From owner-proteins@net.bio.net Tue Feb 01 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!europa.eng.gtefsd.com!darwin.sura.net!fconvx.ncifcrf.gov!fcsparc6!pnh From: pnh@fcsparc6.ncifcrf.gov (Paul N Hengen) Subject: Re: Albumin binding to Nucleic Acids Message-ID: Summary: Why is it acetylated? Sender: Paul N. Hengen Nntp-Posting-Host: fcsparc6.ncifcrf.gov Organization: Frederick Cancer Research and Development Center References: <2ihtn3$g46@charm.magnus.acs.ohio-state.edu> Date: Wed, 2 Feb 1994 23:37:17 GMT Lines: 24 In article <2ihtn3$g46@charm.magnus.acs.ohio-state.edu> mwadhwa@magnus.acs.ohio-state.edu (Manpreet S Wadhwa) writes: >> I know that BSA is routinely used to stabilize or 'cushion' proteins that >> are in dilute buffers and this can lead to increased sp activity for >> enzymes. Could it be that the BSA is favorably affecting the RT rather >> than interacting with the NA? > > bsa apparently stabilizes many enzymes in dilute solution by preventing their > aggregation or non-specific adsorption at interfacial surfaces. stabilization > may also be afforded by glycerol or detergents like triton-x100. NEB supplies acetylated BSA with some of their restriction enzymes so you can add it to reduce spurious star activity if you like. Why is this the acetylated form? Also, FYI, the BSA from them is not exactly the best fraction. I ran some on SDS-PAGE and found several different bands when silver stained. ******************************************************************************* * Paul N. Hengen, Ph.D. /--------------------------/* * National Cancer Institute |Internet: pnh@ncifcrf.gov |* * Laboratory of Mathematical Biology | Phone: (301) 846-5581 |* * Frederick Cancer Research and Development Center| FAX: (301) 846-5598 |* * Frederick, Maryland 21702-1201 USA /--------------------------/* ******************************************************************************* From owner-proteins@net.bio.net Tue Feb 01 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!bcm!cs.utexas.edu!sdd.hp.com!vixen.cso.uiuc.edu!howland.reston.ans.net!europa.eng.gtefsd.com!darwin.sura.net!gatekeeper.es.dupont.com!esds01.es.dupont.com!MILLERTJ@esvx17.es.dupont.com From: millertj@esvx17.es.dupont.com (Thomas Miller, Dupont, Wilm, DE) Subject: Re: Ponceau S staining of PVDF membranes? Message-ID: <1994Feb2.141715.21567@es.dupont.com> Sender: news@es.dupont.com (USENET News System) Nntp-Posting-Host: esvx17.es.dupont.com Reply-To: millertj@esvx17.es.dupont.com Organization: DuPont (Opinions are those of the writer only) References: ,<2io4ka$cbm@elna.ethz.ch> Date: Wed, 2 Feb 1994 14:17:15 GMT Lines: 27 In article <2io4ka$cbm@elna.ethz.ch>, schlaefli@micro.biol.ethz.ch (Hans Rudolf Schläfli) writes: >In article , Bill Nowatzke says: >> Original post: >>I have been trying to blot a tryptic fragment from an 8% SDS PAGE to >>BioRad PVDF membrane. I thought that my blot was unsuccessful after >>attempting to visualize by Ponceau S. I could not even detect my MW >>standards. I decided to coomassie stain after destaining and saw all of >>the protein that I had thought I loaded. Any clues as to why my ponceau S >>did not work? I diluted a fresh Sigam concentrate (20 mL) into 180 mL of >>deionized water. I stained for 5 min and watch as the membrane destained >>in 1% AcOH. At no time did I see anything resembling a protein band. This>problem has occured for the past two consecutive times that I have tried >>the technique. Your comments are appreciated. > Ruedi's reply: >Your Ponceau probably worked, but it is a weak stain, compared to Coomassie, and PVDF >really is a bad blotting membrane and only appropriate for subsequent peptide sequencing. >Try a modified PVDF and/or load more protein or stain with Coomassie in 50% MeOH, this works >for peptide sequencing, too. >The exact procedure is described in "Current Protocols". > >Good luck. >Ruedi >schlaefli@micro.biol.ethz.ch We are using 0.1% Ponceau S in 1% acetic acid staining either ABI ProBlott or Millipore's Immobilon P-SQ membrane. I am not having any problem seeing protein bands but we've been testing it to see if we can do insitu digests off of the blots. So there is plenty of protein on the blot. Tom From owner-proteins@net.bio.net Tue Feb 01 22:00:00 1994 Path: biosci!daresbury!keele!uknet!EU.net!chsun!elna.ethz.ch!usenet From: schlaefli@micro.biol.ethz.ch (Hans Rudolf Schläfli) Newsgroups: bionet.molbio.proteins Subject: Re: Ponceau S staining of PVDF membranes? Date: 2 Feb 1994 12:00:10 GMT Organization: Microbiology ETHZ Lines: 21 Message-ID: <2io4ka$cbm@elna.ethz.ch> References: NNTP-Posting-Host: b26-pro486.ethz.ch X-Newsreader: WinVN 0.83.2 In article , Bill Nowatzke says: > >I have been trying to blot a tryptic fragment from an 8% SDS PAGE to >BioRad PVDF membrane. I thought that my blot was unsuccessful after >attempting to visualize by Ponceau S. I could not even detect my MW >standards. I decided to coomassie stain after destaining and saw all of >the protein that I had thought I loaded. Any clues as to why my ponceau S >did not work? I diluted a fresh Sigam concentrate (20 mL) into 180 mL of >deionized water. I stained for 5 min and watch as the membrane destained >in 1% AcOH. At no time did I see anything resembling a protein band. This>problem has occured for the past two consecutive times that I have tried >the technique. Your comments are appreciated. Your Ponceau probably worked, but it is a weak stain, compared to Coomassie, and PVDF really is a bad blotting membrane and only appropriate for subsequent peptide sequencing. Try a modified PVDF and/or load more protein or stain with Coomassie in 50% MeOH, this works for peptide sequencing, too. The exact procedure is described in "Current Protocols". Good luck. Ruedi schlaefli@micro.biol.ethz.ch From owner-proteins@net.bio.net Tue Feb 01 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!pipex!sunic!EU.net!ub4b!info-sparc1.info.ucl.ac.be!NewsWatcher!user From: marcd@bota.ucl.ac.be (Denis Marc) Subject: plant proteins extraction Message-ID: Followup-To: bionet.molbio.proteins Sender: news@info.ucl.ac.be (News Administrator) Nntp-Posting-Host: 130.104.7.44 Organization: Catholic University of Louvain Date: Wed, 02 Feb 1994 10:32:41 +0100 Lines: 9 I realize for the moment proteins extraction from flower organs grinded in liquid N2 with a boiling SDS buffer (4% SDS, 5% mercaptoethanol, 5% sucrose) followed by incubation in an acetone buffer for one hour a -20¡C. So, question is : Is the acetone step necessary and if it is, what is its interest? Thanks in advance D.M. From owner-proteins@net.bio.net Tue Feb 01 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!pipex!sunic!EU.net!sun4nl!star.cs.vu.nl!balaena!mac136.bio.vu.nl!user From: vdwal@bio.vu.nl (FJ van der Wal) Subject: pasteurella plasmids Message-ID: Followup-To: bionet.molbio.proteins Sender: news@bio.vu.nl Organization: Mol.Micro, Vrije Uni., Amsterdam, NL Date: Wed, 2 Feb 1994 15:09:01 GMT Lines: 15 Hello Does anyone know if plasmids for cloning in Pasteurella, or shuttle vectors also replicating in E.coli, are available somewhere? I would be very obliged if somebody could answer this question, and maybe a handover procedure could be initiated in the near future. I'm mailing this for a collegue; answers can be mailed through oudega@bio.vu.nl Thank in advance! -- Fimme Jan van der Wal dep. Molecular Microbiology Vrije Universiteit Amsterdam de Boelelaan 1087 1081 HV Amsterdam, NL vdwal@bio.vu.nl From owner-proteins@net.bio.net Wed Feb 02 22:00:00 1994 Path: biosci!CS.Arizona.EDU!math.arizona.edu!news.Arizona.EDU!hamblin.math.byu.edu!sol.ctr.columbia.edu!howland.reston.ans.net!vixen.cso.uiuc.edu!sdd.hp.com!col.hp.com!csn!ub!dsinc!netnews.upenn.edu!pharm07.med.upenn.edu!user From: hines@pharm.med.upenn.edu (John Hines) Newsgroups: bionet.molbio.proteins Subject: RIPA - Help?! Followup-To: bionet.molbio.proteins Date: 2 Feb 1994 22:12:40 GMT Organization: University of Pennsylvania Lines: 11 Distribution: world Message-ID: NNTP-Posting-Host: pharm07.med.upenn.edu Does anyone know what the RIPA stands for, as in RIPA lysis buffer/solubilization buffer ?? The recipe for it is in the Harlow and Lane manual on the use of antibodies, and it contains NaCl, Noniodet P-40, SDS, and deoxycholate (I think). I don't know where they get RIPA from and some hard ass is making my life difficult over this little point. Thank you. John Hines Univ. of Penna. sig. pending From owner-proteins@net.bio.net Wed Feb 02 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: Newsgroups: bionet.molbio.proteins Subject: Re: Albumin binding to Nucleic Acids Date: 3 Feb 1994 01:49:48 -0000 Lines: 16 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2ipl7s$9a@mserv1.dl.ac.uk> X-Envelope-To: proteins@net.bio.net X-Vms-To: IN%"proteins@net.bio.net" Original-To: proteins@net.bio.net From: pnh@fcsparc6.ncifcrf.gov (Paul N Hengen): >NEB supplies acetylated BSA with some of their restriction enzymes so you can >add it to reduce spurious star activity if you like. Why is this the acetylated >form? The acetylation doesn't really affect the BSA, but it inactivates nucleases in the not-so-pure prep. - Eric ------------------------------------------------------------------------------ one sorry lobe of the Primordial Undermind "Early in the morning, just before the dawn EARN@portia.caltech.edu I turn my TV on, and watch the fuzz... " - Flaming Lips From owner-proteins@net.bio.net Sat Feb 05 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!europa.eng.gtefsd.com!library.ucla.edu!network.ucsd.edu!sdcc12!jeeves!wsun From: wsun@jeeves.ucsd.edu (Fiberman) Newsgroups: bionet.molbio.proteins Subject: protein extraction from oocytes Message-ID: <60735@sdcc12.ucsd.edu> Date: 6 Feb 94 00:10:35 GMT Sender: news@sdcc12.ucsd.edu Organization: University of California, San Diego Lines: 8 Nntp-Posting-Host: jeeves.ucsd.edu Hello fellow netters, I wish to extract membrane proteins from Xenopus oocytes for a Western blot. Does anyone out there have a quick and simple protocol for extracting membrane proteins from frog eggs? -fm From owner-proteins@net.bio.net Sun Feb 06 22:00:00 1994 Path: biosci!daresbury!doc.ic.ac.uk!uknet!pipex!howland.reston.ans.net!xlink.net!fauern!lrz-muenchen.de!ipp-garching.mpg.de!alf.biochem.mpg.de!krasel From: