ox.ac.uk | WWW: http://www.ocms.ox.ac.uk/~smb/ |____________________________________________________________________________ From owner-proteins@net.bio.net Thu Aug 03 23:00:00 1995 Path: biosci!daresbury!nntp-trd.UNINETT.no!due.unit.no!nac.no!nntp.uio.no!sjreppe From: sjreppe@bioslave.uio.no (Sjur Reppe) Newsgroups: bionet.molbio.proteins,bionet.software Subject: Re: Program to calculate isoelectric points? Followup-To: bionet.molbio.proteins,bionet.software Date: 4 Aug 1995 08:25:50 GMT Organization: University of Oslo Lines: 27 Message-ID: <3vslie$8mk@hermod.uio.no> References: <3tsida$qtg@cwis-20.wayne.edu> NNTP-Posting-Host: bioslave.uio.no X-Newsreader: TIN [version 1.2 PL2] Xref: biosci bionet.molbio.proteins:5246 bionet.software:12924 Angela c. Murphy (acmurphy@helix.nih.gov) wrote: : There is also a program called "isoelectric" in the GCG group of programs : for unix machines. A cruder calculator of isoelectric points may be found : in the DOS and Windows versions of "General Protein Mass Analysis For..." : (DOS or Windows). I got my Windows version from WindowChem Software, and : it was under $200. : Angela C. Murphy : On 11 Jul 1995, Anton Scott Goustin wrote: : > scb1@ellis.uchicago.edu (Samuel C. Blackman) wrote: : > >Does anyone know of a program (Mac, DOS, Unix) that will calculate the : > >isoelectric point of a given peptide? Thanks! : > > : > >-- Sam : > > : > > : > >-- : > >Samuel C. Blackman ! InterNet : scb1@midway.uchicago.edu : > >MD/PhD 2/7 (Pharmacology) ! Disclaimer : Who cares what I say, I'm a student! : > >5513 S. Cornell Ave. #1 ! Quote : I'm not a doctor, but I play one on TV. : > >Chicago, IL 60637-1914 ! Phone : 312/752-1082 (h) Fax: 312/996-1225 : > : > There is a program that does so contained within MacVector 4.5 (Kodak Imaging Technologies)...expensive. Perhaps a neighbor has it there at the University of Chicago. : > : > : > From owner-proteins@net.bio.net Thu Aug 03 23:00:00 1995 Path: biosci!daresbury!nntp-trd.UNINETT.no!Norway.EU.net!nac.no!nntp.uio.no!tolindba From: tolindba@bioslave.uio.no (Toril Lindback) Newsgroups: bionet.molbio.proteins Subject: ribosome binding site Date: 4 Aug 1995 11:20:13 GMT Organization: University of Oslo Lines: 6 Message-ID: <3vsvpd$a35@hermod.uio.no> NNTP-Posting-Host: bioslave.uio.no X-Newsreader: TIN [version 1.2 PL2] Can anyone tell me how to calculate the delta G value of a bacterial ribosome binding site? I will be happy for any sugestions Regards Toril From owner-proteins@net.bio.net Thu Aug 03 23:00:00 1995 Path: biosci!THORIN.UTHSCSA.EDU!SYLVIA From: SYLVIA@THORIN.UTHSCSA.EDU Newsgroups: bionet.molbio.proteins Subject: MAP Kinase inhibitor? Date: 4 Aug 1995 18:09:29 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 5 Sender: daemon@net.bio.net Distribution: world Message-ID: <950804201154.220041@thorin.uthscsa.edu> NNTP-Posting-Host: net.bio.net Hello all. Does anyone know of a good SPECIFIC inhibitor of MAP kinase? I've heard that Wortmannin will work, but it also inhibits PI-3-kinase. Thanks in advance. Vic sylvia@thorin.uthscsa.edu From owner-proteins@net.bio.net Fri Aug 04 23:00:00 1995 Path: biosci!bcm!news.msfc.nasa.gov!newsfeed.internetmci.com!newsxfer.itd.umich.edu!caen!night.primate.wisc.edu!nntp.msstate.edu!fiona.umsmed.edu!fiona.umsmed.edu!hutchins From: hutchins@fiona.umsmed.edu (Jim Hutchins) Newsgroups: bionet.molbio.proteins Subject: Re: Drying 2D SDS PAGE? Date: 5 Aug 1995 14:37:35 GMT Organization: University of Mississippi Medical Center Lines: 31 Distribution: world Message-ID: <3vvvnf$935@fiona.umsmed.edu> References: NNTP-Posting-Host: fiona.umsmed.edu X-Newsreader: TIN [version 1.2 PL1] Elke (egreif@ccu.umanitoba.ca) wrote: : Hi there all you frustrated people doing 2D SDS PAGE. : I have a problem. [stuff deleted] : of a problem is the drying technique. I have tried drying the gels (12% : acrylamide, 1mm thick) on a piece of regular filter paper on a Bio-Rad : dryer at 80'C for 45 to 60 minutes, the results are always the same, : CRACKING! I have adjusted the temps and the times but nothing worked. Then, [more stuff deleted] : Frustrated Winnipegger! : Elke We have had good luck with the BioRad gel dryer, slow ramp setting, 60 deg C for 2 or 3 hours. It's possible that the gel is not completely dry before you release the vacuum. Or, your vacuum may not be strong enough. Many people have trouble with "house vacuum": are you hooked up to a pump with a cold trap in between? BioRad suggests an alternative fixing solution which is (I think) 40% MeOH, 10% HAc, 3% glycerol. (I'm doing this from memory, someone correct me if I'm wrong.) We call it "Gel Fix Intensive Care Formula". This might help reduce cracking, too. If you send me a private note, I'll put you in touch with the person in our lab who does most of the 2D gels. He'd know more than me. -- Jim Hutchins Assoc Prof Anatomy, Asst Prof Neurology (Research), Univ Mississippi Med Ctr http://fiona.umsmed.edu/~hutchins/ *** E-mail: hutchins@umsmed.edu ``It became necessary to destroy the town in order to save it.''--American infantry officer firing on Ben Tre, Vietnam, 2/8/68 From owner-proteins@net.bio.net Fri Aug 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!news.sprintlink.net!tank.news.pipex.net!pipex!sunsite.doc.ic.ac.uk!qmw!orac.sunderland.ac.uk!usenet From: hs0dhr@Sunderland.AC.UK (DALE hreczuk-hirst) Newsgroups: bionet.molbio.proteins Subject: tritium labelling of peptides Date: 4 Aug 1995 07:34:27 GMT Organization: University of Sunderland Lines: 11 Message-ID: <3vsii3$o6t@orac.sunderland.ac.uk> Reply-To: hs0lbe@Sunderland.AC.UK NNTP-Posting-Host: glaxo2.sunderland.ac.uk X-Newsreader: WinVN 0.91.4 Could any one advise me on methods for tritiation of synthetic peptides, references for methods etc. I know that iodo and dehydro amino acid derivatives can be readily incorporated using traditional solid phase methods and can subsequently converted to the tritium labelled form. What i want to know is how this is achieved and details on the effiecency and specificity of this process. thanks, len, sunderland. From owner-proteins@net.bio.net Fri Aug 04 23:00:00 1995 Path: biosci!agate!library.ucla.edu!info.ucla.edu!newsfeed.internetmci.com!EU.net!news.sprintlink.net!tank.news.pipex.net!pipex!howland.reston.ans.net!news-e1a.megaweb.com!newstf01.news.aol.com!newsbf02.news.aol.com!not-for-mail From: erikhom@aol.com (ErikHom) Newsgroups: bionet.molbio.proteins Subject: Re: Looking for George Rose's e-mail address Date: 5 Aug 1995 15:21:45 -0400 Organization: America Online, Inc. (1-800-827-6364) Lines: 18 Sender: root@newsbf02.news.aol.com Message-ID: <400gc9$o6n@newsbf02.news.aol.com> References: Reply-To: erikhom@aol.com (ErikHom) NNTP-Posting-Host: newsbf02.mail.aol.com Greetings, Prof. Roses (who has just relocated to J. Hopkins Med School) is: rose@grserv.med.jhu.edu May I be so nosy and ask why you wish to contact him? He gave a ground-breaking talk on a new protein folding algorithm (LINUS), at U. Delaware and then at Swarthmore College (where I was) this past spring. I believe the work he discussed is now published in the June issue of Proteins: Structure and Function; I haven't had access to a science library that carried that since I graduated so I can't give you the exact details. If you get the citation, would you mind relaying it to me? Thanks a bunch. Prof Rose is a wonderful and warm person to talk to, and I'm sure great to work for. Hope this helps. Cheers, Erik Hom, Swarthmore '95 From owner-proteins@net.bio.net Sat Aug 05 23:00:00 1995 Path: biosci!bloom-beacon.mit.edu!newsfeed.internetmci.com!news.sprintlink.net!dispatch.news.demon.net!demon!tank.news.pipex.net!pipex!howland.reston.ans.net!news.cac.psu.edu!usenet From: Jie Tang Newsgroups: bionet.molbio.proteins Subject: plasmin activity in kidneys Date: 7 Aug 1995 00:51:37 GMT Organization: Penn State University, Center for Academic Computing Lines: 8 Message-ID: <403o2p$gbm@hearst.cac.psu.edu> References: NNTP-Posting-Host: jbpmac1.biochem.hmc.psu.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; PPC) To: thall@cellbio.wustl.edu X-URL: news:thall-3007952117280001@128.252.206.226 Does anybody know if plamin is present in kidney proximal tubular cells? If so, is it active? Thanks Tang From owner-proteins@net.bio.net Sun Aug 06 23:00:00 1995 Path: biosci!rutgers!uwm.edu!cs.utexas.edu!news.sprintlink.net!dispatch.news.demon.net!demon!tank.news.pipex.net!pipex!sunsite.doc.ic.ac.uk!daresbury!bioftp.unibas.ch!citi2.fr!not-for-mail From: camoin@bisance.citi2.fr (Luc Camoin) Newsgroups: bionet.molbio.proteins Subject: BRCN CLEAVAGE Date: 7 Aug 1995 10:48:18 +0200 Organization: CITI2 - Universite Rene Descartes, Paris Lines: 20 Message-ID: <404k0i$6cu@bisance.citi2.fr> NNTP-Posting-Host: bisance.citi2.fr X-Newsreader: TIN [version 1.2 PL2] Dear Netters, The cleavage at methionine residues with CNBR under acidic conditions is well used frequently. The cleavage occurs at Met-X bonds. I would like to know if some Met-X bonds are resistant to this cleavage. For example cleavage of Met-Thr bonds, and Met-Ser bonds, often occurs in low yields. Do you have an idea of Met-Trp cleavage? Luc Camoin ____________________________________________________________________________ _/_/_/_/ _/_/_/_/ _/_/_/_/ _/_/ _/ Luc CAMOIN _/ _/ _/ _/ _/ _/_/ Institut Cochin de Genetique _/ _/ _/ _/_/ _/ _/ _/ Moleculaire CNRS UPR 415 _/ _/ _/ _/ _/ _/ 22 rue Mechain 75014 Paris France _/_/_/_/ _/_/_/_/ _/_/_/_/ _/ _/ camoin@genome.vjf.inserm.fr ____________________________________________________________________________ From owner-proteins@net.bio.net Sun Aug 06 23:00:00 1995 Path: biosci!bcm!news.msfc.nasa.gov!elroy.jpl.nasa.gov!swrinde!tank.news.pipex.net!pipex!howland.reston.ans.net!newsserver.jvnc.net!newsserver2.jvnc.net!netnews.upenn.edu!brass2.med.upenn.edu!user From: obrien@pharm.med.upenn.edu (PJOB) Newsgroups: bionet.molbio.proteins Subject: Protein homology conventions Followup-To: bionet.molbio.proteins Date: Mon, 07 Aug 1995 13:24:06 -0500 Organization: University of Pennsylvania Lines: 18 Message-ID: NNTP-Posting-Host: brass2.med.upenn.edu Are there widely accepted criteria for declaring that amino acid residues in related proteins are homologous? I am aligning a few receptor sequences and wish to express their relatedness in terms of identity and homology. Identity I can handle, but is there a set of amino acids that is generally accepted as homologous? Also, how does one express homology/identity in the case where the proteins are of unequal length? Would it be proper to only refer to overlapping or corresponding regions? For example, if two G-protein coupled receptors are reasonably similar between their putative membrane spanning domains, but one has a much longer n- or c- terminus, how should the homology/identity be expressed? Any references would be greatly appreciated! Peter -- Opinions seen here are channeled to me by a Ring-Tailed Lemur named Oxnyx From owner-proteins@net.bio.net Sun Aug 06 23:00:00 1995 Path: biosci!ns1.faseb.org!darwin.sura.net!vax8.cfsan.fda.gov!pascal.nctr.fda.gov!AJHARRIS.NCTR.FDA.GOV!ajharris Newsgroups: bionet.molbio.proteins Subject: anti CBG antibody Message-ID: From: ajharris@fdant.nctr.fda.gov (Angela J. Harris) Date: Mon, 7 Aug 1995 12:11:55 GMT Organization: National Center for Toxicological Research Keywords: rat, CBG NNTP-Posting-Host: ajharris.nctr.fda.gov X-Newsreader: Trumpet for Windows [Version 1.0 Rev B] Lines: 4 We're looking for a corticosteroid binding globulin antibody which recognizes the rat CBG protein and works on a Western blot. Thanks, Angela From owner-proteins@net.bio.net Sun Aug 06 23:00:00 1995 Path: biosci!bcm!news.msfc.nasa.gov!elroy.jpl.nasa.gov!usc!comserv-f-50.usc.edu!user From: bfry@hsc.usc.edu (Bryan Grieg Fry) Newsgroups: bionet.molbio.proteins Subject: proteins behavior Date: Mon, 07 Aug 1995 16:22:31 -0800 Organization: USC Cancer Center Lines: 15 Sender: bfry@comserv-f-50.usc.edu Message-ID: NNTP-Posting-Host: comserv-f-50.usc.edu Hi!!!!! I am doing a research with proteins and I need to save every little amount but due to protein aggregation I am missing a lot of protein. Can anyone tell that how can this aggregation be prevented when proteins are binded to a big crosslinker which have two aromatic rings. This aggregation is mainly due to its two aromatic rings but the binding is the main research . A lot of protein has been lost already in form of precipitate. Please reply SOON!!!! Bryan Fry University of Third Island on Left (its the oval one with sandy beaches) Castle Anthrax, Somewhat Northwest of Southeast From owner-proteins@net.bio.net Sun Aug 06 23:00:00 1995 Path: biosci!botany.uq.edu.au!J.Marcus From: J.Marcus@botany.uq.edu.au ("Marcus, Dr J.") Newsgroups: bionet.molbio.proteins Subject: Re: CNBr CLEAVAGE Date: 7 Aug 1995 15:28:33 -0700 Organization: Dept of Botany, Univ of Qld Lines: 42 Sender: daemon@net.bio.net Distribution: world Message-ID: <25637AE0A2A@botany.uq.edu.au> NNTP-Posting-Host: net.bio.net Luc, Yes Met-Thr and Met-Ser bonds are resistant to CNBr cleavage. I don't know about Met-Trp, however. If you normally do your cleavage reaction in formic acid you might try trifluoroacetic acid instead for these resistant bonds. There may be some other problems with TFA so you'll have to play it by ear. You might want to look at the book edited by A. Darbre called "Practical Protein Chemistry" published by Wiley-Interscience Publications (1986) as this has some good discussion of the chemistry involved. Shively's book called something like "Methods of Protein Microcharacterization" also has some pointers with regard to the use of TFA. If you don't have much to loose give TFA a try. Let me know if you need more information on Shively's book and I can try to find out the publisher etc. Hope that helps. Regards, John > The cleavage at methionine residues with CNBR under acidic conditions is well > used frequently. The cleavage occurs at Met-X bonds. I would like > to know if some Met-X bonds are resistant to this cleavage. For example > cleavage of Met-Thr bonds, and Met-Ser bonds, often occurs in low yields. Do > you have an idea of Met-Trp cleavage? > > Luc Camoin > > _________________________________________________________ John Marcus Marcus@tpp.uq.edu.au (Dr J.Marcus) Cooperative Research Centre for Tropical Plant Pathology 5th Level John Hines Building University of Queensland St. Lucia, QLD 4072 AUSTRALIA Fax: 61-7-365-4771 Phone: 61-7-365-4764 From owner-proteins@net.bio.net Mon Aug 07 23:00:00 1995 Path: biosci!bcm!pendragon!news.msfc.nasa.gov!newsfeed.internetmci.com!news.sprintlink.net!howland.reston.ans.net!tank.news.pipex.net!pipex!oleane!jussieu.fr!univ-lyon1.fr!swidir.switch.ch!scsing.switch.ch!news.belwue.de!news.dfn.de!uni-muenster.de!news From: Torsten Boerchers Newsgroups: bionet.molbio.proteins Subject: Re: FLAG epitope tagging ??? Date: 8 Aug 1995 17:33:34 GMT Organization: ICB Lines: 26 Message-ID: <40875e$1802@majestix.uni-muenster.de> References: <3voo40$e67@vixen.cso.uiuc.edu> NNTP-Posting-Host: arnheim.uni-muenster.de Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Windows; I; 16bit) To: dclayton@uiuc.edu David Clayton wrote: >I want to stick an epitope on a recombinant protein to allow detection >with a commercial antibody (ideally, via immunofluorescence against >cultured cells and perhaps tissues). Questions: >1) is there a commercial source for the FLAG epitope and antibody? >2) can anyone offer advice regarding the utility of the FLAG epitope vs. >other possibilities (e.g., His, myc, influenza)? > Any advice appreciated! >>David Clayton dclayton@uiuc.edu 217-244-4525 > > David, the Flag Epitope system is available from Kodak,seems that Kodak distributes it in US itself. Since I only plan to use it, I cannot comment on its utility. Kind regards ------------------------------------------------------------------------/ Torsten Boerchers / _/ _/_/_/ _/_/_/ Institute of Chemical- and Biochemical Sensor/ _/ _/ _/ _/ Research (Molecular Biology Group) / _/ _/ _/_/_/ Mendelstr 7, D-48149 Muenster, Germany / _/ _/ _/ _/ Fax: +49-251-980 2890 Phone: +49-251-980 2876/ _/ _/_/_/ _/_/_/_/ E-Mail: borcher@uni-muenster.de / -----------------------------------------------------------------/ From owner-proteins@net.bio.net Mon Aug 07 23:00:00 1995 Path: biosci!agate!kos5mac24.berkeley.edu!user From: spenson@nature.berkeley.edu (Simon Penson) Newsgroups: bionet.molbio.proteins Subject: How does ATP gamma S work? Date: Tue, 08 Aug 1995 16:00:43 -0800 Organization: UC Berkeley Lines: 11 Message-ID: NNTP-Posting-Host: kos5mac24.berkeley.edu X-Newsreader: Value-Added NewsWatcher 2.0b24.0+ Dear Netters, I need a substrate which will bind irreversibly and specifically to the active site of a protein kinase. I don't want to get involved in photoaffinity labelling if I can avoid it. So, my question is: Does ATP gamma S bind irreversibly to protein kinases? If not, how does it inhibit ATP-requiring enzymes/processes? Thanks, Simon. From owner-proteins@net.bio.net Mon Aug 07 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!ns1.faseb.org!darwin.sura.net!nih-csl!postman From: Mary Karczewski Subject: Re: FLAG epitope tagging ??? Content-Type: text/plain; charset=us-ascii Message-ID: <1995Aug8.203431.29573@alw.nih.gov> To: Sender: postman@alw.nih.gov (AMDS Postmaster) Nntp-Posting-Host: db4101.niaid.nih.gov Content-Transfer-Encoding: 7bit Organization: NIAID/NIH References: <3voo40$e67@vixen.cso.uiuc.edu> Mime-Version: 1.0 Date: Tue, 8 Aug 1995 20:34:31 GMT X-Mailer: Mozilla 1.1N (Windows; I; 16bit) Lines: 7 I have used the flag epitope to tag several proteins, and have had success using the monoclonal antibody available from Kodak in immunoprecipitation and in immunofluorescence, but not in western. I have never used any of the other epitope tags you mentioned, so I can't compare for you- hope this helps! marykk From owner-proteins@net.bio.net Mon Aug 07 23:00:00 1995 Path: biosci!bcm!news.msfc.nasa.gov!newsfeed.internetmci.com!gatech!swrinde!tank.news.pipex.net!pipex!oleane!jussieu.fr!univ-lyon1.fr!swidir.switch.ch!scsing.switch.ch!news.belwue.de!news.dfn.de!uni-muenster.de!news From: Torsten Boerchers Newsgroups: bionet.molbio.proteins Subject: Re: Fragment B of staphylococcal protein A Date: 8 Aug 1995 17:25:04 GMT Organization: ICB Lines: 36 Message-ID: <4086lg$1802@majestix.uni-muenster.de> References: <3vn3h6$4kd@hpg30a.csc.cuhk.hk> NNTP-Posting-Host: arnheim.uni-muenster.de Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Windows; I; 16bit) To: b535762@mailserv.cuhk.hk b535762@mailserv.cuhk.hk (Chung Yuk Ka) wrote: >Hi all, > >I am looking for the 3d structure of the Fragment B of >staphylococcal protein A in PDB but I can't find it. >I have check from the web site www.pdb.bnl.gov that it >should have the id 1ESF but I can't found it in the >ftp site. I would like to know if I got the wrong id >or not. Anyone who has this information please post >the relevant information here or email to >yukkachung@cuhk.hk. > >Thank you very much. > >Chung Yuk Ka >(Johnnie Walker) >Statistics Dept., The Chinese University of Hong Kong I only found a rather old (1981) entry of Deisenhofer: 1fc2 : IMMUNOGLOBULIN FC AND FRAGMENT B OF PROTEIN A COMPLEX and nothing under id 1esf. Hope this helps Torsten ------------------------------------------------------------------------- / Torsten Boerchers / _/ _/_/_/ _/_/_/ Institute of Chemical- and Biochemical Sensor/ _/ _/ _/ _/ Research (Molecular Biology Group) / _/ _/ _/_/_/ Mendelstr 7, D-48149 Muenster, Germany / _/ _/ _/ _/ Fax: +49-251-980 2890 Phone: +49-251-980 2876/ _/ _/_/_/ _/_/_/_/ E-Mail: borcher@uni-muenster.de / ------------------------------------------------------------------/ From owner-proteins@net.bio.net Mon Aug 07 23:00:00 1995 Path: biosci!bcm!pendragon!news.msfc.nasa.gov!newsfeed.internetmci.com!news.sprintlink.net!uunet!in1.uu.net!demos!dnews-server From: mashko@vnigen.msk.su (Lab 13) Newsgroups: bionet.software,bionet.molbio.proteins Subject: Needs: a program for isoelectric point calculation Date: 8 Aug 1995 19:28:49 +0400 Organization: Union Institute for Genetics Lines: 12 Sender: news-server@news.demos.su Distribution: world Message-ID: Reply-To: mashko@vnigen.msk.su NNTP-Posting-Host: news.demos.su X-Return-Path: news.demos.su!kremvax.demos.su!kiae!vnigen!mashko Xref: biosci bionet.software:12948 bionet.molbio.proteins:5262 Dear Colleagues, Does anybody know a software to calculate the isoelectric point for a protein? Could you please write to my address: MASHKO@VNIGEN.MSK.SU Thank you in advance. Sincerely, Katya Rogozhkina From owner-proteins@net.bio.net Mon Aug 07 23:00:00 1995 Path: biosci!bcm!news.msfc.nasa.gov!newsfeed.internetmci.com!bloom-beacon.mit.edu!news.starnet.net!wupost!ukma!hermes.louisville.edu!news From: Elias Klein Newsgroups: bionet.molbio.proteins Subject: Murine monoclonals on Hollow Fibers Date: 8 Aug 1995 13:37:58 GMT Organization: University of Louisville, Louisville KY USA Lines: 12 Message-ID: <407pbm$10a@hermes.louisville.edu> NNTP-Posting-Host: eklein1.kdp-baptist.louisville.edu Would like to exchange experiences with anyone growing murine monoclonals We use about 5% fetal bovine in the media together with phenol red. Both these additives complicate the eventual purification (Pr A). Would like to minimize the processing between haravest and purification to reduce losses on tubing, walls, etc. Our purification - temporarily - consists of dialyzing the media against 0.5-0.8 M Na2SO4 and