krasel@alf.biochem.mpg.de (Cornelius Krasel) Newsgroups: bionet.molbio.proteins Subject: Re: Elution of GST fusion protein Date: 7 Feb 1994 12:01:36 GMT Organization: Rechenzentrum der Max-Planck-Gesellschaft in Garching Lines: 31 Distribution: bionet Message-ID: <2j5aj0INN1aug@sat.ipp-garching.mpg.de> References: <2j3kud$csv@mserv1.dl.ac.uk> NNTP-Posting-Host: alf.biochem.mpg.de X-Newsreader: TIN [version 1.1 PL8] Tupy.Jon (tupy@rapids.tularik.com) wrote: : I have produced a GST fusion protein via T7 promotor in e. coli (BL21) that is : about 70 Kd (including GST). After an initial fractionation on SP Sepharose, : I have : attempted a secondary purification on Glutathione Sepharose 4B (Pharmacia). : While I have obtained some pure fusion protein in eluting this column, : the vast : majority (>90%) of this protein remains bound to the column. My elution : buffer : contains 20 mM reduced glutathione,100 mM KCl, and 25 mM HEPES (pH 7.9). Maybe this is a trivial point, but do you add your glutathione _freshly_ to the buffer? I found that even during elution, quite a proportion of GSH starts to oxidize as evident by UV absorption. The elution buffer I used (three years ago) contained 20 mM Tris/HCl, pH 8.0 200 mM NaCl 1 mM EDTA 2 mM DTT 0.02% Na-azide 10 mM reduced glutathione (freshly added) Hope that helps, --Cornelius. -- /* Cornelius Krasel, Abt. Lohse, Genzentrum, D-82152 Martinsried, Germany */ /* email: krasel@alf.biochem.mpg.de fax: +49 89 8578 3795 */ /* "People are DNA's way of making more DNA." (Edward O. Wilson, 1975) */ From owner-proteins@net.bio.net Sun Feb 06 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: "Tupy.Jon" Newsgroups: bionet.molbio.proteins Subject: Elution of GST fusion protein Date: 6 Feb 1994 20:46:05 -0000 Lines: 17 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2j3kud$csv@mserv1.dl.ac.uk> Original-To: "methods-and-reagents@net.bio.net " , proteins@net.bio.net Hi netland, I have produced a GST fusion protein via T7 promotor in e. coli (BL21) that is about 70 Kd (including GST). After an initial fractionation on SP Sepharose, I have attempted a secondary purification on Glutathione Sepharose 4B (Pharmacia). While I have obtained some pure fusion protein in eluting this column, the vast majority (>90%) of this protein remains bound to the column. My elution buffer contains 20 mM reduced glutathione,100 mM KCl, and 25 mM HEPES (pH 7.9). I first applied this buffer at 4 degrees C, which didn't work. Then I attempted an elution at room temp, which also didn't yield additonal protein. Finally, in an effort to batch-elute the fusion protein from the resin, I added two column volumes of elution buffer to the resin, capped the column at both ends, and incubated the whole thing on a rocker at RT for an hour. *Still* no elution! Anyone have any tricks? Thanks! Jon From owner-proteins@net.bio.net Sun Feb 06 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: Michal Harel Newsgroups: bionet.molbio.proteins Subject: Statistics on acidic vs. basic residues Date: 7 Feb 1994 10:31:00 -0000 Lines: 6 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2j5594$hqt@mserv1.dl.ac.uk> Original-To: proteins@dl.ac.uk Hi Netters, We are looking for a program which will scan a library of protein sequences and print out the percentage of acidic and basic residues in each. Any suggestions? Thanks. Michal Harel csharel2@weizmann.weizmann.ac.il From owner-proteins@net.bio.net Sun Feb 06 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!daresbury!doc.ic.ac.uk!warwick!pipex!howland.reston.ans.net!paladin.american.edu!news.univie.ac.at!edvz.sbg.ac.at!wst!ortnerm From: ortnerm@wst.edvz.sbg.ac.at (Maria Ortner) Subject: PROSA II - PROtein Structure Analysis available Message-ID: Keywords: protein structure, modelling, X-Ray, NMR Lines: 67 Sender: ortnerm@wst (Maria Ortner) Reply-To: ortnerm@wst.edvz.sbg.ac.at (Maria Ortner) Organization: University of Salzburg / Austria Date: Mon, 7 Feb 1994 19:06:54 GMT PROSA II - PROtein Structure Analysis ===================================== Version 2.0 for SGI Iris/Indigo Here is Prosa II. Prosa II is a powerful tool in protein structure research. Prosa II supports and guides your studies aimed at the determination of a protein's native fold. It is helpful for experimental structure determinations and you will find it indispensable in modeling studies. Suppose you are confronted with the three-dimensional structure of a protein. The structure may have been determined by X-ray analysis or NMR-spectroscopy, it may be the result of modeling studies or it could be a nice looking structure deliberately misfolded to fool specialists in protein structure theory. Obvious questions are: Is the structure correct? Are there faulty parts? Prosa II helps you to answer these questions. Prosa II assists you in protein structure quality control. It calculates a score for your input-structure. Scores of native protein folds are in a characteristic range. If your score is outside this range then your structure may have problems. Prosa II's energy graphs provide diagnostic tools to spot problematic sections in your structure. Energy graphs display the energetic architecture of protein folds as a function of amino acid sequence position. High energies correspond to stressed or strained sections of the chain and may point to problematic parts of a fold. As long as scores and energy graphs are non native like chances are that the structure can be improved. Prosa II is applicable to a particular range of problems. Consult the manual and references on the interpretation of results, on the range of applicability and further details Prosa II's staff is not responsible for your applications. There is no warranty for the consequences of your actions. Proper usage is at your hands and you are responsible for your results and their proper interpretation. AVAILABILITY: ============ Prosa II runs on Silicon Graphics Workstations with IRIX 4.0.5 or higher. You can get the demo version of Prosa II by anonymous ftp from Gundi.came.sbg.ac.at (141.201.27.11) You can use this version of Prosa II until March 27, 1994. After this date Prosa II will refuse to start - unless you reset the date on your computer - or you crack the programm. We do not recommend any of these or related actions. Commercialized versions of Prosa II and its successors will be available in the near future. For further details please contact M.J.Sippl at e-mail address sippl@agnes.came.sbg.ac.at or if this fails sippl@dsb835.edvz.sbg.ac.at (the former address is preferred). From owner-proteins@net.bio.net Sun Feb 06 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!daresbury!doc.ic.ac.uk!uknet!EU.net!sun4nl!star.cs.vu.nl!balaena!mac131.bio.vu.nl!user From: vdwal@bio.vu.nl (FJ van der Wal) Subject: is there somebody working on pasteurella? Message-ID: Followup-To: bionet.molbio.proteins Sender: news@bio.vu.nl Organization: Mol.Micro, Vrije Uni., Amsterdam, NL Date: Mon, 7 Feb 1994 08:47:33 GMT Lines: 11 Is there someone out there working on pasteurella? We are looking for a plasmid to clone genes in pasteurella. You can reply through oudega@bio.vu.nl -- Fimme Jan van der Wal dep. Molecular Microbiology Vrije Universiteit Amsterdam de Boelelaan 1087 1081 HV Amsterdam, NL vdwal@bio.vu.nl From owner-proteins@net.bio.net Sun Feb 06 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!daresbury!keele!uknet!EU.net!news.funet.fi!luotsi.uku.fi!messi.uku.fi!pentikai From: pentikai@messi.uku.fi (Jaana Pentikainen) Subject: dissolving of KLH? Sender: news@luotsi.uku.fi Message-ID: Date: 7 Feb 94 14:43:33 GMT Organization: University of Kuopio, Finland Lines: 8 We are trying to dissolve Keyhole limpet hemocyanin but it has proven to be difficult. Has anyone experience in dissolving KLH to normal buffers as PBS? We are waiting for good advices. Jaana Pentik{inen pentikai@messi.uku.fi From owner-proteins@net.bio.net Mon Feb 07 22:00:00 1994 Path: biosci!rutgers!concert!news.duke.edu!galactose.mc.duke.edu.uucp!srps From: srps@galactose.mc.duke.edu (Steven Pirie-Shepherd) Newsgroups: bionet.molbio.proteins Subject: Re: protein extraction from oocytes Message-ID: <2j8pg8$ri2@news.duke.edu> Date: 8 Feb 94 19:34:32 GMT References: <60735@sdcc12.ucsd.edu> Organization: Duke University; Durham, N.C. Lines: 30 NNTP-Posting-Host: galactose.mc.duke.edu In article <60735@sdcc12.ucsd.edu> wsun@jeeves.ucsd.edu (Fiberman) writes: >Hello fellow netters, > >I wish to extract membrane proteins from Xenopus oocytes for a >Western blot. Does anyone out there have a quick and simple >protocol for extracting membrane proteins from frog eggs? > >-fm > Internal membranes or plasmallemma ?????. If internal, then homogenise teh eggs in the tube and spin briefly (10 minutes) at low spped to remove the cell debris, cell membrane. Take the supernatant and spin hard and long to pellet the organelle membrane (ER, Golgi). Adding PreTreatment buffer to all fractions (ie SDS/2-Mercaptoethanol) should solubilise everything, if all you want to look at is a Western Blot. The crucial part is separating internal from external membrane, and differential centrifugation should do this. Check the 'g' generated by your microfuge, a rough guide; big stuff (outer and debris) comes down at 10,000g for 10 minutes, internal comes down at 100,000g for 30-60 minutes. These times are for big centrifuges, but take into account the characteristics of your benchtop or eppendorf centrifuge, and reduce the time. Hope this helps steven -- __________________________________________________ Steven Pirie-Shepherd srps@galactose.mc.duke.edu -=insert your own pithy phrase just about here=- From owner-proteins@net.bio.net Mon Feb 07 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: MJ Duggan Newsgroups: bionet.molbio.proteins Subject: dissolving of KLH? (fwd) Date: 8 Feb 1994 12:01:58 -0000 Lines: 22 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2j7uvm$t1r@mserv1.dl.ac.uk> Original-To: proteins@dl.ac.uk (proteins news-group) stuff deleted > > We are trying to dissolve Keyhole limpet hemocyanin but it has proven to be > difficult. Has anyone experience in dissolving KLH to normal buffers as PBS? > We are waiting for good advices. > > Jaana Pentik{inen > pentikai@messi.uku.fi > > Try changing te pH, actually I forget whether it is more soluble at acid