then loading the Ab soln onto a Pr A bed. We elute with low salt and low pH. If we don't dialyze off the phenol red it sticks to the Pr A matrix and elutes with the Ab. Any suggstions on how to reduce the phenol red problem? Unfortunately, the medium we use comes only with the indicator in it. My E-mail is: e0klei01@ULKYVM.LOUISVILLE.EDU From owner-proteins@net.bio.net Mon Aug 07 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!usc!howland.reston.ans.net!tank.news.pipex.net!pipex!oleane!jussieu.fr!univ-lille1.fr!cict.fr!ecstasy!natalie From: natalie@ecstasy.cemes.fr (Natalie Chinardet) Newsgroups: bionet.molbio.proteins Subject: native page recipe ? Date: 8 Aug 1995 10:54:11 GMT Lines: 18 Message-ID: <407foj$4ae@news.cict.fr> NNTP-Posting-Host: ecstasy.cemes.fr X-Newsreader: TIN [version 1.2 PL2] Dear netters, I'm looking for a native page recipe which would allow my protein to migrate as a cation, because it is not really soluble above its pI (8.3-8.4). Thanks in advance, natalie ---------------------------------------------------------------------------- Natalie Chinardet e-mail : natalie@ecstasy.cemes.fr Groupe de Cristallographie Biologique du LPTF Toulouse France ---------------------------------------------------------------------------- From owner-proteins@net.bio.net Mon Aug 07 23:00:00 1995 Path: biosci!bcm!pendragon!news.msfc.nasa.gov!newsfeed.internetmci.com!gatech!concert!bigblue.oit.unc.edu!mmlds1.pha.unc.edu!iiv From: Iosif Vaisman Newsgroups: bionet.molbio.proteins,bionet.molec-model,bionet.software,bionet.software.gcg Subject: Computational Molecular Biology Workshop Date: Tue, 8 Aug 1995 13:17:47 -0400 Organization: The University of North Carolina at Chapel Hill Lines: 52 Message-ID: NNTP-Posting-Host: mmlds1.pha.unc.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII Xref: biosci bionet.molbio.proteins:5265 bionet.molec-model:531 bionet.software:12949 bionet.software.gcg:1340 CAROLINA WORKSHOPS Computational Molecular Biology October 22 -- October 27, 1995 University of North Carolina at Chapel Hill This course is designed for scientists with limited prior experience in computational molecular biology. The topics to be covered include: biomolecular informatics and computer networks, protein and nucleic acid databases, protein and nucleic acid sequence analysis and alignment, 3D protein structure analysis and prediction, molecular modeling and dynamic simulations of proteins and nucleic acids, and structure-based drug design. The workshop will consist of the in-depth theoretical lectures and intensive hands-on laboratory sessions. The course will be taught at the training facility of the North Carolina Supercomputing Center, Research Triangle Park, North Carolina. CAROLINA WORKSHOPS are intensive hands-on courses designed to teach cutting edge methods in molecular biology and biotechnology. Four or five courses on different topics in molecular biology and/or biotechnology are offered each year. The courses are designed for novice students as well as for individuals with prior experience. All students benefit from in-depth interaction with instructors. To apply, send a curriculum vitae and a brief letter describing your research interests and their relevance to the Workshop. Applicants should contact the program office as soon as possible. Please indicate your complete mailing address and telephone/fax number. Application Deadline-September 7, 1995. Tuition - $ 1,200.00. Participation is limited to 15 people, please apply early. COURSE DIRECTOR: Alexander Tropsha, University of North Carolina at Chapel Hill INSTRUCTORS: Frank K. Brown (Glaxo-Wellcome) Wayne Litaker (UNC-Chapel Hill) Michael Mitchell (Becton-Dickinson) David C. Richardson (Duke University) Alexander Tropsha (UNC-Chapel Hill) Iosif Vaisman (UNC-Chapel Hill) For further information or to apply, contact: Dr. Wayne Litaker, Facility Director University of North Carolina at Chapel Hill Program in Molecular Biology & Biotechnology 402 Taylor Hall, CB 7100 Chapel Hill, North Carolina 27599-7100 TELEPHONE (919) 966-1730, FAX (919) 966-6821 Email Address: litaker@med.unc.edu From owner-proteins@net.bio.net Mon Aug 07 23:00:00 1995 Path: biosci!biosci!not-for-mail From: communications@asmusa.org (ASM Communications) Newsgroups: bionet.announce,bionet.celegans,bionet.cellbiol,bionet.general,bionet.immunology,bionet.microbiology,bionet.molbio.hiv,bionet.molbio.proteins,bionet.molbio.yeast,bionet.parasitology,bionet.protista,bionet.virology Subject: ASM Homepage Date: 8 Aug 1995 11:20:26 -0700 Organization: American Society for Microbiology Lines: 30 Sender: biohelp@net.bio.net Approved: bionews-moderator@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Xref: biosci bionet.announce:2371 bionet.celegans:506 bionet.cellbiol:2659 bionet.general:16454 bionet.immunology:5030 bionet.microbiology:2909 bionet.molbio.hiv:1516 bionet.molbio.proteins:5264 bionet.molbio.yeast:3473 bionet.parasitology:942 bionet.protista:343 bionet.virology:4136 Microbiology has a new domain on the Web. The American Society for Microbiology (ASM) Homepage premiered on the World Wide Web August 1 at http://www.asmusa.org. Designed as a comprehensive, easy to navigate, information source on the microbiological sciences, the ASM homepage will appeal to everyone >From scientists to the lay public. The multimedia environment contains information and activities useful to ASM Members, other scientists and the general public: * Financial aid: Information on scholarships, fellowships and travel grants online. * Downloadable forms will allow users to do everything from joining the Society and ordering publications to searching for a job in microbiology. * Keep up to date on the latest research being published in the Society's 11 peer-reviewed journals with tables of contents online. Also online: press releases, meeting programs, position statements and congressional testimony on key issues affecting microbiology. * Lots of links connect users to a wealth of information on the microbiological sciences including universities, government agencies, newsgroups, electronic journals and the latest search engines for the web. Work has already begun on a future ftp site of Society documents as well as inline forms and credit card transactions. # # # The American Society for Microbiology, headquartered in Washington, D.C., is the oldest and largest single biological membership organization, with nearly 40,000 members worldwide. From owner-proteins@net.bio.net Mon Aug 07 23:00:00 1995 Path: biosci!bcm!news.msfc.nasa.gov!newsfeed.internetmci.com!usenet.eel.ufl.edu!noc.netcom.net!netcomsv!uu3news.netcom.com!netcomsv!uucp3.netcom.com!gatekeeper.genencor.com!usenet From: Greg Nudelman Newsgroups: bionet.molbio.proteins Subject: HIV Protease Inhibitor for Hire Date: 9 Aug 1995 00:58:28 GMT Organization: Genencor International, Inc. Lines: 28 Message-ID: <40917k$lg@gatekeeper.genencor.com> NNTP-Posting-Host: 10.1.1.100 X-Newsreader: AIR News 3.X (SPRY, Inc.) Hi! I have spent the last 8 months designing an HIV-Protease 1 inhibitor, and was successful at designing a structure with a better binding energy then the DuPont-Merck Cyclic Urea Inhibitor as tested on five available from bnl.pdb.pdb.gov x-ray crystal structures of this enzyme. GN213 is a cyclic, highly constrained diol which is specific to the flaps' water molecule, all four pockets and both catalitic aspartates. I've spoken with some synthetic Ph.D. chemists at San Francisco State University and they say that the synthesis should be trivial although don't seem to be that excited about making it. Does anyone (please!) know where I can test the binding constant, toxicity, e.t.c. once I have completed the synthesis? I'd like to work on this project further during my Ph.D.. Does anyone know someone I can talk to about it? Please reply inthis news group or e-mail me at: nudelman@lewis.sfsu.edu or nudelma@genencor.com In advance eternaly greatful, Greg Nudelman Senior in Chemistry/Biochemistry San Francisco State University P.S. Genencor Intl., a company where I have a summer internship, makes detergent enzymes and refuses to deal with pharmaceuticals. From owner-proteins@net.bio.net Mon Aug 07 23:00:00 1995 Path: biosci!daresbury!nntp-trd.UNINETT.no!Norway.EU.net!EU.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.proteins Subject: Re: How does ATP gamma S work? Date: 9 Aug 1995 02:31:49 GMT Organization: University of Illinois at Urbana Lines: 34 Message-ID: <4096ml$3no@vixen.cso.uiuc.edu> References: NNTP-Posting-Host: ux1.cso.uiuc.edu spenson@nature.berkeley.edu (Simon Penson) writes: >Dear Netters, >I need a substrate which will bind irreversibly and specifically to the >active site of a protein kinase. I don't want to get involved in >photoaffinity labelling if I can avoid it. So, my question is: > Does ATP gamma S bind irreversibly to protein kinases? If not, how does it > inhibit ATP-requiring enzymes/processes? =============== ATP gamma S does not bind irreversibly -- binding varies across the board. This compound was first touted as a non-hydrolyzable substrate, but close inspection has shown it can be hydrolyzed. You'd probably get further investingating 2'-azido-ATP, or any of a bevy of other ATP analogs that are commercially available (or can be synthesized -- some hard). Photoaffinity labelling really isn't that tough, getting the proper substrate probably will be. There are ATP derivatives with the attaching group on the gamma phosphate -- these might be of interest. You might also consider the dialdehyde derivative of ADP (periodate treated ADP) for labelling. Almost any labelling project gets involved if you do it properly, so it's usually best to consider this a reasonable effort endeavour. -- Eric Larson | University of Illinois at Urbana-Champaign USDA/Agronomy | 190 ERML; 1201 W. Gregory; Urbana, IL 61801 elarson@ux1.cso.uiuc.edu | Voice 217.244.3079 Fax 217.244.4419 Fidonet: 1:233/4.1 | My opinions are my own, but correct :-) From owner-proteins@net.bio.net Mon Aug 07 23:00:00 1995 Path: biosci!agate!news.mindlink.net!vanbc.wimsey.com!unixg.ubc.ca!hoekstra From: hoekstra@unixg.ubc.ca (Sucking Gippy) Newsgroups: bionet.molbio.proteins Subject: Storage and Reprobing Westerns Date: 9 Aug 1995 03:40:31 GMT Organization: University of British Columbia, Vancouver, B.C., Canada Lines: 15 Message-ID: <409anf$hce@nnrp.ucs.ubc.ca> NNTP-Posting-Host: interchg.ubc.ca X-Newsreader: TIN [version 1.2 PL2] In the last few months there have been a couple of threads concerning the storage of transferred membranes for later immunoblotting and reprobing westerns. I did read these posts but much to quickly...and wouldn't you know it...now I would like to try these techniques! First. After transferring a gel