or alkali pH but I know that it is better in one direction and worse in the other than at neutral. Or try adding some SDS. Both of theese speed up the process and you can generally dialyse the buffers back to what you want without it coming out of solution. good luck Mike Duggan From owner-proteins@net.bio.net Mon Feb 07 22:00:00 1994 Path: biosci!rutgers!concert!news.duke.edu!galactose.mc.duke.edu.uucp!srps From: srps@galactose.mc.duke.edu (Steven Pirie-Shepherd) Newsgroups: bionet.molbio.proteins Subject: Re: Ponceau S staining of PVDF membranes? Message-ID: <2j8p4k$reo@news.duke.edu> Date: 8 Feb 94 19:28:20 GMT References: <2io4ka$cbm@elna.ethz.ch> Organization: Duke University; Durham, N.C. Lines: 23 NNTP-Posting-Host: galactose.mc.duke.edu In article <2io4ka$cbm@elna.ethz.ch> schlaefli@micro.biol.ethz.ch (Hans Rudolf Schldfli) writes: >In article , Bill Nowatzke says: >>the protein that I had thought I loaded. Any clues as to why my ponceau S >>did not work? I diluted a fresh Sigam concentrate (20 mL) into 180 mL of >>deionized water. I stained for 5 min and watch as the membrane destained >>in 1% AcOH. At no time did I see anything resembling a protein band. This>problem has occured for the past two consecutive times that I have tried >>the technique. Your comments are appreciated. You are probably washinbg the Poncy pink off the protein (this is not a permenant stain). When I use Ponceau, I stain once , then rinse in distilled water. The bands usually appear. If they do not appear 1st time, try restaining. The weak AcOH will knock the stain off, I had this problem once as well, but it is easily solved. -- __________________________________________________ Steven Pirie-Shepherd srps@galactose.mc.duke.edu -=insert your own pithy phrase just about here=- From owner-proteins@net.bio.net Mon Feb 07 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!agate!msuinfo!Richard.Ransom From: rransom@msu.edu (Parcheesi Man) Newsgroups: bionet.molbio.proteins Subject: AUT electrophoresis of histones...HELP! Date: 8 Feb 1994 21:12:18 GMT Organization: Michigan State University Lines: 10 Sender: -Not-Authenticated-[4628] Message-ID: <2j8v7i$g5j@msuinfo.cl.msu.edu> References: NNTP-Posting-Host: 35.8.197.59 X-Posted-From: InterNews 1.0@msuinfo.cl.msu.edu. Xdisclaimer: No attempt was made to authenticate the sender's name. I have been trying to run acetic acid/urea/triton gels of histones as described by Bonner et al. (and also in _Gel electrophoresis of proteins_ ed. Hames and Rickwood) and I've been unable to get a decent electrophoresis run. Does anyone out there know about AUT gels? If so, email me and I'll pour out my tale o' woe. Thanks in advance...Richard. rransom@msu.edu From owner-proteins@net.bio.net Mon Feb 07 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: MJ Duggan Newsgroups: bionet.molbio.proteins Subject: liposomal protein delivery Date: 8 Feb 1994 14:10:05 -0000 Lines: 12 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2j86ft$5md@mserv1.dl.ac.uk> Original-To: proteins@dl.ac.uk (proteins news-group) Dear Netters, Does anybody have any experience on using liposomes to deliver proteins to the cytoplasm of cells growing in culture (ordinary cell lines e.g. 3T3 etc.)? I would be grateful for any methods references etc. thanks in advance. Mike Duggan sgex400@sghms.lon.ac.uk From owner-proteins@net.bio.net Wed Feb 09 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!europa.eng.gtefsd.com!library.ucla.edu!csulb.edu!paris.ics.uci.edu!news.claremont.edu!nntp-server.caltech.edu!rickert From: rickert@cco.caltech.edu (Keith Warren Rickert) Newsgroups: bionet.molbio.proteins Subject: Re: peptide sequencing Date: 10 Feb 1994 20:28:17 GMT Organization: California Institute of Technology, Pasadena Lines: 24 Message-ID: <2je5d1$nah@gap.caltech.edu> References: <2jdkio$ot1@news.nd.edu> NNTP-Posting-Host: sandman.caltech.edu In <2jdkio$ot1@news.nd.edu> (dave bennatt) writes: >I've worked out a scheme where I biotinylate membrane proteins which is >useful for both detection and concentration of affinity column eluts >on a strep-agarose column.. now I would like to get a peptide sequence on >my protein of interest, and I'm wondering if the biotin on all the primary >amines will interfere with good sequencing results? >has anyone else tried to sequence biotinylated proteins? Not that I've tried that as such, but most of the papers I've seen on sequencing of other modified proteins (phosphorylated or glycosylated) the modified residues just arent identifiable, but they can usually get the sequence around it. This still assumes that you have a free amine at the N-terminus; if you've capped that, then Edman degradation type chemistry is going to have some real problems... Keith -- Keith Rickert | "That was only one of the many occasions on which rickert@cco.caltech.edu | I met my death - an experience I don't hesitate keith@imppig.caltech.edu | strongly to recommend" - Baron von Munchchausen From owner-proteins@net.bio.net Wed Feb 09 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!noc.near.net!news.delphi.com!usenet From: newittja@delphi.com Newsgroups: bionet.molbio.proteins Subject: Re: dissolving of KLH? Date: Thu, 10 Feb 94 19:13:33 -0500 Organization: Delphi (info@delphi.com email, 800-695-4005 voice) Lines: 21 Message-ID: References: NNTP-Posting-Host: bos1c.delphi.com X-To: Jaana Pentikainen Jaana Pentikainen writes: > >We are trying to dissolve Keyhole limpet hemocyanin but it has proven to be >difficult. Has anyone experience in dissolving KLH to normal buffers as PBS? >We are waiting for good advices. > >Jaana Pentik{inen >pentikai@messi.uku.fi > Sometimes it is the quality of KLH that determines how well it dissolves. Try the KLH from Pierce; it dissolves well. Also, Pierce recommends dissolving it in 0.9 M NaCl, 50-100 mM NaPi pH 7, 10 mM EDTA (this is for a peptide coupling procedure). Hope this helps. John newittja@delphi.com From owner-proteins@net.bio.net Wed Feb 09 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!spool.mu.edu!news.nd.edu!news From: (dave bennatt) Newsgroups: bionet.molbio.proteins Subject: peptide sequencing Followup-To: bionet.molbio.proteins Date: 10 Feb 1994 15:41:12 GMT Organization: University of Notre Dame Lines: 18 Distribution: world Message-ID: <2jdkio$ot1@news.nd.edu> NNTP-Posting-Host: mcabee134.bio.nd.edu I've worked out a scheme where I biotinylate membrane proteins which is useful for both detection and concentration of affinity column eluts on a strep-agarose column.. now I would like to get a peptide sequence on my protein of interest, and I'm wondering if the biotin on all the primary amines will interfere with good sequencing results? has anyone else tried to sequence biotinylated proteins? ************************ dave bennatt university of notre dame dbennatt@darwin.cc.nd.edu onca@mead.u.washington.edu **************************** From owner-proteins@net.bio.net Thu Feb 10 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: "Andrew, Tel. +39-6-91093434" Newsgroups: bionet.molbio.proteins Subject: RE: Presence of disulphide bond in protein - HELP!! Date: 11 Feb 1994 20:11:05 -0000 Lines: 70 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2jgoop$8jl@mserv1.dl.ac.uk> Original-To: proteins@dl.ac.uk From: IRBMV1::WALLACE "Andrew, Tel. +39-6-91093434" 11-FEB-1994 21:03:12.04 To: SMTP%"ps44@namaste.cc.columbia.edu" CC: WALLACE Subj: RE: Presence of disulphide bond in protein - HELP!! Dear Pavel, Another approach you could try is to measure the number of free thiols present using the Ellman assay (Ellman, G.L. (1959) Arch. Biochem. Biophys. 82, 70), a good protocol for which is described in the IRL practical approach series book on protein structure by T.E. Creighton. In chapter 7 on page 157 he writes as follows: "The Ellman assay (ref) measures the nitrothiobenzoate (NTB) released upon reaction of a thiol with DTNB (5,5'-dithionitrobenzoic acid which you can buy from Pierce or Sigma). The procedure described here uses GdmCl to unfold the protein and make all its thiols reactive, but the denaturant need not be included. 1) Prepare a suitable protein solution of known concentration in 6 M GdmCl with 0.1 M phosphate buffer (pH 7.3) and 1 mM EDTA, plus a suitable reference mixture without the protein. If the protein has been dialysed or gel-filtered, use the same volume of the dialysis or chromatography buffer in the reference mixture. Use sufficient protein in the assay so that at least one thiol per protein molecule could be detected; usually, at least 2 nmol is required. 2) Measure, or set to zero, the absorbance difference at 412 nm between defined volumes of each of the protein and reference samples. Maintain the temperature of the samples near to 25 deg C. 3) For each ml of sample, add 50 ul of 3 mM DTNB (in 0.1 M phosphate buffer, pH 7.3) to each cuvette, followed by thorough mixing. 4) Immediately follow the increase in absorbance at 412 nm of the protein sample. 5) When the absorbance stops increasing, measure the absorbance difference between the protein and the reference samples. 6) From the increased absorbance in the protein sample caused by the reaction of DTNB, calculate the molar concentration of thiols present from the molar absorbance of the TNB anion {extinction coefficient = 13700/M cm in 6 M GdmCl, 14150/M cm without GdmCl} Method adapted from: Riddles et al., Methods in Enzymology (1983) 91, 49ff. Hope it works for you. Andrew =============================================================================== Andrew Wallace, IRBM P. Angeletti, Pomezia, Italia. Voice: +39-6-91093434 Fax: +39-6-91093225 Email: wallace@irbm.it IIIIIIIIII RRRRRRRRR BBBBBBBB MMM MMM IIIIIIIIII RRRR RRRRR BBB BBBB MMMM MMMM IIII RRR RRRR BBB BBBB MMMMM MMMMM IIII RRRR RRRR BBB BBBB MMMMMM MMMMMM IIII RRRRRRRRR BBBBBBBB MMMMMMMMMMMMM IIII RRRR RRRRR BBB BBBB MMMM MMMM IIII RRRR RRRRR BBB BBBB MMMM MMMM IIIIIIIIII RRRR RRRRR BBB BBBB MMMM MMMM IIIIIIIIII RRRR RRRRR BBBBBBBB MMMM MMMM "The home of the Minibody" Questa programma e' stata presentata da "Minibodao Meravigliao". "Minibodao Meravigliao - for the latest in designer protein fashion." From owner-proteins@net.bio.net Thu Feb 10 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!sol.ctr.columbia.edu!news.columbia.edu!namaste.cc.columbia.edu!ps44 From: ps44@namaste.cc.columbia.edu (Pavel Sova) Newsgroups: bionet.molbio.proteins Subject: Presence of disulphide bond in protein - HELP!! Date: 11 Feb 1994 17:52:01 GMT Organization: Columbia University Lines: 49 Message-ID: <2jggk1$jq9@apakabar.cc.columbia.edu> NNTP-Posting-Host: namaste.cc.columbia.edu Hi netters, I have a question regarding analysis of presence/absence of cysteine bridge in protein. The protein of my interest is virally encoded, small (23kD) and located inside of the mammalian cell, partially in cytoplasm, partially associated with membrane. Intracellular localization would suggest this protein does not create a cysteine bond; -> there are two cysteine residues 11 aa apart inside of it. Functional analysis studies showed that either or both cysteines is esential for the function. (We did this by removing of cysteine(s) by site-directed mutagenesis) To find out if there is a cysteine bond, I lysed infected cells in RIPA buffer in the presence of Iodoacetamide (0-10-25-50mM) and then I mixed the lysates with SDS-PAGE sample buffer (-DTT or -MeOH), and ran a SDS-PAGE, followed by WB with specific polyclonal antiserum. The wild type protein in lysates in the presence of IAA was of the same mobility as mutated (Cys-) proteins (where I don't expect creation of disulphide bond - thus shift in mobility). However, when cells were lysed without IAA, I could detect double-band of my protein in a wild-type form - with one additional "faster" band. That would suggest me that without adding IAA, I detected a product of partial oxidation and that this oxidation is and artefact of sample preparation. In conclusion, the result that, in the presence of IAA, wild-type protein (two cysteine res. present) is of the *same mobility* as mutated proteins (one or none cysteine res.) or proteins run under presence of DTT tells me that there is actually no intrachain cysteine bond. Sorry for this long explanation, but I want to go out with this for a publication and I am not sure (not being a biochemist) if my thinking and conclusions are right. Does anyone have a comment? - Please e-mail me (or hit R/F key). Thanks. Pavel Sova = ps44@columbia.edu Molecular Virology Laboratory Columbia University New York P.S. I apologize for my wrong tip regarding the databaze search for amino acid composition of protein I posted ~14 days ago - I mixed the terms "composition/sequence". Thanks for your e-mail straighting it up to me. Pavel From owner-proteins@net.bio.net Thu Feb 10 22:00:00 1994 Path: biosci!NET.BIO.NET!biosci-help From: biosci-help@NET.BIO.NET (BIOSCI Administrator) Newsgroups: bionet.molbio.proteins Subject: test of proteins@net.bio.net Date: 12 Feb 1994 02:44:10 -0000 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 2 Sender: daemon@net.bio.net Distribution: bionet Message-ID: Reply-To: biosci-help@net.bio.net NNTP-Posting-Host: net.bio.net test of proteins@net.bio.net From owner-proteins@net.bio.net Fri Feb 11 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!agate!ames!decwrl!pa.dec.com!jac.zko.dec.com!gemgrp.enet.dec.com!winalski From: winalski@gemgrp.enet.dec.com (Paul S. Winalski) Newsgroups: bionet.molbio.proteins Subject: Fe and S linkage in nonheme iron proteins? Date: 12 Feb 1994 19:18:16 GMT Organization: Digital Equipment Corporation, Nashua NH Lines: 12 Distribution: world Message-ID: <2jja1o$oms@jac.zko.dec.com> Reply-To: winalski@gemgrp.enet.dec.com (Paul S. Winalski) NNTP-Posting-Host: GEMGRP X-Newsreader: mxrn 6.18-8 My 20-year-old copy of Lehninger's BIOCHEMISTRY says about ferredoxin and the other nonheme iron proteins involved in oxidation and electron transport that the structure of the Fe linkage to the protein is not known. All that was certain at that time is that the proteins contain the same number of cysteine residues as Fe atoms, and that the sulfur is acid-labile and released as H2S. What's been discovered about this since 1970? Do we know the structure of the Fe linkage in the nonheme iron proteins now? --PSW From owner-proteins@net.bio.net Fri Feb 11 22:00:00 1994 Path: biosci!daresbury!doc.ic.ac.uk!agate!usenet.ins.cwru.edu!po.CWRU.Edu!rdh5 From: rdh5@po.CWRU.Edu (Robert D. Hoffman) Newsgroups: bionet.molbio.proteins Subject: Re: Presence of disulphide bond in protein - HELP!! Date: 12 Feb 1994 17:54:36 GMT Organization: Case Western Reserve University, Cleveland, Ohio (USA) Lines: 12 Message-ID: <2jj54s$qlb@usenet.INS.CWRU.Edu> NNTP-Posting-Host: slc8.ins.cwru.edu Pavel- Have you tried 2-D electrophoresis to demonstrate the SS bond? Lysis in RIPA->alkylation with iodoacetamide->nonreducing SDS PAGE 1st dimension->reducing SDS PAGE 2nd dimension. Anything with an internal or external SS bond is expected to fall off the diagonal.-Bob -- Robert D. Hoffman, M.D., Ph.D.//Director, Autopsy Pathology Institute of Pathology//Case Western Reserve University 2085 Adelbert Road//Cleveland, Ohio 44106, USA rdh5@po.cwru.edu//HOFFMAN@ncifcrf.gov From owner-proteins@net.bio.net Fri Feb 11 22:00:00 1994 Path: biosci!daresbury!doc.ic.ac.uk!agate!howland.reston.ans.net!news.moneng.mei.com!uwm.edu!fnnews.fnal.gov!nntp-server.caltech.edu!rickert From: rickert@cco.caltech.edu (Keith Warren Rickert) Newsgroups: bionet.molbio.proteins Subject: Re: Fe and S linkage in nonheme iron proteins? Date: 12 Feb 1994 21:10:12 GMT Organization: California Institute of Technology, Pasadena Lines: 27 Message-ID: <2jjgjk$8fh@gap.cco.caltech.edu> References: <2jja1o$oms@jac.zko.dec.com> NNTP-Posting-Host: sandman.caltech.edu In <2jja1o$oms@jac.zko.dec.com> winalski@gemgrp.enet.dec.com (Paul S. Winalski) writes: >My 20-year-old copy of Lehninger's BIOCHEMISTRY says about ferredoxin and >the other nonheme iron proteins involved in oxidation and electron transport >that the structure of the Fe linkage to the protein is not known. All that >was certain at that time is that the proteins contain the same number of >cysteine residues as Fe atoms, and that the sulfur is acid-labile and released >as H2S. >What's been discovered about this since 1970? Do we know the structure of >the Fe linkage in the nonheme iron proteins now? I believe that the clusters involved in ferredoxin are Fe4S4 clusters - imagine a cube, with 4 of the corners being S and 4 being Fe, and arranged so that every Fe has 3 nearest neighbors are S, and vice versa. Cysteines attach to the irons. There are other varieties of non-heme irons, for quite a number of proteins. Keith -- Keith Rickert | "That was only one of the many occasions on which rickert@cco.caltech.edu | I met my death - an experience I don't hesitate keith@imppig.caltech.edu | strongly to recommend" - Baron von Munchchausen From owner-proteins@net.bio.net Sat Feb 12 22:00:00 1994 Path: biosci!FOX.NSTN.NS.CA!ewright From: ewright@FOX.NSTN.NS.CA ("Edwin Wright") Newsgroups: bionet.molbio.proteins Subject: Gaps (Protein Sequences) Date: 13 Feb 1994 21:43:43 -0000 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 48 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <63805.ewright@fox.nstn.ns.ca> NNTP-Posting-Host: net.bio.net Reference: Gaston H. Gonnet, Mark A. Cohen, Steven A. Benner, SCIENCE Vol. 256, P. 1443 (1992) I quote the following statement from the above reference: "The difficulties in contructing alignment routines are further complicated by the requirement that they handle deletions and insertions. Even random sequences can be aligned if gaps are introduced at no penalty. Most alignment programs therefore assign penalties to gaps of the form (ak + b), where a and b are arbitrarily chosen constants. There is no justification, either theoretical or empirical, for this treatment. Indeed, many of the questionable conclusions drawn from alignments arise because of inappropriately placed gaps. Conversely, correctly placed gaps provide information that is critical to the de novo prediction of the folded structure of proteins from sequence data alone (4-6). Such information led to the remarkably accurate predictions of the folded structures of tryptophan synthase and protein kinase before crystallographic information was avilable (4-6)." I pose the following question to the network: Given the increasingly apparent importance of "correctly placed gaps" do you know of any researchers taking the concept of gaps even further, i.e. by discriminating between "conserved" positions (i.e. specific residues always present at a particular location in a group of aligned sequences) and "non-conserved" or "degenerate" positions (i.e. not only gap positions in the traditional sense of insertions/deletions but also mutation positions). The point here is that a position is either conserved or it is not; the only essential difference between a gap (insertion/deletion) and a mutation of a specific length is that the presence of a gap indicates that in at least one sequence in the group of aligned sequences the mutation is present as "the empty or null set" (to borrow a concept from set theory). I believe the real challenge to analysts is in establishing this discrimination between conserved and degenerate sequence position. It may be that in broadening the idea of mutations to include gaps, such a generalization would require nested or recursive analysis. I would appreciate any comments from the network in this regard. Edwin Wright 85 Spinnaker Drive, A-602 Halifax, Nova Scotia CANADA B3N 3E3 Telephone: (902) 477-5037 Email: ewright@fox.nstn.ns.ca =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= From owner-proteins@net.bio.net Sat Feb 12 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!wupost!waikato!canterbury.ac.nz!otago.ac.nz!sanger.otago.ac.nz!craigm From: craigm@sanger.otago.ac.nz (Craig Marshall) Subject: Classification of proteases Message-ID: Sender: usenet@news.otago.ac.nz (News stuff) Nntp-Posting-Host: sanger.otago.ac.nz Organization: University of Otago X-Newsreader: TIN [version 1.1 PL8] Date: Sun, 13 Feb 1994 23:25:35 GMT Lines: 13 Can anyone help me with a recent review about the classification of proteases? I am a bit out of touch with recent thinking about how proteases should be classified (and named) and would like to make good the deficiency. Many thanks -- Craig Marshall craigm@sanger.otago.ac.nz or Biochemistry Department bioc07@otago.ac.nz University of Otago Phone 64 3 479 7849 P.O. Box 56 Fax 64 3 479 7866 