to nitrocellulose and blocking it with a protein solution..say 2% skim milk...air dry it..what are some probable storage conditions for later immunoblotting? Second. After probing for a specific protein and using the enzymatic reaction with NBT/BCIP, what could I use to effectively "strip off" the first and secondary Ab leaving the protein intact on the blot for another blotting procedure? Help is greatly appreciated. From owner-proteins@net.bio.net Mon Aug 07 23:00:00 1995 Path: biosci!bcm!news.msfc.nasa.gov!newsfeed.internetmci.com!usenet.eel.ufl.edu!noc.netcom.net!netcomsv!uu3news.netcom.com!netcomsv!uucp3.netcom.com!gatekeeper.genencor.com!usenet From: Greg Nudelman Newsgroups: bionet.molbio.proteins Subject: HIV Protease Inhibitor for Hire (0/1) Date: 9 Aug 1995 00:54:26 GMT Organization: Genencor International, Inc. Lines: 2 Message-ID: <409102$lg@gatekeeper.genencor.com> NNTP-Posting-Host: 10.1.1.100 X-Newsreader: AIR News 3.X (SPRY, Inc.) From owner-proteins@net.bio.net Mon Aug 07 23:00:00 1995 Path: biosci!daresbury!nntp-trd.UNINETT.no!Norway.EU.net!EU.net!howland.reston.ans.net!swrinde!elroy.jpl.nasa.gov!lll-winken.llnl.gov!fnnews.fnal.gov!unixhub!news.Stanford.EDU!tip-mp8-ncs-10.stanford.edu!user From: wridgway.leland.stanford.edu (ridgway) Newsgroups: bionet.molbio.proteins Subject: .PDB Database? Date: Tue, 08 Aug 1995 22:09:40 -0700 Organization: Stanford University Lines: 5 Message-ID: NNTP-Posting-Host: tip-mp8-ncs-10.stanford.edu I would be grateful if anyone could post an ftp address for a .pdb protein structure database. I've poked around with archie and webworm and haven't gotten much. I looked at the Brookhaven www site, but was surprised to find only .gif and .rgb formats. Any help would be much appreciated. From owner-proteins@net.bio.net Mon Aug 07 23:00:00 1995 Path: biosci!bcm!news.msfc.nasa.gov!elroy.jpl.nasa.gov!swrinde!howland.reston.ans.net!torn!news.bc.net!info.ucla.edu!news.ucdavis.edu!dale.ucdavis.edu!ez007089 From: Alejandro Ortiz-Acevedo Newsgroups: bionet.molbio.proteins Subject: photoactive compound Date: Tue, 8 Aug 1995 16:51:37 -0700 Organization: University of California, Davis Lines: 7 Message-ID: NNTP-Posting-Host: dale.ucdavis.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII Content-Transfer-Encoding: QUOTED-PRINTABLE X-Sender: ez007089@dale.ucdavis.edu I am working with a photoactive amiloride analogue called Nenmba. Does=20 anyone know what UV light power (=B5W/cm2) to use in order to photoactivate= =20 it? I need to perform the photoactivation in a 1.5% hematocrit solution=20 of red blood cells. Please send reply to alejandro@hph.ucdavis.edu Thanks=20 Alejandro From owner-proteins@net.bio.net Mon Aug 07 23:00:00 1995 Path: biosci!bcm!news.msfc.nasa.gov!elroy.jpl.nasa.gov!swrinde!howland.reston.ans.net!newsjunkie.ans.net!news.agt.net!news.planet.eon.net!news From: brian fitzsimons Newsgroups: bionet.molbio.proteins Subject: Information on medical research Date: 9 Aug 1995 02:53:36 GMT Organization: Public Live Access Network (PLAnet) Lines: 7 Message-ID: <4097vg$o31@tigger.planet.eon.net> NNTP-Posting-Host: 204.209.176.228 Would you like to know about some of the latest research on painful knees? Living Wills? The Genetics of Cancer? Or other health concerns? Look up the new Alberta Heritage Foundation for Medical Research website. The AHFMR is dedicated to research and development in the medical and scientific fields. http://www.sas.ab.ca/ahfmr From owner-proteins@net.bio.net Mon Aug 07 23:00:00 1995 Path: biosci!daresbury!not-for-mail From: lsprilus@inherit1.weizmann.ac.il (Jaime Prilusky) Newsgroups: bionet.molbio.proteins Subject: Re: .PDB Database? Date: 9 Aug 1995 07:00:12 +0100 Lines: 28 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <409itc$6o@mserv1.dl.ac.uk> Original-To: wridgway.leland.stanford.edu@dl.ac.uk, proteins@dl.ac.uk > I would be grateful if anyone could post an ftp address for a .pdb > protein structure database. I've poked around with archie and webworm and > haven't gotten much. I looked at the Brookhaven www site, but was > surprised to find only .gif and .rgb formats. Any help would be much > appreciated. I think you will find faster access to our local PDB database, URL ftp://bioinformatics.weizmann.ac.il/pub/databases/pdb or the EBI retrieval mechanism, if you already have the PDB ID (xxxx) URL http://www.ebi.ac.uk/htbin/pdbfetch?xxxx but you may also access the BNL source URL ftp://ftp.pdb.bnl.gov URL http://www.pdb.bnl.gov -- Dr Jaime Prilusky ! lsprilus@weizmann.weizmann.ac.il Head Bioinformatics Unit ! lsprilus@bioinformatics.weizmann.ac.il Dep. of Biological Services ! Weizmann Institute of Science ! fax: 972-8-344113 76100 Rehovot - Israel ! tel: 972-8-343456 info URL http://bioinformatics.weizmann.ac.il/jaime_prilusky.html From owner-proteins@net.bio.net Tue Aug 08 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!daresbury!nntp-trd.UNINETT.no!Norway.EU.net!EU.net!howland.reston.ans.net!news.starnet.net!wupost!news.utdallas.edu!corpgate!crchh327.rich.bnr.ca!brtph500.bnr.ca!bcarh189.bnr.ca!nott!cunews!freenet.carleton.ca!FreeNet.Carleton.CA!at332 From: at332@FreeNet.Carleton.CA (Matt Parker) Subject: Proteins with one tyrosine Message-ID: Sender: at332@freenet.carleton.ca (Matt Parker) Reply-To: at332@FreeNet.Carleton.CA (Matt Parker) Organization: The National Capital FreeNet Date: Wed, 9 Aug 1995 15:54:12 GMT Lines: 12 Hi! Does anyone know of a commercially - available (and preferably cheap) protein which has only ONE tyrosine and no tryptophans? We are trying to follow denaturation of a protein which fits that description, but we are having a lot of trouble getting reproducable results with our K-Mart fluorometer. My boss wants me to find out why it's not working well, and she suggested I use a similar protein to ours, instead of wasting huge quantities of our own, difficult to purify, protein. Thanks, Matt From owner-proteins@net.bio.net Tue Aug 08 23:00:00 1995 Path: biosci!CS.Arizona.EDU!news.Arizona.EDU!hamblin.math.byu.edu!park.uvsc.edu!news.provo.novell.com!news.cs.utah.edu!news.cc.utah.edu!news From: brad@corona.med.utah.edu (Brad Nicholson) Newsgroups: bionet.molbio.proteins Subject: Re: replacement of plasmid by another Date: 9 Aug 1995 22:05:38 GMT Organization: Department of Pathology Lines: 45 Message-ID: <40bbfi$5op@news.cc.utah.edu> References: <3vq74l$17ue@sat.ipp-garching.mpg.de> NNTP-Posting-Host: mac64_14.med.utah.edu X-Newsreader: Internet News version 1.0 In article <3vq74l$17ue@sat.ipp-garching.mpg.de>, jiang@assel.biochem.mpg.de (Weiping Jiang) wrote: >snip< >Is it possible to replace one plasmid by another one? >snip< >Weiping Hi Weiping, If I understand you question correctly, you want to get rid of the first plasmid that you put into your E. coli so that you are able to identify a mutation? Furthermore, you want to know if you can do this by adding a second plasmid? The answer is that this can be done easily, with a few caveats. 1) The first plasmid must be dispensible for the survival of the bacteria. 2) You need to have a plamsid of the same incompatability group with a unique selectable marker. Most of the common high copy # plasmids all use the same replicon. (pBR series, pUC series, or anything made from them use the pMB1 replicon). Alternatively, pACYC177 or 184, the broad host range plasmids or F plasmids all use different replicons. How to do it. Find a plasmid that is the same incompatability group as the plasmid you want to remove, but has a different selectable marker. Transform said plasmid into your strain of E. coli and select for resistance marker that is carried on the second plasmid. Let the colonies grow up and streak for singles, take the singles and test them for sensitivity to the antibiotic that the original plamsid gave them resistance to. Now you have a strain of E. coli that doesn't have the plasmid that you didn't want but now has another plasmid in it. If you are really lucky you can find a second plasmid with a temperature sensitive origin of replication, so that you can get rid of the second plasmid by raising the temperature. Alternatively, can you move the chromosomal mutation into a new strain with phage P1? Sorry this got so long, write me if you have questions. Brad =========================================================================== Brad Nicholson |"If it worked the first time, it wouldn't be Department of Pathology | research."...Brad Nicholson University of Utah | Live from behind the Zion Curtian. Salt Lake City, UT 84132 | brad@corona.med.utah.edu | or: (801)-581-4365 | My opinions are solely my own. =========================================================================== From owner-proteins@net.bio.net Tue Aug 08 23:00:00 1995 Path: biosci!daresbury!nntp-trd.UNINETT.no!Norway.EU.net!EU.net!howland.reston.ans.net!newsfeed.internetmci.com!newsxfer.itd.umich.edu!news.itd.umich.edu!usenet From: @ Newsgroups: bionet.molbio.proteins Subject: Re: Reduction/Pyridethylation/HPLC Purification of proteins(?) Date: 9 Aug 1995 15:54:51 GMT Organization: University of Michigan Lines: 6 Message-ID: <40alob$6qg@lastactionhero.rs.itd.umich.edu> References: <1F7AF6B2027@botany.uq.edu.au> Reply-To: @ NNTP-Posting-Host: 50.33.med.umich.edu X-Newsreader: IBM NewsReader/2 v1.03 I would assume that your reason for the red/alk is that you are preparing to sequence this protein. I am also assuming that you have a protocol for S- pyridylethylation of Cystine. I normally do the reaction, without the presence of argon, and simpley desalt the sample by RP-HPLC using the small C-4 column by Brownlee/ From owner-proteins@net.bio.net Tue Aug 08 23:00:00 1995 Path: biosci!daresbury!bioftp.unibas.ch!citi2.fr!jussieu.fr!univ-lille1.fr!cict.fr!ecstasy!natalie From: natalie@ecstasy.cemes.fr (Natalie Chinardet) Newsgroups: bionet.molbio.proteins Subject: IPG-IEF with Multiphor II Date: 9 Aug 1995 16:09:20 GMT Lines: 25 Message-ID: <40amjg$mo0@news.cict.fr> NNTP-Posting-Host: ecstasy.cemes.fr X-Newsreader: TIN [version 1.2 PL2] Dear Netters, I would like to get in touch with people PEPARING and running IPG-IEF with Multiphor II (Pharmacia). After several trials we're still unable to obtain good results : The gel became wavy near the electrodes and sometimes burnt at the sample loading position... We're not sure that is correctly dried and we're wondering if the running conditions are good. It seems that for homemade gels, the protocols given in user manual should be adapted. Any information would be appreciated, thanks in advance. natalie ------------------------------------------------------------------- Natalie Chinardet natalie@ectasy.cemes.fr Groupe de Cristallographie Biologique du LPTF Toulouse FRANCE ------------------------------------------------------------------------------------------------------------------------------------- From owner-proteins@net.bio.net Tue Aug 08 23:00:00 1995 Path: biosci!rutgers!uwm.edu!cs.utexas.edu!howland.reston.ans.net!tank.news.pipex.net!pipex!dispatch.news.demon.net!demon!uknet!bhamcs!news.ox.ac.uk!oxpath!rpgrant From: rpgrant@molbiol.ox.ac.uk (Mad Dan Eccles) Newsgroups: bionet.molbio.proteins Subject: Re: Storage and Reprobing Westerns Date: 9 Aug 95 15:24:59 GMT Organization: Oxford University Molecular Biology Data Centre Lines: 35 Message-ID: <1995Aug9.152459@ania.path.ox.ac.uk> References: <409anf$hce@nnrp.ucs.ubc.ca> <409q6r$837@phunn1.sb.com> NNTP-Posting-Host: ania.path.ox.ac.uk In article <409q6r$837@phunn1.sb.com>, Steven_Mcclue-1%notes@sb.com (Steve Mcclue) writes: > In article <409anf$hce@nnrp.ucs.ubc.ca>, hoekstra@unixg.ubc.ca says... > >>Second. After probing for a specific protein and using the enzymatic >>reaction with NBT/BCIP, what could I use to effectively "strip off" the >>first and secondary Ab leaving the protein intact on the blot for another >>blotting procedure? > > I haven't tried this myself, but one method for stripping blots involved washing with 2%SDS + > 100mM b-mercaptoethanol for 30 min at 70oC. > See Kaufmann, Anal Biochem, 161:89-95 (1987) From my thesis.. After detection, filters were wetted with dH2O and incubated in 62.5 mM Tris-ClpH 6.8, 2% SDS, 100 mM [[beta]]-mercaptoethanol at 50oC for 30 min, with occasional agitation. Filters were placed into fresh buffer and the incubation repeated. The filters were then washed twice at room temperature for 10 min in 500 ml PBS-T, blocked as in 2.20.2 (a) and reprobed. --------------------------------------------------------------- Works a treat. For further details, see my Thesis, Bodleian Library, Oxford, email me, or follow http://www.molbiol.ox.ac.uk/www/users/rpgrant/thesis :) -- Richard P. Grant MA DPhil rpgrant@molbiol.ox.ac.uk Nuffield Department of Obstetrics and Gynaecology, University of Oxford. http://sable.ox.ac.uk/~lady0266 Call yourself a dog? I've seen better hairs on a lavatory brush! From owner-proteins@net.bio.net Tue Aug 08 23:00:00 1995 Path: biosci!bcm!news.msfc.nasa.gov!newsfeed.internetmci.com!news.sprintlink.net!dispatch.news.demon.net!demon!uknet!bhamcs!news.ox.ac.uk!news From: Geoff Barton Newsgroups: bionet.molbio.proteins Subject: Re: Protein homology conventions Date: 9 Aug 1995 13:02:26 GMT Organization: Laboratory of Molecular Biophysics Lines: 55 Message-ID: <40abl2$rs3@news.ox.ac.uk> References: NNTP-Posting-Host: weevil.biop.ox.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (X11; I; IRIX 5.3 IP22) X-URL: news:obrien-070895132406@brass2.med.upenn.edu obrien@pharm.med.upenn.edu (PJOB) wrote: >Are there widely accepted criteria for declaring that amino acid residues >in related proteins are homologous? I am aligning a few receptor sequences >and wish to express their relatedness in terms of identity and homology. >Identity I can handle, but is there a set of amino acids that is generally >accepted as homologous? Doubtless others will have someting to say about this, but here is my 2d worth. There is a lot of misuse and abuse of the word homology in the literature relating to sequence comparison. You should read the letter by Reeck et al that was published in Cell back in 1987 for guidance. The bottom line is that homology should not be used to mean "similarity". There is no such thing as a set of amino acids that are "homologous". HOMOLOGY IN PROTEINS AND NUCLEIC-ACIDS - A TERMINOLOGY MUDDLE AND A WAY OUT OF IT REECK_GR, DEHAEN_C, TELLER_DC, DOOLITTLE_RF, FITCH_WM, DICKERSON_RE, CHAMBON_P, MCLACHLAN_AD, MARGOLIASH_E, JUKES_TH, ZUCKERKANDL_E CELL, 1987, Vol.50, No.5, p.667 In general it is safest to quote percentage identity together with the length of the alignment and how that identity was calculated (e.g. divide by shortest sequence length). You need the length since percentage identity expected by chance is strongly length dependent. (see http://geoff.biop.ox.ac.uk/papers/rev93_1/rev93_1.html and references therein for guidance). You should also quote S.D. scores from randomisation of the sequences since this corrects in part for composition bias. There are several programs around that will give you this. You can see our home page for one! It is also useful to quote BLAST probabilities for the comparison of your sequences. This will give an idea of how unusual the sequence similarity you are describing is compared to the current sequence database. >Also, how does one express homology/identity in the case where the proteins >are of unequal length? Would it be proper to only refer to overlapping or >corresponding regions? For example, if two G-protein coupled receptors are >reasonably similar between their putative membrane spanning domains, but >one has a much longer n- or c- terminus, how should the homology/identity >be expressed? It is common practice to express similarities for multidomain proteins on a domain by domain basis. Geoff. -- Geoffrey J. Barton, Laboratory of Molecular Biophysics, University of Oxford, Rex Richards Building, South Parks Road, Oxford OX1 3QU, U.K. email: gjb@bioch.ox.ac.uk, Tel: +44 1865 275368, Fax: +44 1865 510454, ftp://geoff.biop.ox.ac.uk, http://geoff.biop.ox.ac.uk From owner-proteins@net.bio.net Tue Aug 08 23:00:00 1995 Path: biosci!bcm!news.tamu.edu!mac2wild2.tamu.edu!user From: Yasha@bioch.tamu.edu (Yasha Hartberg) Newsgroups: bionet.molbio.proteins Subject: Zn Fingers Date: 9 Aug 1995 17:03:45 GMT Organization: Texas A&M University Lines: 4 Message-ID: NNTP-Posting-Host: mac2wild2.tamu.edu I was curious to know the range of Zn binding constants for known Zn fingers. Any help would be greatly appreciated. "Now ritual is the husk of faith and loyalty, the beginning of confusion." Lao Tsu From owner-proteins@net.bio.net Tue Aug 08 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!usc!howland.reston.ans.net!newsserver.jvnc.net!netnews.sb.com!news From: Steven_Mcclue-1%notes@sb.com (Steve Mcclue) Newsgroups: bionet.molbio.proteins Subject: Re: Storage and Reprobing Westerns Date: 9 Aug 1995 08:04:43 GMT Organization: Smithkline Beecham Pharmaceuticals Lines: 13 Message-ID: <409q6r$837@phunn1.sb.com> References: <409anf$hce@nnrp.ucs.ubc.ca> NNTP-Posting-Host: had542.uk.sb.com Mime-Version: 1.0 X-Newsreader: WinVN 0.99.2 In article <409anf$hce@nnrp.ucs.ubc.ca>, hoekstra@unixg.ubc.ca says... >Second. After probing for a specific protein and using the enzymatic >reaction with NBT/BCIP, what could I use to effectively "strip off" the >first and secondary Ab leaving the protein intact on the blot for another >blotting procedure? I haven't tried this myself, but one method for stripping blots involved washing with 2%SDS + 100mM b-mercaptoethanol for 30 min at 70oC. See Kaufmann, Anal Biochem, 161:89-95 (1987) Steve From owner-proteins@net.bio.net Tue Aug 08 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!newsfeed.internetmci.com!newsxfer.itd.umich.edu!news.itd.umich.edu!usenet From: @ Newsgroups: bionet.molbio.proteins Subject: Re: Reduction/Pyridethylation/HPLC Purification of proteins(?) Date: 9 Aug 1995 16:05:37 GMT Organization: University of Michigan Lines: 16 Message-ID: <40amch$6qg@lastactionhero.rs.itd.umich.edu> References: <1F7AF6B2027@botany.uq.edu.au> Reply-To: @ NNTP-Posting-Host: 50.33.med.umich.edu X-Newsreader: IBM NewsReader/2 v1.03 Yes it is common to get several peaks without protein present. It is not neccessary to carry the reaction out under argon, as I have found no "oxidation byproducts" which would interfere with Edman sequencing. I am assuming that sequencing is the goal of your red/alk/purification. Desalting is simpley carried out on a narrow-bore RP-HPLC. I use a Brownlee/Perkin Elmer C-4 column .21 x 4 cm, pumping aqeous for 40', followed by high solvent (80% MeCN/0.1% TFA) wash to remove the bound protein. The protocol for the red/alk I routinely use is a modified version of Applied Biosystems User Bulletin #28, and should you require a copy just drop me an e-mail. Hope this helps. Jake Tropea Protein & Carbohydrate Structure Facility University of Michigan (313)763-6710 tropea@brcf.med.umich.edu From owner-proteins@net.bio.net Tue Aug 08 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!news.sprintlink.net!uunet!in2.uu.net!nih-csl!postman From: knierman@ncifcrf.gov (Michael D. Knierman) Subject: Re: HPLC Troubleshooting question Message-ID: <1995Aug9.183821.18166@alw.nih.gov> Sender: postman@alw.nih.gov (AMDS Postmaster) Nntp-Posting-Host: knierman-home.ncifcrf.gov Organization: National Institutes of Health X-Newsreader: Forte Free Agent v0.55 References: <11EF24B23F5@botany.uq.edu.au> <3v5l1q$6v2@fu-berlin.de> <3viqua$cva@amcnix.amc.uva.nl> Date: Wed, 9 Aug 1995 21:39:56 GMT Lines: 55 valeryt@ag.arizona.edu (Valery F. Thompson) wrote: >In article <3viqua$cva@amcnix.amc.uva.nl> seppen@amc.uva.nl writes: >>From: seppen@amc.uva.nl >>Subject: Re: HPLC Troubleshooting question >>Date: Mon, 31 Jul 95 16:56:07 -100 >>In Article <3v5l1q$6v2@fu-berlin.de> >>strecker@chemie.fu-berlin.de (Andreas Strecker) writes: >>>In article <11EF24B23F5@botany.uq.edu.au>, >>> J.Marcus@botany.uq.edu.au ("Marcus, Dr J.") wrote: >>>>Dear Netters, >>>> I have been struggling with our Waters HPLC >>>>for over a month now and would appreciate ANY clues >>>>that might help sort out the problem. If there is any >>>>interest, I will post a summary of responses. >>>> The problem manifests itself as an extremely wavy >>>>baseline which coincides with the pump cycle of pump B >>>>(6000A Series pump with dual piston design). After >>>>changing the pump seals and check valves, the problem >>>>remains. Every indication now seems to point to a bubble in >>>>one of the pump heads; but, try as I may, I have not been >>>>able to get rid of it. I have tried pumping methanol; I >>>>have used the solvent draw-off valve to pressurize the >>>>intake line while the pump is going at 10ml/min; I have >>>>tried tapping the pump head. >>> >>> >>>>John Marcus >>> >I have another possibility that I haven't seen mentioned. I have a Waters 625 >pump. I have had many problems with air bubbles. They are coming from the >mixer. It doesn't necessarily happen only when mixing solvents. On the 625 >pump, the tubing going into the check valves is white (semi-clear) plastic. I >can see the bubbles going from the mixer to the check valves. Waters has >replaced the mixer once a couple of years ago and it seemed to work for a >while. But the problem is back. I haven't quite decided what to do about it. >It is no longer under warranty and we don't have a service contract for it. >I'm debating whether or not the problem is bad enough to warrant spending the >money for a service