Dunedin, New Zealand From owner-proteins@net.bio.net Sat Feb 12 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!daresbury!doc.ic.ac.uk!agate!howland.reston.ans.net!wupost!waikato!canterbury.ac.nz!otago.ac.nz!sanger.otago.ac.nz!craigm From: craigm@sanger.otago.ac.nz (Craig Marshall) Subject: Re: AUT electrophoresis of histones...HELP! Message-ID: Sender: usenet@news.otago.ac.nz (News stuff) Nntp-Posting-Host: sanger.otago.ac.nz Organization: University of Otago X-Newsreader: TIN [version 1.1 PL8] References: <2j8v7i$g5j@msuinfo.cl.msu.edu> Date: Sun, 13 Feb 1994 23:08:15 GMT Lines: 27 I the past I have had great trouble with AUT gels. One problem that I did find was with the Triton. This can apparently degrade with the production of peroxides, ketones and carboxylic acids. The test for this is the pH of a 10% solution which should be neutral according to Merck. If it is not, try and get another fresh bottle of Triton and see if that helps. Cheers Parcheesi Man (rransom@msu.edu) wrote: > I have been trying to run acetic acid/urea/triton gels of histones as > described by Bonner et al. (and also in _Gel electrophoresis of > proteins_ ed. Hames and Rickwood) and I've been unable to get a decent > electrophoresis run. Does anyone out there know about AUT gels? If > so, email me and I'll pour out my tale o' woe. Thanks in > advance...Richard. > rransom@msu.edu -- Craig Marshall craigm@sanger.otago.ac.nz or Biochemistry Department bioc07@otago.ac.nz University of Otago Phone 64 3 479 7849 P.O. Box 56 Fax 64 3 479 7866 Dunedin, New Zealand From owner-proteins@net.bio.net Sat Feb 12 22:00:00 1994 Path: biosci!s.u-tokyo!news.tisn.ad.jp!news.u-tokyo.ac.jp!sinetnews!daffy!uwvax!uwm.edu!vixen.cso.uiuc.edu!howland.reston.ans.net!xlink.net!zib-berlin.de!netmbx.de!Germany.EU.net!EU.net!chsun!elna.ethz.ch!usenet From: schlaefli@micro.biol.ethz.ch (Hans Rudolf Schlaefli) Newsgroups: bionet.molbio.proteins Subject: Re: Fe and S linkage in nonheme iron proteins? Date: 13 Feb 1994 11:06:58 GMT Organization: Microbiology ETHZ Lines: 17 Message-ID: <2jl1ki$2a2@elna.ethz.ch> References: <2jja1o$oms@jac.zko.dec.com> NNTP-Posting-Host: b26-pro486.ethz.ch X-Newsreader: WinVN 0.83.2 In article <2jja1o$oms@jac.zko.dec.com>, winalski@gemgrp.enet.dec.com (Paul S. Winalski) says: > > >What's been discovered about this since 1970? Do we know the structure of >the Fe linkage in the nonheme iron proteins now? > >--PSW Most non-heme iron-sulfur proteins of the mentioned types contain simple (2Fe-2S) centers of the "plant-ferredoxin" or the "Rieske" type. In the plant-ferredoxin type proteins each Fe-atom is linked to the two inorganic S and to two organic (Cys) S. In the Rieske type (2Fe-2S) centers one Fe is coordinated in the same way, the second is linked to the inorganic S and to two His- residues of the protein. Annu.Rev.Microbiol. (1992) 46: 277-305 gives a good overview. HRS From owner-proteins@net.bio.net Sun Feb 13 22:00:00 1994 Path: biosci!parc!barrnet.net!sgiblab!sgigate.sgi.com!olivea!news.bu.edu!lll-winken.llnl.gov!fnnews.fnal.gov!nntp-server.caltech.edu!folding!wqc From: wqc@hood.caltech.edu (Wangqin Chen) Newsgroups: bionet.molbio.proteins Subject: Swiss protein sequence Date: 14 Feb 1994 19:10:17 GMT Organization: Bio-computational center, Caltech. Lines: 5 Distribution: world Message-ID: <2joiap$dm8@gap.cco.caltech.edu> Reply-To: wqc@hood.caltech.edu NNTP-Posting-Host: folding.hood.caltech.edu How do I access to the SWISS:PROT protein sequence data base? If anybody knows, please help! Thank. -Qin Chen From owner-proteins@net.bio.net Sun Feb 13 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!headwall.Stanford.EDU!ames!sgiblab!swrinde!cs.utexas.edu!howland.reston.ans.net!EU.net!Germany.EU.net!netmbx.de!zib-berlin.de!fauern!news.dfn.de!gwdu03.gwdg.de!leptin-lab.mpib-tuebingen.mpg.de!user From: --- (Leptin lab) Subject: protease site prediction Message-ID: <----140294212521@leptin-lab.mpib-tuebingen.mpg.de> Followup-To: bionet.molbio.methds-reagnts Sender: news@gwdu03.gwdg.de (USENET News System) Nntp-Posting-Host: leptin-lab.mpib-tuebingen.mpg.de Organization: MPI f. Entwicklungsbiologie Date: Mon, 14 Feb 1994 21:25:21 +0100 Lines: 6 I would very much appreciate if anyone could send me information about the prediction of consensus protease cleavage sites. Thanks for the the help. Cristina Stella, MPI fuer Entwicklungsbiologie, TŸbingen Stella@MPBVAX.MPIB-Tuebingen.MPG.DE From owner-proteins@net.bio.net Sun Feb 13 22:00:00 1994 Path: biosci!parc!decwrl!ames!elroy.jpl.nasa.gov!swrinde!cs.utexas.edu!news.unt.edu!puck.daily.unt.edu!puck From: puck@daily.unt.edu (Puck) Newsgroups: bionet.molbio.proteins Subject: Hormone Bovine Somatotropin Date: Mon, 14 Feb 1994 21:29:10 GMT Organization: North Texas Daily Newspaper, UNT Lines: 12 Message-ID: NNTP-Posting-Host: puck.daily.unt.edu Keywords: Bovine I am looking for information on the use of a biengineered protein to increase dair cow milk yield. Is the hormone released in the cow's milk? Does pasteurization (or any other process) kill the hormone? If the hormone is present in commercial milk, what are the health consequences for those who drink it? Are there any discussion groups or gophers where I could find this information? From owner-proteins@net.bio.net Sun Feb 13 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!noc.near.net!news.cs.brandeis.edu!brenner From: brenner@rose.brandeis.edu (Charles Brenner) Subject: Re: Classification of proteases Message-ID: <1994Feb15.043331.4836@news.cs.brandeis.edu> Sender: news@news.cs.brandeis.edu (USENET News System) Organization: Brandeis University References: Date: Tue, 15 Feb 1994 04:33:31 GMT Lines: 18 craigm@sanger.otago.ac.nz (Craig Marshall) writes: >Can anyone help me with a recent review about the classification of >proteases? I am a bit out of touch with recent thinking about how >proteases should be classified (and named) and would like to make good >the deficiency. There are going to be two volumes of MEth Enz coming out soon. Alan J. Barrett is the editor and he has written a chapter on classification. Until the arrival of those volumes, look for the report of the INt. Union of Biochem that A. J. Barrett headed up. It is the definitive word on classification and nomenclature. -- Charles Brenner, Ph.D. Fellow of the Leukemia Society of America Rosenstiel Center, rm 610 phone (617) 736-4944 Brandeis University fax (617) 736-2405 Waltham, MA 02254-9110 brenner@auriga.rose.brandeis.edu From owner-proteins@net.bio.net Sun Feb 13 22:00:00 1994 Path: biosci!parc!barrnet.net!sgiblab!swrinde!cs.utexas.edu!howland.reston.ans.net!europa.eng.gtefsd.com!library.ucla.edu!csulb.edu!paris.ics.uci.edu!news.claremont.edu!nntp-server.caltech.edu!speicher From: speicher@cco.caltech.edu (Stephen Speicher) Newsgroups: bionet.molbio.proteins Subject: Re: Swiss protein sequence Date: 15 Feb 1994 02:07:35 GMT Organization: California Institute of Technology, Pasadena Lines: 14 Message-ID: <2jpap8$s4o@gap.cco.caltech.edu> References: <2joiap$dm8@gap.cco.caltech.edu> NNTP-Posting-Host: punisher.caltech.edu wqc@hood.caltech.edu (Wangqin Chen) writes: >How do I access to the SWISS:PROT protein sequence data base? If anybody >knows, please help! Thank. > -Qin Chen Hi, Swiss-prot can be found at ncbi.nlm.nih.gov in directory repository/swiss-prot. Stephen sjs@moon.hood.caltech.edu From owner-proteins@net.bio.net Sun Feb 13 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!daresbury!bioftp.unibas.ch!comp.bioz.unibas.ch!doelz From: doelz@comp.bioz.unibas.ch (Reinhard Doelz) Subject: Re: Swiss protein sequence Message-ID: <1994Feb15.072107.24971@comp.bioz.unibas.ch> Sender: usenet@comp.bioz.unibas.ch (NEWS transaction account) Nntp-Posting-Host: biox.embnet.unibas.ch Organization: EMBnet Switzerland [Basel] References: <2joiap$dm8@gap.cco.caltech.edu> Date: Tue, 15 Feb 1994 07:21:07 GMT Lines: 83 In article <2joiap$dm8@gap.cco.caltech.edu>, wqc@hood.caltech.edu (Wangqin Chen) writes: |> |> How do I access to the SWISS:PROT protein sequence data base? If anybody |> knows, please help! Thank. The current release information is THE SWISS-PROT PROTEIN SEQUENCE DATA BANK Release 27, October 1993 33329 entries, comprising 11,484,420 amino acids The original author is Amos Bairoch Medical Biochemistry Department Centre Medicale Universitaire 1211 Geneva 4 Switzerland You may access SWISSPROT via - purchased media EMBL's CD ROM pack, including access sw for Mac and DOS systems Entrez from NCBI, including access sw for Mac,Windows,Unix systems (GUI) - public domain clients/Network servers FTP (various sites, including NCBI and others in the US), GOPHER (see GOPHER FAQ what GOPHER is), amongst other sites, on Name=About SWISS-PROT Type=0 Port=70 Path=0/molbio/aswiss Host=helix.nih.gov WWW (World Wide Web) , (see also, some WWW FAQ) on http://expasy.hcuge.ch/sprot/sprot-top.html - special client software Browsing with BLAST via WWW or netblast, Browsing with BLAST or FASTA via EMBnet HASSLE servers, - electronic Mail servers BLAST (NCBI) BLITZ (EMBL) FASTA (EMBL) - other software of interest , on "mainframes" or workstations GCG (Genetics Computer Group) - SW package [commercial] IG Suite (Intelligenetics) - SW package [commercial] many others on PC, Mac, or other OS [partially commercial] Maybe this helps Regards Reinhard ===================================== DISCLAIMER Note that the software mentioned resembles Computer Program(s) which require a license in order to be run unless stated otherwise in a state- ment codistributed with the software. The use of the program(s) was men- tioned within a specific problem or example and must not be used to con- clude that other software products cannot possibly do a similar job. +---------------------------+-------------------------------------------+ | Dr. Reinhard Doelz | Tel. x41 61 2672247 Fax x41 61 2672078 | | Biocomputing | electronic Mail doelz@urz.unibas.ch | |Biozentrum der Universitaet+-------------------------------------------+ | Klingelbergstrasse 70 | EMBnet embnet@comp.bioz.unibas.ch | |CH 4056 Basel SWITZERLAND | Switzerland gopher.embnet.unibas.ch | +---------------------------+------------- http://beta.embnet.unibas.ch/ From owner-proteins@net.bio.net Mon Feb 14 22:00:00 1994 Newsgroups: bionet.software,bionet.molbio.genbank,bionet.molbio.embldatabank,bionet.molbio.pir,bionet.molbio.proteins