call. >Other than this annoyance I have been really please with the system. >Good luck with your problem. >Valery F. Thompson >Sr. Research Specialist >Muscle Biology Group >University of Arizona >valeryt@ag.arizona.edu You don't mention the solvents you are using. I found that I needed to sparge my solvents or they would degas in the mixing valve when using acetronitrile/water. From owner-proteins@net.bio.net Tue Aug 08 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!news.sprintlink.net!uunet!in2.uu.net!nih-csl!postman From: knierman@ncifcrf.gov (Michael D. Knierman) Subject: Re: Murine monoclonals on Hollow Fibers Message-ID: <1995Aug9.183357.18007@alw.nih.gov> Sender: postman@alw.nih.gov (AMDS Postmaster) Nntp-Posting-Host: knierman-home.ncifcrf.gov Organization: National Institutes of Health X-Newsreader: Forte Free Agent v0.55 References: (none) <407pbm$10a@hermes.louisville.edu> Date: Wed, 9 Aug 1995 21:35:32 GMT Lines: 37 Elias Klein wrote: >Would like to exchange experiences with anyone growing murine monoclonals >We use about 5% fetal bovine in the media together with phenol red. Both >these additives complicate the eventual purification (Pr A). Would like >to minimize the processing between haravest and purification to reduce >losses on tubing, walls, etc. > Our purification - temporarily - consists of dialyzing the media >against 0.5-0.8 M Na2SO4 and then loading the Ab soln onto a Pr A bed. We >elute with low salt and low pH. If we don't dialyze off the phenol red >it sticks to the Pr A matrix and elutes with the Ab. Any suggstions on >how to reduce the phenol red problem? Unfortunately, the medium we use >comes only with the indicator in it. > My E-mail is: e0klei01@ULKYVM.LOUISVILLE.EDU My name is Mike Knierman and I work in a contract facility for the NCI making monoclonal antibodies and recombinant proteins for Phase I clinical trials. I showed your article to our antibody production people and this is thier reply. " Currently we are growing murine hybridomas in a Cellex hollow fiber bioreactor. We have our media manufactured by Biowhittaker without phenol red. We have had succuss growing cells serum free in the bioreactor using Biowhittaker Ultraculture prepared without phenol red. We anticipate most of our runs this way. In cases where FBS is necessary we have found 2% to be a sufficient concentration. The only other additive used is an increased concentration of glutamine to 4mM final. What media are you using? " If you would like to reply to them you can send me an email. They are intersted in knowing which bioreactor you are using. I work in a group that develops purification methods for the monoclonal antibodies as well as manufacture, refold and purify recombinant proteins from e.coli inclusion bodies. We have a number of diffrent approaches for Ab if you are interested. From owner-proteins@net.bio.net Tue Aug 08 23:00:00 1995 Path: biosci!ns1.faseb.org!darwin.sura.net!mother.usf.edu!news From: tabibzadeh@rics.moffitt.usf.edu (SIAMAK TABIBZADEH) Newsgroups: bionet.molbio.proteins Subject: Frontiers in Bioscience, an electronic journal and virtual library Date: 9 Aug 1995 16:36:42 GMT Organization: Moffitt Cancer Center at USF Lines: 40 Message-ID: <40ao6q$rp6@mother.usf.edu> NNTP-Posting-Host: pc3160.moffitt.usf.edu Mime-Version: 1.0 X-Newsreader: WinVN 0.93.11 Frontiers in Bioscience, an electronic journal and virtual library An electronic journal and virtual library has been created in order to facilitate rapid dissemination of scientific data as well as to provide investigators with numerous online tools for use in their day-to-day research activities. The publication cost is minimized or completely eliminated. A section of the journal is dedicated to publishing manuscripts that contain real time events. Access to a large number of databases is quite easy from the journal. These include databases for analysis of scientific data, search strategies, dictionaries, atlases, tutorials, conferences, information on products of various manufacturers, links to online journals and many other valuable information. Access to the journal and these services is free. The staff members of the journal are in the process of creation of various databases. One such database on gene knockout is already online. The journal can be accessed at the following address on WWW: http://bayanet.com/bioscinece Although submission of data for publication in electronic platforms has just begun, this method of distribution of scientific information would certainly be the logical route of the future. The first volume of the journal to be published around Jan 1996 will contain excellent manuscripts. Please take a moment to examine the journal and consider to send manuscripts for publication in this new and novel forum. The address of the editorial office is as follows: Frontiers in Bioscience S Tabibzadeh, MD, Dept of Pathology University of South Florida 12901 Bruce B Downs Blvd Tampa, FL 33612 Tel: 813-979-7237 Fax: 813-979-3085 E-mail: tabibzadeh@rics.moffitt.usf.edu From owner-proteins@net.bio.net Tue Aug 08 23:00:00 1995 Path: biosci!bcm!pendragon.jsc.nasa.gov!news.msfc.nasa.gov!newsfeed.internetmci.com!usenet.eel.ufl.edu!ukma!hermes.louisville.edu!news From: Elias Klein Newsgroups: bionet.molbio.proteins Subject: Time on the newsgroup Date: 9 Aug 1995 17:12:29 GMT Organization: University of Louisville, Louisville KY USA Lines: 3 Message-ID: <40aq9t$rhn@hermes.louisville.edu> NNTP-Posting-Host: eklein1.kdp-baptist.louisville.edu Can anyone tell me how long a message stays active on this newsgroup ? I placed a query here yesterday about monoclonals, and today it's gone from the list. Thanks for your help. EKLEIN e0klei01@ULKYVM.LOUISVILLE.EDU From owner-proteins@net.bio.net Tue Aug 08 23:00:00 1995 Path: biosci!CS.Arizona.EDU!news.Arizona.EDU!hamblin.math.byu.edu!park.uvsc.edu!news.provo.novell.com!news.cs.utah.edu!news.cc.utah.edu!zifi.genetics.utah.edu!zinc From: zinc@zifi.genetics.utah.edu (zinc) Newsgroups: bionet.molbio.proteins Subject: Re: Time on the newsgroup Date: 9 Aug 1995 20:57:42 GMT Organization: assorted dsRNAs and lotsa zinc Lines: 35 Message-ID: <40b7g6$539@news.cc.utah.edu> References: <40aq9t$rhn@hermes.louisville.edu> NNTP-Posting-Host: zifi.genetics.utah.edu -----BEGIN PGP SIGNED MESSAGE----- In article <40aq9t$rhn@hermes.louisville.edu>, Elias Klein wrote: >Can anyone tell me how long a message stays active on this newsgroup ? >I placed a query here yesterday about monoclonals, and today it's gone from the list. >Thanks for your help. EKLEIN e0klei01@ULKYVM.LOUISVILLE.EDU howdy, the length of time a mesg 'lasts' is solely dependent on how your network news site is configured. there is not a central storage location for news. if you think articles are expiring too fast at your site then you should talk to your administrator. it appears you are getting news from hermes.louisville.edu, a mail mesg to news@hermes.louisville.edu or perhaps to root@hermes.louisville.edu regarding this might be appropriate. - -pjf - -- patrick finerty = zinc@zifi.genetics.utah.edu = pfinerty@nyx.cs.du.edu U of Utah biochem grad student in the Bass lab - zinc fingers + dsRNA! ** FINGER zinc-pgp@zifi.genetics.utah.edu for pgp public key - CRYPTO! zifi runs LINUX 1.2.11 -=-=-=WEB=-=-=-> http://zifi.genetics.utah.edu -----BEGIN PGP SIGNATURE----- Version: 2.6.2 iQCVAwUBMCkhlU3Qo/lG0AH5AQGqqwQAs1POhD7Tz50CRq+H5TTzxclwyEXBizbr CFBz0YZvNE2TF6yD0X7LFfuF0Md660beZ8Zs9QqhSMwauyTBA+2Oj4ekPgLMiJp/ jD69NbS+uLlbn/scHCrvyjkc6icx9hN2+TScHcnRkD2l8Jl39J7mV7RaNRNYsc0D Jqpm8YyOyoQ= =AgcA -----END PGP SIGNATURE----- From owner-proteins@net.bio.net Wed Aug 09 23:00:00 1995 Path: biosci!rutgers!uwm.edu!vixen.cso.uiuc.edu!news.uoregon.edu!newsfeed.internetmci.com!EU.net!Germany.EU.net!news.dfn.de!news.belwue.de!news.uni-stuttgart.de!uni-regensburg.de!lrz-muenchen.de!fauern!winx03!wpxx02.toxi.uni-wuerzburg.de!krasel From: krasel@wpxx02.toxi.uni-wuerzburg.de (Cornelius Krasel) Newsgroups: bionet.molbio.proteins Subject: Re: Time on the newsgroup Date: 10 Aug 1995 11:11:02 GMT Organization: Dept. of Pharmacology, U Wuerzburg Lines: 18 Message-ID: <40cpg6$7m6@winx03.informatik.uni-wuerzburg.de> References: <40aq9t$rhn@hermes.louisville.edu> NNTP-Posting-Host: wpxx02.toxi.uni-wuerzburg.de X-Newsreader: TIN [version 1.2 PL2] Elias Klein (e0klei01@ulkyvm.louisville.edu) wrote: > Can anyone tell me how long a message stays active on this newsgroup ? It depends on your local news server. You could ask your local administrator to lengthen the expire time for articles. However, if an article disappears from your local news server, this does not mean at all that it has disappeared from all servers around the world. For example, your question on monoclonals still is available here at the University of Wuerzburg, Germany, together with one response. Hope that helps, --Cornelius. -- /* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */ /* D-97078 Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de */ /* "Science is the game we play with God to find out what His rules are." */ From owner-proteins@net.bio.net Wed Aug 09 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!rutgers!gatech!news.sprintlink.net!dispatch.news.demon.net!demon!uknet!newsfeed.ed.ac.uk!leeds.ac.uk!news From: bmbrl@biovax.leeds.ac.uk Subject: Scatchard Analysis of Ligand Binding Message-ID: <1995Aug10.113031.26610@leeds.ac.uk> Reply-To: bmbrl@biovax.leeds.ac.uk NNTP-Posting-Host: bmbvax.leeds.ac.uk Organization: The University of Leeds, UK Date: Thu, 10 Aug 1995 12:30:30 +0100 (BST) Lines: 12 I recently had a (hurried) discussion with a colleague who had investigated ligand binding to receptors which left me with some doubts over how far Scatchard analysis of ligand binding can be trusted. What are the common problems/pitfalls when using this method of analysis? Are there any assumptions about receptor behaviour that have to be made before carrying out a Scatchard analysis? As I said, it was a quick chat and I didn't have time for an in depth discussion, which is why I'm turning to the group for outside help. Finally, he mentioned something about non-specific binding that behaved in a way that might be confused with specific binding. Can this happen and how can it be avoided? Thanks in advance, Rob From owner-proteins@net.bio.net Wed Aug 09 23:00:00 1995 Path: biosci!bcm!news.msfc.nasa.gov!newsfeed.internetmci.com!tank.news.pipex.net!pipex!oleane!jussieu.fr!univ-lyon1.fr!swidir.switch.ch!cisun2000.unil.ch!news From: ROCHAT Bertrand Newsgroups: bionet.molbio.proteins Subject: looking for CYP - P450 newsgroups Date: 10 Aug 1995 11:18:54 GMT Organization: Hospvd Lines: 25 Message-ID: <40cpuu$5qc@cisun2000.unil.ch> NNTP-Posting-Host: pc2cerybiochim.hospvd.ch Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Windows; I; 16bit) I AM LOKING FOR NEWSGROUPS ON CYTOCHROMES P-450 TOPICS. THANX VERY MUCH TO SEND ME THESE ADDRESSES TO THE NEXT E-MAIL: Bertrand.Rochat@inst.hospvd.ch IF IT DOES NOT EXIST, HOW IS IT POSSIBLE TO CREATE P-450 NEWSGROUPS ? -- Bertrand.Rochat@inst.hospvd.ch Unite de biochimie et psychopharmacologie clinique Hopital de Cery CH-1008 PRILLY - Switzerland phone: +21. 643. 65. 05. fax: +21. 643. 64. 69. From owner-proteins@net.bio.net Wed Aug 09 23:00:00 1995 Path: biosci!rutgers!oitnews.harvard.edu!purdue!mozo.cc.purdue.edu!rhphmac11.cc.purdue.edu!user From: () Newsgroups: bionet.molbio.proteins Subject: help:immunoprecipitation of membrane protein oligomer Followup-To: bionet.molbio.proteins Date: 10 Aug 1995 08:09:44 GMT Organization: Purdue University Lines: 16 Distribution: world Message-ID: <40ces8$mj0@mozo.cc.purdue.edu> NNTP-Posting-Host: rhphmac11.cc.purdue.edu I study the gene of a membrane protein which has a role in immortalization of some B cell lines when it is transfected into the cells. The membrane protein has an ion channel like structure (six membrane spanning motiff and cytoplasmic 34 aa N-terminal domain and 199 aa C-terminal domain). The protein seems to form hetero and/or homo oligomers. In order to address the oligomerization of the protein, I would like to do immunoprecipitation with a mutant gene which expresses an antigen tagged protein of the membrane protein. The mutant gene expresses at high level enough to detect the protein by western blot. I think that this protein forms oligomers by non-cobalent interaction. I would like to have an optimal condition for the purification of the protein without disturbing it's quaternay sturcture. I am designing the protocol for the isolatation of the putative protein oligomer from membrane lipid to do immunoprecipiation. If you have some experience about immunoprecipitation of membrane protein oligomers, please advise me. I will appreciate your help. From owner-proteins@net.bio.net Wed Aug 09 23:00:00 1995 Path: biosci!bcm!news.msfc.nasa.gov!newsfeed.internetmci.com!news.sprintlink.net!howland.reston.ans.net!xlink.net!rz.uni-karlsruhe.de!news.urz.uni-heidelberg.de!usenet From: Houthaeve Tony Newsgroups: bionet.molbio.proteins Subject: Re: BRCN CLEAVAGE Date: 10 Aug 1995 07:51:56 GMT Organization: EMBL Lines: 14 Message-ID: <40cdqs$6cp@sun0.urz.uni-heidelberg.de> References: <404k0i$6cu@bisance.citi2.fr> NNTP-Posting-Host: mac-mann2.embl-heidelberg.de Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) X-URL: news:404k0i$6cu@bisance.citi2.fr If you perform the CNBr digest in TFA instead of HCOOH, you get Met-Ser and Met-Thr cleavage in higher yield. The W might oxidise if kept to long in acidic/aerobic conditions, creating cleavage problems. Purge before digest start with Argon or Nitrogen helps. Even then the message with CNBr cleavage is: that it does not always work so good as with say a Trypsin digest. Especially when working in the lower pmol protein range, it might become difficult. This is also, because all proteins are quit individual in behaviour and an all-problem-solving solution does not exist. But anyway, good trials. Tony Houthaeve PPG - EMBL-HD From owner-proteins@net.bio.net Wed Aug 09 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!daresbury!nntp-trd.UNINETT.no!Norway.EU.net!EU.net!uknet!newsfeed.ed.ac.uk!udcf.gla.ac.uk!nobody From: William Sands Subject: Re: MAP Kinase inhibitor? Content-Type: text/plain; charset=us-ascii Message-ID: To: SYLVIA@THORIN.UTHSCSA.EDU Sender: nobody@udcf.gla.ac.uk (uid no body) Content-Transfer-Encoding: 7bit Organization: University of Glasgow References: <950804201154.220041@thorin.uthscsa.edu> Mime-Version: 1.0 Date: Thu, 10 Aug 1995 14:08:14 GMT X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) X-Url: news:950804201154.220041@thorin.uthscsa.edu Lines: 20 SYLVIA@THORIN.UTHSCSA.EDU wrote: >Hello all. Does anyone know of a good SPECIFIC inhibitor of MAP kinase? >I've heard that Wortmannin will work, but it also inhibits PI-3-kinase. >Thanks in advance. >Vic >sylvia@thorin.uthscsa.edu Dear Vic, I don't know of any specific inhibitors of MAP kinase, but Tyrphostin A10 is reported to inhibit p42 MAPK phosphorylation (and presumably activation). As you are aware, inhibitors are only specific until someone shows otherwise, and it is probably a reasonable assumption that all inhibitors are at best only selective. Wortmannin will also inhibit PI-4-kinase and Myosin light chain kinase. Cheers, Billy From owner-proteins@net.bio.net Wed Aug 09 23:00:00 1995 Path: biosci!daresbury!nntp-trd.UNINETT.no!Norway.EU.net!EU.net!Portugal.EU.net!news.rccn.net!iris02.itqb.unl.pt!iris02.itqb.unl.pt!martel From: martel@doom.itqb.unl.pt (Paulo Martel) Newsgroups: bionet.molbio.proteins Subject: Re: Modeling Software Date: 10 Aug 1995 14:23:38 GMT Organization: Instituto de Tecnologia Quimica e Biologica, Portugal Lines: 42 Message-ID: References: <3ugpaf$hh6@newsbf02.news.aol.com> <3vl1dv$agf@sun4.bham.ac.uk> <1995Aug1.163717.20977@alw.nih.gov> <3vnmsi$239@news.ox.ac.uk> <1995Aug2.210613.12760@alw.nih.gov> <3vqjbl$6te@news.ox.ac.uk> <1995Aug3.181013.709@alw.nih.gov> NNTP-Posting-Host: doom.itqb.unl.pt In-reply-to: johnk@spasm.niddk.nih.gov's message of Thu, 3 Aug 1995 18:10:13 GMT In article <1995Aug3.181013.709@alw.nih.gov> johnk@spasm.niddk.nih.gov (John Kuszewski) writes: > Because xplor doesn't have any energy terms for modeling data like > MODELER's. It doesn't even have a solvation energy term. > MD simulations of normal force fields (CHARMm, AMBER, etc) > produce garbage for anything more complicated than ideal gasses, unless > you add in energy terms that force the structure to look like > experimental terms (eg., the NOE, chemical shift, and xray terms in > xplor, many of which are my code). With all due respect, I think you are wrong here. If you mean that the forcefields used in protein MD/Minimization are not good enough to do ab initio folding, I agree with you (we all know that, don't we ? :-)). A different thing is to start from a physically plausible conformation for a protein (either Xray, NMR or experimental data), and use it as starting conformation for an MD run. It seems to me that you are saying that you cannot get anything meaningful in this later case unless you use some kind of restraint based on experimental data _throughout_ the simulation. I disagree completely with you here: there are number of articles by the groups of Karplus, van Gunsteren and Levitt (among others) that show clearly how a long (in the nanosecond range) protein MD run can reproduce some dynamical properties of proteins and how the properties computed from trajectories agree well with the experiment (and the RMS from the experimental structure stays low throughout the simulation). Of course these are simulations in water. As for solvation terms, I don't think they add any level of realism to the system ; on the contrary, they are a cruder approximation of the solvent than the one given by in-water MD. Their real advantage is the speed up in CPU time you get by not having to treat water-protein interactions explicitly. |) /\/\ | e-mail: martel@doom.itqb.unl.pt | ._ O |aulo / \artel | phone: +351-1-4426171 Ext.235 | / //\. I.T.Q.B. | FAX: +351-1- | \>> | 2780 Oeiras, PORTUGAL | | \\ -- |) /\/\ | e-mail: martel@doom.itqb.unl.pt | ._ O |aulo / \artel | phone: +351-1-4426171 Ext.235 | / //\. I.T.Q.B. | FAX: +351-1- | \>> | 2780 Oeiras, PORTUGAL | | \\ From owner-proteins@net.bio.net Wed Aug 09 23:00:00 1995 Path: biosci!bcm!news.msfc.nasa.gov!newsfeed.internetmci.com!usenet.eel.ufl.edu!ukma!hermes.louisville.edu!news From: Elias Klein Newsgroups: bionet.molbio.proteins Subject: Re: Time on the newsgroup Date: 10 Aug 1995 16:19:10 GMT Organization: University of Louisville, Louisville KY USA Lines: 4 Message-ID: <40dbhu$qnb@hermes.louisville.edu> References: <40aq9t$rhn@hermes.louisville.edu> <40cpg6$7m6@winx03.informatik.uni-wuerzburg.de> NNTP-Posting-Host: eklein1.kdp-baptist.louisville.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Windows; I; 16bit) To: C.,Krasel Thanks for your explanation. But what really interested me was the quote at the bottom of your message. Who is it from ? EKlein E-Mail: e0klei01@ULKYVM.LOUISVILLE.EDU From owner-proteins@net.bio.net Wed Aug 09 23:00:00 1995 Path: biosci!rutgers!gatech!news.sprintlink.net!moonbeam.aecom.yu.edu!usenet From: gottfrie@alsys1.aecom.yu.edu (David S. Gottfried) Newsgroups: bionet.molbio.proteins Subject: Re: Scatchard Analysis of Ligand Binding Date: 10 Aug 1995 13:06:13 GMT Organization: Albert Einstein College of Medicine - Biophysics Lines: 19 Message-ID: <40d085$f7f@moonbeam.aecom.yu.edu> References: <1995Aug10.113031.26610@leeds.ac.uk> NNTP-Posting-Host: marcus.aecom.yu.edu X-Posted-From: InterNews 1.0.6@marcus.aecom.yu.edu X-Authenticated: gottfrie on POP host alsys1.aecom.yu.edu In article <1995Aug10.113031.26610@leeds.ac.uk> bmbrl@biovax.leeds.ac.uk writes: > I recently had a (hurried) discussion with a colleague who had investigated > ligand binding to receptors which left me with some doubts over how far > Scatchard analysis of ligand binding can be trusted. > What are the common problems/pitfalls when using this method of analysis? > Are there any assumptions about receptor behaviour that have to be made before > carrying out a Scatchard analysis? I reccomend that you take a look at the article "Numbers of Receptor Sites from Scatchard Graphs: Facts and Fantasies", Klotz, I. M., Science, 217, 1247 (1982). In general, with the advent of computers for doing non-linear curve fitting, there is no reason to transform binding data to Scatchard-type linear plots for extracting binding constants. Best wishes, David From owner-proteins@net.bio.net Wed Aug 09 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!bcm!news.msfc.nasa.gov!newsfeed.internetmci.com!news.sprintlink.net!in2.uu.net!utcsri!utnut!nott!cunews!freenet.carleton.ca!FreeNet.Carleton.CA!at332 From: at332@FreeNet.Carleton.CA (Matt Parker) Subject: Standards for native PAGE Message-ID: Sender: at332@freenet2.carleton.ca (Matt Parker) Reply-To: at332@FreeNet.Carleton.CA (Matt Parker) Organization: The National Capital FreeNet Date: Thu, 10 Aug 1995 20:22:35 GMT Lines: 15 Hi! I am planning to try to do some native PAGE. I was wondering if anybody knows of any proteins which I could use as rough standards. Since I am looking at a protein with a calculated (but not necessarily actual) pI of about 7.7, and it appears to exist as a mixture of monomers of about 11 kD and dimers, I am looking for cheap, readily available, slightly basic proteins of about 10 - 15 and 20-30 kD. Thanks! Matt PS...Please reply directly to