Path: biosci!parc!decwrl!ames!agate!howland.reston.ans.net!cs.utexas.edu!uunet!hearst.acc.Virginia.EDU!murdoch!dayhoff.med.Virginia.EDU!wrp From: wrp@dayhoff.med.Virginia.EDU (William R. Pearson) Subject: New FASTA versions available Message-ID: Sender: usenet@murdoch.acc.Virginia.EDU Organization: University of Virginia Date: Sat, 12 Feb 1994 16:55:29 GMT Lines: 29 Xref: biosci bionet.software:7267 bionet.molbio.genbank:1535 bionet.molbio.embldatabank:285 bionet.molbio.pir:3 bionet.molbio.proteins:1364 A new release of the FASTA program package, version 1.7, is now available from virginia.EDU in pub/fasta as fasta17.shar(.Z). This version replaces the "rdf2" and "rss" programs with "prdf" and "prss", which calculate more accurate estimates for the statistical significance of a similarity score based on the scores of randomly shuffled sequences. The earlier "rdf2" and "rss" programs calculated a "z-value," which is not very informative if the distribution of similarity scores is not normal. Sequence similarity scores for random sequences are distributed according to the extreme value distribution, which is quite different from the normal distribution, especially for high scores. Prss and prdf estimate the parameters of the extreme value distribution and use these parameters to calculate the probability that a score as good or better than the unshuffled sequence score will be obtained. I appreciate the help of Stephen Altschul, who showed me the error of calculating "z-values", and the help of Phil Green, who provided the extreme value distribution estimation routine. Statistical estimates based on the extreme value distribution are usually more conservative than earlier estimates based on "z-values." In addition, a bug in the alignment routines that caused error messages and core dumps on some machines has been fixed. This bug has also been fixed in version 1.6. The new 1.6 version is available as fasta16c32.shar(.Z). Bill Pearson From owner-proteins@net.bio.net Mon Feb 14 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!parc!barrnet.net!sgiblab!sgigate.sgi.com!olivea!charnel!psgrain!library.ucla.edu!europa.eng.gtefsd.com!howland.reston.ans.net!newsserver.jvnc.net!cyanamid!igate.cyanamid.com!sandy From: sandy@nmr1.pt.cyanamid.COM (Sandy Silverman) Subject: Re: Hormone Bovine Somatotropin In-Reply-To: puck@daily.unt.edu's message of Mon, 14 Feb 1994 21:29:10 GMT Message-ID: Sender: news@cyanamid.uucp Organization: American Cyanamid Company References: Date: Tue, 15 Feb 1994 17:59:51 GMT Lines: 6 The protein is normally present in milk. See discussion in sci.agriculture recently. -- Sanford Silverman >Opinions expressed here are my own< American Cyanamid sandy@pt.cyanamid.com, silvermans@pt.cyanamid.com "Yeast is Best" From owner-proteins@net.bio.net Tue Feb 15 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: MJ Duggan Newsgroups: bionet.molbio.proteins Subject: streptolysin-o Date: 16 Feb 1994 13:40:31 -0000 Lines: 24 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2jt7of$i52@mserv1.dl.ac.uk> Original-To: methods@dl.ac.uk (molbiol. methods newsgroup) Dear all, We need some streptolysin-o rather urgently for some work on permeablising cells. We had thought to obtain it from Sigma, but thei are having some problems with their production at the moment (i.e. the stuff is not active when it reaches us the customer). We are, therefore, looking for some alternative source of supply. If anybody knows of a company which can supply reliable streptolysin-o, preferably with a UK/European agent, we would be most grateful. We have heard that it can be (or used to be) obtained from WBAG Resource Inc. in Switzerland and The Institut Pasteur Production Company (?) but cannot find phone/fax numbers for these organisations, again any help would be most welcome. thanks Mike Duggan From owner-proteins@net.bio.net Tue Feb 15 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!sdd.hp.com!saimiri.primate.wisc.edu!news.doit.wisc.edu!bromo.bocklabs.wisc.edu!user From: rphershb@facstaff.wisc.edu (Rick Hershberger) Newsgroups: bionet.molbio.proteins Subject: Floating Amm.SO4 ppts. Followup-To: bionet.molbio.proteins Date: 16 Feb 1994 21:01:58 GMT Organization: University of Wisconsin-Madison Lines: 26 Distribution: world Message-ID: References: <761426472snz@genesys.demon.co.uk> NNTP-Posting-Host: bromo.bocklabs.wisc.edu In article <761426472snz@genesys.demon.co.uk>, Duncan@genesys.demon.co.uk (Duncan Clark) wrote: > Hi Netters, > > Does anyone know why one sometimes gets Amm.SO4 ppts of proteins that float > rather than pellet? Is there anyway to get preps that do this to pellet? In > my case the prep is E.coli, sonicated, spun down, addition of polymin P (to > ppt nucleic acids) plus 0.2M NaCl (to keep my protein in solution) and the > ppt spun down and the supernatant kept. Addition of Amm.SO4 to 70%, left O/N > at 4C then spun at 50,000g. Floating pellicle. Why? > > ----------------------------------------------------------------------------- > Duncan Clark | Internet: duncan@genesys.demon.co.uk > GeneSys Ltd. | Compuserve: 100015.1406@compuserve.com > ----------------------------------------------------------------------------- I've wondered the same thing. Though in my case, I've tried to Amm.SO4-ppt my protein from a solution containing 0.3% sarkosyl. I thought that it was because of the detergent, but that's just a guess. Any detergent in your sample? I'll be eager to hear what others say! _ _ |_ RICK HERSHBERGER, PH.D. * Postdoctoral Fellow | |_| | | Inst. for Molecular Virology * UW-Madison | rphershb@facstaff.wisc.edu * RickOnline@aol.com From owner-proteins@net.bio.net Tue Feb 15 22:00:00 1994 Newsgroups: bionet.molbio.proteins From: Duncan@genesys.demon.co.uk (Duncan Clark) Path: biosci!bcm!news.msfc.nasa.gov!europa.eng.gtefsd.com!howland.reston.ans.net!pipex!demon!genesys.demon.co.uk!Duncan Subject: Floating Amm.SO4 ppts. Distribution: world Organization: GeneSys Ltd. Reply-To: Duncan@genesys.demon.co.uk X-Newsreader: Simple NEWS 1.90 (ka9q DIS 1.21) Lines: 17 Date: Wed, 16 Feb 1994 19:21:12 +0000 Message-ID: <761426472snz@genesys.demon.co.uk> Sender: usenet@demon.co.uk Hi Netters, Does anyone know why one sometimes gets Amm.SO4 ppts of proteins that float rather than pellet? Is there anyway to get preps that do this to pellet? In my case the prep is E.coli, sonicated, spun down, addition of polymin P (to ppt nucleic acids) plus 0.2M NaCl (to keep my protein in solution) and the ppt spun down and the supernatant kept. Addition of Amm.SO4 to 70%, left O/N at 4C then spun at 50,000g. Floating pellicle. Why? Thanks Duncan -- ----------------------------------------------------------------------------- Duncan Clark | Internet: duncan@genesys.demon.co.uk GeneSys Ltd. | Compuserve: 100015.1406@compuserve.com ----------------------------------------------------------------------------- From owner-proteins@net.bio.net Tue Feb 15 22:00:00 1994 Path: biosci!JHUIGF.BITNET!GEETHA From: GEETHA@JHUIGF.BITNET Newsgroups: bionet.molbio.proteins Subject: Re: protein sequence and structure Date: 16 Feb 1994 17:24:15 -0000 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 13 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <5DB304BCEDFF2082F6@JHUIGF.BITNET> NNTP-Posting-Host: net.bio.net Hello netters, Following are the references for structural alignments. 1. A review of methods for protein str. comparison, C.A.Orengo, in "Patterns in Protein Sequence and Structure", ed. W.R.Taylor, 1992 2. Quantification of Secondary Str. prediction improvement using multiple alignments, Levin etal., Prot.Eng.,vol.6,pp.849, 1993 3. Limits of Prot.Sec.Str. prediction accuracy from multiple seq. alignment, Russell etal., J.MOl.Biol., vol.234, pp.951, 1993 4. A structural basis for sequence comparisons-an evaluation of scoring methodlogies, Johnson etal., vol.233,pp.716, 1993 The review (1) covers almost all the existing methods. Hope this helps. --------Geetha Vasudevan. From owner-proteins@net.bio.net Tue Feb 15 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!uunet!zib-berlin.de!news.belwue.de!news.uni-freiburg.de!bio5.chemie.uni-freiburg.de!bio5.chemie.uni-freiburg.de!dreyer From: dreyer@bio5.chemie.uni-freiburg.de (Matthias Dreyer) Newsgroups: bionet.xtallography,bionet.molbio.proteins Subject: P-glycolohydroxamate Date: 16 Feb 1994 15:09:02 GMT Organization: Inst. f. Organische Chemie u. Biochemie, Freibu Lines: 33 Distribution: world Message-ID: <2jtcueINN6k03@bio5.chemie.uni-freiburg.de> NNTP-Posting-Host: bio5.chemie.uni-freiburg.de Xref: biosci bionet.xtallography:736 bionet.molbio.proteins:1367 Dear Netters, I am looking for a commercial or non-commercial source for phospho-glycolohydramate: __ __ | | 3- | O | | | | | O-P-O-CH2-C=N-OH | | | | | | O O | | | __ __ I need this as a transition state analogue of the ene-diolate form of dihydroxyacetone phosphate. Thanks for any help, Matthias ********************************************* *Matthias Dreyer * *Institut fuer Org. Chemie und Biochemie * *Universitaet Freiburg * *Alberstr. 21 * *D- 79104 Freiburg * *Germany * *e-mail: dreyer@bio5.chemie.uni-freiburg.de * ********************************************* From owner-proteins@net.bio.net Tue Feb 15 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!daresbury!doc.ic.ac.uk!warwick!pavo.csi.cam.ac.uk!mbfs.bio.cam.ac.uk!smb18 From: smb18@mbfs.bio.cam.ac.uk (Simon Brocklehurst (Bioc)) Subject: Re: Gaps (Protein Sequences) Message-ID: <1994Feb16.132328.16637@infodev.cam.ac.uk> Sender: news@infodev.cam.ac.uk (USENET news) Nntp-Posting-Host: mbfs.bio.cam.ac.uk Organization: U of Cambridge, England References: <63805.ewright@fox.nstn.ns.ca> Distribution: bionet Date: Wed, 16 Feb 1994 13:23:28 GMT Lines: 37 ewright@FOX.NSTN.NS.CA ("Edwin Wright") writes: >Given the increasingly apparent importance of "correctly placed gaps" do >you know of any researchers taking the concept of gaps even further, i.e. by >discriminating between "conserved" positions (i.e. specific residues always >present at a particular location in a group of aligned sequences) and >"non-conserved" or "degenerate" positions (i.e. not only gap positions in >the traditional sense of insertions/deletions but also mutation positions). This kind of thing is being considered in the modelling group of Tom Blundell, at Birkbeck College, Universty of London, UK. They use so-called "subsitution tables" which are multidimensional matrices where each element is the probability that one residue (or a gap) will mutate to another residue (or a gap) when the the residue (or gap) is in a given structural environment. The probabilities are obtained by analysis of sequence alignments derived from "GOOD" structural alignments. I'm afraid I don't have their references to hand. Those who want to do a literature search, should check the following authors : T. L. Blundell, J. P. Overington, M. S. Johnson, C. M. Topham, A. Sali. I think the first papers on this probably appeared in 1990 or 91. The alignments produced by this group are probably the best in the field, and thus the data they derive is likely to be those most rich in information. !