me by e-mail. I am going on vacation soon, and I don't think these postings stay around for very long before they get deleted. From owner-proteins@net.bio.net Wed Aug 09 23:00:00 1995 Newsgroups: bionet.cellbiol,bionet.molbio.methds-reagnts,bionet.molbio.proteins Path: biosci!bcm!news.msfc.nasa.gov!newsfeed.internetmci.com!news.sprintlink.net!in1.uu.net!nih-csl!NewsWatcher!user From: newitt@nih.gov (John A. Newitt) Subject: Need GTPase for positive control Message-ID: Sender: postman@alw.nih.gov (AMDS Postmaster) Nntp-Posting-Host: 128.231.113.35 Organization: National Institutes of Health Date: Thu, 10 Aug 1995 21:34:29 GMT Lines: 12 Xref: biosci bionet.cellbiol:2690 bionet.molbio.methds-reagnts:32032 bionet.molbio.proteins:5305 I am doing some GTPase assays and would like to obtain an enzyme with intrinsic GTPase activity to use as a positive control. Does anyone know of a commercially available GTPase that I could use (or a source of a non-commercial enzyme). If it requires a GAP, I would also need to obtain that as well, but I would prefer a GTPase that has significant activity (Kcat.GTP ~ 1-5 per min.) by itself. Regards, John A. Newitt, Ph.D. | National Institutes of Health | FAX: 301-402-0387 Bethesda, Maryland USA | From owner-proteins@net.bio.net Wed Aug 09 23:00:00 1995 Path: biosci!KISTMAIL.KIST.RE.KR!hyunpark From: hyunpark@KISTMAIL.KIST.RE.KR (Park Hyun) Newsgroups: bionet.molbio.proteins Subject: (none) Date: 10 Aug 1995 23:57:04 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 1 Sender: daemon@net.bio.net Distribution: world Message-ID: <9508110652.AA19120@kistmail.kist.re.kr> NNTP-Posting-Host: net.bio.net unsubscribe list From owner-proteins@net.bio.net Wed Aug 09 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!news.tamu.edu!news.utdallas.edu!corpgate!bcarh189.bnr.ca!nott!nrcnet0.nrc.ca!BRI.NRC.CA!James.Fethiere From: James.Fethiere@BRI.NRC.CA (James Fethiere) Newsgroups: bionet.molbio.proteins Subject: Re: Scatchard Analysis of Ligand Binding Date: 11 Aug 1995 04:02:34 GMT Organization: Institut de recherche en biotechnologie Lines: 6 Message-ID: <40ekoq$snt@nrcnet0.nrc.ca> References: <1995Aug10.113031.26610@leeds.ac.uk> <40d085$f7f@moonbeam.aecom.yu.edu> NNTP-Posting-Host: hermes.bri.nrc.ca X-Newsreader: TIN [version 1.2 PL2] This is exactly the answer i was going to give. Actually i try as much as i can to discourage people around me from using scatchard analysis, specially to detect receptor heterogenity. However this method of analysis is still published quite heavily. Oh well.... From owner-proteins@net.bio.net Wed Aug 09 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!daresbury!nntp-trd.UNINETT.no!Norway.EU.net!EU.net!news.sprintlink.net!in2.uu.net!newsfeed.ACO.net!edvz.sbg.ac.at!news From: Anton Hutticher Subject: Help: Small peptides and protein identification Message-ID: Sender: news@wst.edvz.sbg.ac.at (USENET News System) Organization: University of Salzburg / Austria Date: Fri, 11 Aug 1995 04:31:38 GMT Lines: 62 Can anyone give me advice on evaluating our sequence data: Problem: We have used an antibody raised against Protein 1 in one species to identify a possibly related protein (protein 2) in another species ( by crossreaction on a western blot after SDS-PAGE). We purified and partially sequenced protein 2. Due to the method we used, we got a lot of rather short peptides, mostly 5 to 8 aa. Because the protein in question is evolving rather quickly and the species are not that close, the sequence homology is rather low. Searching the protein data bases with these peptides using the NCBIs BLASTP program gave a lot of hits (= proteins found) with low scores and a high probability of being produced just by chance for every peptide. The list of hits looks different for every peptide. There are several proteins which occur on more than one list, usually way down. No protein occurs on all lists and none occurs decidedly more often than the others. There is no obvious way to combine the scores of one protein from different lists ( that is, when the same protein has been found by searching with different peptides ) into one overall score, which could be compared to the overall scores of other proteins. What I would want is a program, which permits me to search the data base using several peptides at the same time. In effect I would use -say - four pentapetides as if they were one 20 aa long peptide with variable gaps between the pentapeptides and variable ordering. Of course the discriminatory power of four pentapetides is smaller because of the unknown gaps and order, but we still should see an improvement over searches with one pentapeptid only. The closest I have come to this is to use the convention of BLASTP that - means a variable gap. However this still means several to many queries for even small numbers of peptides. Worse; the scores are still given separately for each peptide separated by a gap instead of being combined into one score. My questions are : a) Is this the correct news: group? If not, where should I go. b) Is there a way to make BLASTP perform as wished. c) Is there any software which can deal with the situation of having several small peptides scattered over a sequence instead of having one or two sufficient long ones. d) Is there any other way which enables us to say in effect: "Protein 2 is probably the homolog of protein 1 in this species" or " Protein 2 is most related to Protein 3, not to Protein 1". Please post and e-mail me, since on my site the posts age off the system in about four days and I may not catch all posts. Thank you for your help Anton Hutticher e-mail: huttich@edvz.sbg.ac.at snail mail: Dr. Anton Hutticher Univ. Salzburg Inst. Zoologie, Abt. Tierphysiologie Hellbrunnerstr. 34 A-5020 Salzburg Austria, EUROPE From owner-proteins@net.bio.net Wed Aug 09 23:00:00 1995 Path: biosci!daresbury!nntp-trd.UNINETT.no!Norway.EU.net!EU.net!news.sprintlink.net!cs.utexas.edu!news.tamu.edu!news.utdallas.edu!news.starnet.net!wupost!waikato!canterbury.ac.nz!chmeds.ac.nz!gjacobs From: gjacobs@chmeds.ac.nz Newsgroups: bionet.molbio.proteins Subject: Locating the coords of human serum albumin Message-ID: <1995Aug11.100509.260@chmeds.ac.nz> Date: 11 Aug 95 10:05:09 +1200 References: <3uvvb1$rie@n.ruf.uni-freiburg.de> Lines: 20 Hello, I am trying to locate the coordinates of human serum albumin for someone else in my lab. This was originally published in Nature in 1992 (358:209-15) by He and Carter. The coordinates are not in PDB, nor in their waiting/pending/hold lists... Likewise Carter's email is not in my version of Teeter's list (which admittedly is a little out of date, but then the reference is 3 years ago). Daniel Carter address is given as the Space Science lab., Marshall Space Flight Center, Huntsville, Alabama. All the researcher concerned would like to do is to map a particular residue with respect to the active site -- a five minute job if the structure were available... sigh. Any ideas/help gratefully welcomed. Grant Jacobs. From owner-proteins@net.bio.net Wed Aug 09 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!rutgers!uwm.edu!cs.utexas.edu!usc!sdd.hp.com!vixen.cso.uiuc.edu!uchinews!gi-mac4.bsd.uchicago.edu!user From: krabenau@quads.uchicago.edu (karen elizabeth rabenau) Subject: G418 resistance of NIH 3T3 cells X-Nntp-Posting-Host: gi-mac4.bsd.uchicago.edu Message-ID: Sender: news@midway.uchicago.edu (News Administrator) Organization: university of chicago Date: Thu, 10 Aug 1995 06:28:00 GMT Lines: 3 I am a student doing a summer project, pressed for time, and hence need approx. [G418] for selection of NIH 3T3 cells transfected with E-cadherin- thanks, C.R. From owner-proteins@net.bio.net Wed Aug 09 23:00:00 1995 Path: biosci!bcm!news.msfc.nasa.gov!newsfeed.internetmci.com!usenet.eel.ufl.edu!ukma!hermes.louisville.edu!news From: Elias Klein Newsgroups: bionet.molbio.proteins Subject: Re: Time on the newsgroup Date: 10 Aug 1995 16:22:48 GMT Organization: University of Louisville, Louisville KY USA Lines: 5 Message-ID: <40dboo$qnb@hermes.louisville.edu> References: <40aq9t$rhn@hermes.louisville.edu> <40cpg6$7m6@winx03.informatik.uni-wuerzburg.de> NNTP-Posting-Host: eklein1.kdp-baptist.louisville.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Windows; I; 16bit) To: phak004@rzbox.uni-wuerzburg.de Thanks for your explanation. I'll talk to our mainframe administrator. But the real purpose of this message is to find the source of the quote at the end of the message. Where did it come from ? EKLEIN E-mail: e0klei01@ULKYVM.LOUISVILLE.EDU From owner-proteins@net.bio.net Wed Aug 09 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!convex!darwin.sura.net!mother.usf.edu!news From: tabibzadeh@rics.moffitt.usf.edu (SIAMAK TABIBZADEH) Newsgroups: bionet.molbio.proteins Subject: Frontiers in Bioscience, a journal and virtual library Date: 10 Aug 1995 14:29:13 GMT Organization: Moffitt Cancer Center at USF Lines: 40 Message-ID: <40d53p$73c@mother.usf.edu> NNTP-Posting-Host: pc3160.moffitt.usf.edu Mime-Version: 1.0 X-Newsreader: WinVN 0.93.11 Frontiers in Bioscience, a journal and virtual library An electronic journal and virtual library has been created in order to facilitate rapid dissemination of scientific data as well as to provide investigators with numerous online tools for use in their day-to-day research activities. The publication cost is minimized or completely eliminated. A section of the journal is dedicated to publishing manuscripts that contain real time events. Access to a large number of databases is quite easy from the journal. These include databases for analysis of scientific data, search strategies, dictionaries, atlases, tutorials, conferences, information on products of various manufacturers, links to online journals and many other valuable information. Access to the journal and these services is free. The staff members of the journal are in the process of creation of various databases. One such database on gene knockout is already online. The journal can be accessed at the following address on WWW: http://bayanet.com/bioscience Although submission of data for publication in electronic platforms has just begun, this method of distribution of scientific information would certainly be the logical route of the future. The first volume of the journal to be published around Jan 1996 will contain excellent manuscripts. Please take a moment to examine the journal and consider to send manuscripts for publication in this new and novel forum. The address of the editorial office is as follows: Frontiers in Bioscience S Tabibzadeh, MD, Dept of Pathology University of South Florida 12901 Bruce B Downs Blvd Tampa, FL 33612 Tel: 813-979-7237 Fax: 813-979-3085 E-mail: tabibzadeh@rics.moffitt.usf.edu From owne