----------------------------------------------------------------------- Simon M. Brocklehurst Cambridge Centre for Molecular Recognition Department of Biochemistry University of Cambridge Cambridge UK E-mail: s.m.brocklehurst@bioc.cam.ac.uk From owner-proteins@net.bio.net Tue Feb 15 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!news.unt.edu!news.oc.com!news.kei.com!yeshua.marcam.com!zip.eecs.umich.edu!newsxfer.itd.umich.edu!nntp.cs.ubc.ca!alberta!quartz.ucs.ualberta.ca!unixg.ubc.ca!bpgm From: bpgm@unixg.ubc.ca (Brent Pollock) Newsgroups: bionet.molbio.proteins Subject: Re: Floating Amm.SO4 ppts. Date: 16 Feb 1994 21:00:09 GMT Organization: University of British Columbia, Vancouver, B.C., Canada Lines: 23 Message-ID: <2ju1gp$kfa@nnrp.ucs.ubc.ca> References: <761426472snz@genesys.demon.co.uk> NNTP-Posting-Host: unixg.ubc.ca Duncan: Perhaps it is some membrane component like a lipopolysaccharide? We often get the same thing with our preps but we haven't investigated it to any great degree. It doesn't seem to interfere with the preps. Ciao, Brent Pollock >Hi Netters, > >Does anyone know why one sometimes gets Amm.SO4 ppts of proteins that float >rather than pellet? Is there anyway to get preps that do this to pellet? In >my case the prep is E.coli, sonicated, spun down, addition of polymin P (to >ppt nucleic acids) plus 0.2M NaCl (to keep my protein in solution) and the >ppt spun down and the supernatant kept. Addition of Amm.SO4 to 70%, left O/N >at 4C then spun at 50,000g. Floating pellicle. Why? > >Thanks > >Duncan From owner-proteins@net.bio.net Tue Feb 15 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!europa.eng.gtefsd.com!news.umbc.edu!haven.umd.edu!purdue!news.bu.edu!pfoster From: pfoster@bu.edu (Patricia Foster) Newsgroups: bionet.molbio.proteins Subject: Specificity of antibodies for Conformation Date: 17 Feb 1994 00:02:58 GMT Organization: Boston University Lines: 25 Message-ID: <2juc7i$7b3@news.bu.edu> NNTP-Posting-Host: acs3.bu.edu X-Newsreader: TIN [version 1.2 PL0] [ Article crossposted from bionet.molbio.methds-reagnts ] [ Author was Patricia Foster ] [ Posted on 17 Feb 1994 00:00:27 GMT ] Dear Netters, We have to decide whether to have antibodies raised against our already purified denatured protein, or to try to purify it under native conditions. So, how likely is it that polyclonal antibodies raised against a denatured protein will immunoabsorb the native form of the protein, or a complex of it with other proteins, in a cell lysate? Thanks, Pat -- Patricia L. Foster Boston University School of Medicine Boston, MA USA pfoster@bu.edu -- Patricia L. Foster Boston University School of Medicine Boston, MA USA pfoster@bu.edu From owner-proteins@net.bio.net Wed Feb 16 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!daresbury!doc.ic.ac.uk!uknet!pipex!pavo.csi.cam.ac.uk!nntp-serv.cam.ac.uk!sre From: sre@al.cam.ac.uk (Sean Eddy) Subject: Re: Gaps (Protein Sequences) In-Reply-To: ewright@FOX.NSTN.NS.CA's message of 13 Feb 1994 21:43:43 -0000 Message-ID: Sender: news@infodev.cam.ac.uk (USENET news) Nntp-Posting-Host: al.mrc-lmb.cam.ac.uk Organization: Laboratory of Molecular Biology, MRC, Cambridge UK References: <63805.ewright@fox.nstn.ns.ca> Distribution: bionet Date: Thu, 17 Feb 1994 08:22:17 GMT Lines: 62 In article <63805.ewright@fox.nstn.ns.ca> ewright@FOX.NSTN.NS.CA ("Edwin Wright") writes: > (quoting from Gonnet et. al.) >"The difficulties in contructing alignment routines are further complicated >by the requirement that they handle deletions and insertions. Even random >sequences can be aligned if gaps are introduced at no penalty. Most >alignment programs therefore assign penalties to gaps of the form (ak + b), >where a and b are arbitrarily chosen constants. There is no justification, >either theoretical or empirical, for this treatment." I've always found this statement faintly amusing. If you read carefully, you find that after this very forceful (and quite correct, except for the "arbitrarily chosen" bit) paragraph, they turn right around and use a gap penalty of form (ak + b) for their automatic alignments. Why? There *is* a theoretical justification for using affine gap costs, though it's a weak one -- the algorithms are fast :) >Given the increasingly apparent importance of "correctly placed gaps" do >you know of any researchers taking the concept of gaps even further, i.e. by >discriminating between "conserved" positions (i.e. specific residues always >present at a particular location in a group of aligned sequences) and >"non-conserved" or "degenerate" positions (i.e. not only gap positions in >the traditional sense of insertions/deletions but also mutation positions). Yes. If I understand, you want to use statistics from multiple sequence alignments -- some positions should get more weight than others because they're more conserved, and insertion/deletion costs should be variable per position because we know that insertions are more likely in some places (loops) than others (secondary structure elements). There's a lot of nice work on this problem. Here's a couple of references. Gribskov et. al. introduced "sequence profiles" which roughly do what you're suggesting. Krogh et. al., in what may become a very influential paper, have formalized sequence profiles in terms of hidden Markov models; one interesting advantage of HMMs is that they are adaptive models, which can "learn" their parameters from initially unaligned sequence data. @Article{Gribskov90, author = "Michael Gribskov and Roland Luthy and David Eisenberg", title = "Profile Analysis", journal = "Meth. Enzymol.", year = 1990, volume = 183, pages = "146-159" } @Article{Krogh94, author = "Anders Krogh and Michael Brown and I. Saira Mian and Kimmen Sjolander and David Haussler", title = "Hidden {M}arkov Models in Computational Biology: Applications to Protein Modeling", journal = "J. Mol. Biol.", year = 1994, volume = 235, pages = "1501-1531" } -- - Sean Eddy - MRC Laboratory of Molecular Biology, Cambridge, England - sre@mrc-lmb.cam.ac.uk From owner-proteins@net.bio.net Wed Feb 16 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!headwall.Stanford.EDU!agate!howland.reston.ans.net!usenet.ins.cwru.edu!lerc.nasa.gov!purdue!mentor.cc.purdue.edu!aclcb.purdue.edu!MURIANA From: muriana@aclcb.purdue.edu (Peter M. Muriana) Subject: Re: Floating Amm.SO4 ppts. Message-ID: Sender: news@mentor.cc.purdue.edu (USENET News) Reply-To: muriana@aclcb.purdue.edu Organization: Purdue University AIDS Center References: <761426472snz@genesys.demon.co.uk>, Date: Fri, 18 Feb 1994 00:31:17 GMT Lines: 57 In article , rphershb@facstaff.wisc.edu (Rick Hershberger) writes: >In article <761426472snz@genesys.demon.co.uk>, Duncan@genesys.demon.co.uk >(Duncan Clark) wrote: > >> Hi Netters, >> >> Does anyone know why one sometimes gets Amm.SO4 ppts of proteins that float >> rather than pellet? Is there anyway to get preps that do this to pellet? In >> my case the prep is E.coli, sonicated, spun down, addition of polymin P (to >> ppt nucleic acids) plus 0.2M NaCl (to keep my protein in solution) and the >> ppt spun down and the supernatant kept. Addition of Amm.SO4 to 70%, left O/N >> at 4C then spun at 50,000g. Floating pellicle. Why? >> >> ----------------------------------------------------------------------------- >> Duncan Clark >I've wondered the same thing. Though in my case, I've tried to Amm.SO4-ppt >my protein from a solution containing 0.3% sarkosyl. I thought that it was >because of the detergent, but that's just a guess. Any detergent in your >sample? I'll be eager to hear what others say! > > > _ _ |_ RICK HERSHBERGER, PH.D. Yes, most readily during the purification of bacteriocins (antimicrobial peptides) from various lactic acid bacteria. The media that is commonly used to grow lactobacilli, etc., often contains the detergent, Tween 80. During precipitation with ammonium sulfate, we often (rather, always) see the formation of a floating flocculate which forms a tighter pellicle upon centrifugation. It appears that the hydrophobic bacteriocins are associating with Tween micelles that forms the bulk of the floating layer when ammonium sulfate is applied (this has been noticed in AEM 57:114(1991) ;AEM 57:1829(1991); J.Appl.Bact 74:67(1993); Curr. Microbiol.27:35(1993)). We've found that the Tween actually facilitates purification of the bacteriocin from culture supernatant broths at relatively low saturation levels; use a Solid Phase extraction cartridge with stepwise organic phase elution to further purify large quantities (0%, 25%, 50%, 75% isopropanol); most of the contaminating proteins come off in the 25% eluent, whereas our bacteriocins come off with the 50% wash. However, they strongly associate with the Tween, but you can resolve to homogeniety on HPLC reversed-phase. Armon et al., 1988; Can. J. Microbiol. purposely added Tween and beef extract to bacteriophage preps, followed by ammonium sulfate flocculation to concentrate/recover bacteriophage in the floating pellicle . (Tween and beef extract are both components of MRS broth used to culture lactobacilli for production of bacteriocins!). Oh, Can.J. Microbiol.34:651, 1988. Regards, Peter Muriana ********************************************************************* * Peter M. Muriana, Ph.D. Phone = (317)-494-8284 * * Dept. of Food Science FAX = (317)-494-7953 * * Purdue University E-mail = muriana@aclcb.purdue.edu * * W. Lafayette, IN 47907 * ********************************************************************* From owner-proteins@net.bio.net Wed Feb 16 22:00:00 1994 Path: biosci!bcm!news.msfc.nasa.gov!europa.eng.gtefsd.com!howland.reston.ans.net!wupost!sdd.hp.com!saimiri.primate.wisc.edu!news.doit.wisc.edu!sweetprotein.ahabs.wisc.edu!user From: ming@ahabs.wisc.edu (Ding Ming) Newsgroups: bionet.molbio.proteins Subject: Re: Sine-like bands in isoelectric focussing Followup-To: bionet.molbio.proteins Date: Thu, 17 Feb 1994 11:24:25 -0600 Organization: University of Wisconsing-Madison Lines: 26 Distribution: bionet Message-ID: References: NNTP-Posting-Host: sweetprotein.ahabs.wisc.edu In article , harris@POSSUM.MURDOCH.EDU.AU (leon harris) wrote: > of a nice straight band I am getting a curvey one (~ instead of -). What > causes it? Dear Leon, Based on my experience, IEF is the trickiest one in all kinds of page. So be patient and careful. The curved band you mentioned here may be caused by your sample rather than the electrophoresis device and setup. It seems to me that your sample has a relatively high concentration of salts and you should dialyze your samples thoroughly before loading them on the gel. In addition, if there is anything you found which is related to gel batch, try to make the gel in a hour or so, polymerization should not be either too slow or too fast. The bending problem in native page was callled "edge effect"(:)! Try to avoid using the wellls next to both edges. Good luck! ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Ding Ming Telephone: (608)265-3544 University of Wisconsin-Madison Fax: (608)262-7420 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ From owner-proteins@net.bio.net Wed Feb 16 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!headwall.Stanford.EDU!agate!howland.reston.ans.net!vixen.cso.uiuc.edu!uchinews!quads!rjl4 From: rjl4@quads.uchicago.edu (Bob Litt) Subject: Trouble binding GST fusion protein to glutathione column Message-ID: <1994Feb18.014008.4188@midway.uchicago.edu> Keywords: GST fusion proteins Sender: news@uchinews.uchicago.edu (News System) Reply-To: rjl4@midway.uchicago.edu Organization: University of Chicago Date: Fri, 18 Feb 1994 01:40:08 GMT Lines: 35 I am having trouble binding a GST fusion protein to a glutathione column. I am attempting to purify fusion proteins with Ypt1, a yeast GTP binding protein. I have succesfully purified the wild-type protein and two different mutants of this protein using fusions to GST. My third mutant (which is, of course, the most interesting) expresses at high levels in E. coli (checked by Western) at similar levels to the wild-type Ypt1 fusion. The protein is soluble (not in inclusion bodies). This troublesome GST fusion does not bind well to the glutathione column. This troublesome fusion protein binds 20 times less than the analogous wild-type fusion protein. My yields are abysmal. I need more protein. I have tried: 1) Extending the binding time to the glutahtione dolumn from 1 hour to overnight. This has no effect on the amount of protein bound to the beads. 2) Decreasing or eliminating the amount of detergent in the bacterial lysate and in the washes after binding. This has no effect. 3) Changing the time and temperature and amounts of IPTG during the induction period of the fusion protein. This has helped "some", but is probably optimized now. I am at a loss. Has anyone ever had similar problems with GST fusions, or heard of similar problems? Any and all help is greatly appreciatted. Thanks in advance, Bob Litt -- Ypt1Sec4Rab3ARab1ARasGTPBindingConsensusMumboJumboARFNSFGTPgammaS ER | Bob Litt (rjl4@midway.uchicago.edu) \/ | Segev Lab -- "if it's secreted, we like it" Golgi | Univ of Chicago Dept of Molec Genetics and Cell Biol From owner-proteins@net.bio.net Wed Feb 16 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!daresbury!doc.ic.ac.uk!uknet!pipex!howland.reston.ans.net!agate!msuinfo!harbinger.cc.monash.edu.au!bruce.cs.monash.edu.au!lloyd From: lloyd@cs.monash.edu.au (Lloyd Allison) Subject: Re: Gaps (Protein Sequences) Message-ID: Sender: news@bruce.cs.monash.edu.au (USENET News System) Organization: Computer Science, Monash University, Australia References: <63805.ewright@fox.nstn.ns.ca> Distribution: bionet Date: Fri, 18 Feb 1994 00:42:17 GMT Lines: 33 sre@al.cam.ac.uk (Sean Eddy) writes: > >where a and b are arbitrarily chosen constants. There is no justification, > >either theoretical or empirical, for this treatment." >I've always found this statement faintly amusing. If you read >carefully, you find that after this very forceful (and quite correct, >except for the "arbitrarily chosen" bit) paragraph, they turn right >around and use a gap penalty of form (ak + b) for their automatic >alignments. Why? There *is* a theoretical justification for using >affine gap costs, though it's a weak one -- the algorithms are fast :) for a general philosophy of comparing models of gaps (and other parameters) in models of evolving sequences: %A L. Allison %A C. S. Wallace %A C. N. Yee %T Finite-state models in the alignment of macromolecules %J J. Mol. Evol %V 35 %P 77-89 %D 1992 %K JME, finite state, model, FSM, probabilistic, PFSM, hidden Markov model, HMM, insert, delete, indel, gap, gaps, cost function, penalty, string, sequence, homology, similarity Lloyd ALLISON Central Inductive Agency, Department of Computer Science, email: lloyd@cs.monash.edu.au Monash University, Clayton, tel: 61 3 905 5205 Victoria 3168, AUSTRALIA fax: 61 3 905 5146 From owner-proteins@net.bio.net Wed Feb 16 22:00:00 1994 Path: biosci!headwall.Stanford.EDU!agate!library.ucla.edu!europa.eng.gtefsd.com!howland.reston.ans.net!cs.utexas.edu!uunet!noc.near.net!news.delphi.com!usenet From: newittja@delphi.com Newsgroups: bionet.molbio.proteins Subject: Re: Floating Amm.SO4 ppts. Date: Thu, 17 Feb 94 19:56:06 -0500 Organization: Delphi (info@delphi.com email, 800-695-4005 voice) Lines: 19 Message-ID: References: <761426472snz@genesys.demon.co.uk> NNTP-Posting-Host: bos1b.delphi.com X-To: Duncan Clark Duncan Clark writes: >Does anyone know why one sometimes gets Amm.SO4 ppts of proteins that float >rather than pellet? Is there anyway to get preps that do this to pellet? In >my case the prep is E.coli, sonicated, spun down, addition of polymin P (to >ppt nucleic acids) plus 0.2M NaCl (to keep my protein in solution) and the >ppt spun down and the supernatant kept. Addition of Amm.SO4 to 70%, left O/N >at 4C then spun at 50,000g. Floating pellicle. Why? If it is indeed protein in your precipitate, the problem is that your solution density is to great. That is the salt and other components in your solution combined with the added ammonium sulfate create a solution that has a density greater than that of the precipitated protein. The only way to get it to pellet is to reduce the density of the solution, i.e. try precipitating with less ammonium sulfate if you can get away with it, or reduce the solute concentration in your protein solution if you can. Hope this helps. - John From owner-proteins@net.bio.net Wed Feb 16 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!lhc!ray!bryant From: bryant@ray.nlm.nih.gov (Steve Bryant) Subject: Sequences from PDB via Entrez Message-ID: <1994Feb17.150013.4056@nlm.nih.gov> Sender: news@nlm.nih.gov Organization: National Library of Medicine X-Newsreader: Tin 1.1 PL4 Date: Thu, 17 Feb 94 15:00:13 GMT Lines: 40 I've in the past seen messages on this board concering access to sequences derived from the Brookhaven Protein Data Bank. I've recently finished putting the latest PDB sequences into the Entrez database distributed by NCBI, and I thought I'd take the opportunity to remind folks that Entrez can be an easy way to retrieve a sequence from PDB. PDB-derived sequences can be identified within Entrez by using the keyword "pdb-structure". This will find either all PDB-derived protein sequences or all PDB-derived nucleic acid sequences, depending on which category one selects. Particular sequences within these groups may be found by pdb id-code, "accession number" in Entrez, or by looking for protein names and the like in "text terms". The pdb-derived entries contain "text-terms" derived from PDB COMPOUND and SOURCE records, as well as from other PDB record types. One can also find PDB-derived sequences by searching for descriptive names in the Medline abstracts included with Entrez. About 90% of the citations in PDB are linked to the corresponding Medline citation, and if you can find the paper that reported a structure, you can then ask for the associated sequence. Sequences may be written out of Entrez in different formats, including FASTA sequence files. The pdb sequence reports in Entrez combine information provided on pdb ATOM and/or HETATM records with the explicit sequence given on SEQRES. (In about 1% of cases ATOM/HETATM and SEQRES cannot be linked unambiguously, due to missing data or inconsistencies. In these cases biopolymer sequences are derived from ATOM records.) Because of this linking the sequence reports contain a fairly rich annotation, including secondary structure, disulfide bonds, bonds to nonpolymer groups, and descriptions of modified biopolymer residues. They also contain the residue numbers assigned by pdb on ATOM/HETATM records, so that one can unambiguously identify the coordinates in the pdb file that go with each residue in the sequence. PDB-derived sequence reports in Entrez are derived automatically from pdb files, and I update the collection with each new release of pdb. Network Entrez version 9.0 came out on February 10, and its database includes all polypeptide and nucleic acid sequences on the "October, 1993" Brookhaven CD, which I received in late January. Steve Bryant 2/16/94 From owner-proteins@net.bio.net Wed Feb 16 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!bcm!news.msfc.nasa.gov!europa.eng.gtefsd.com!howland.reston.ans.net!torn!watserv2.uwaterloo.ca!peptidarus.UWaterloo.ca!ltaylor From: ltaylor@peptidarus.UWaterloo.ca (Lorne Taylor) Subject: PD Distance Geometry Software?? Message-ID: Keywords: Greetings; Sender: news@watserv2.uwaterloo.ca Organization: University of Waterloo Date: Thu, 17 Feb 1994 17:40:54 GMT Lines: 12 Sorry if this is a FAQ but is there a public domain distance geometry package (for protein nmr data) available via the net. Cheers; Lorne Taylor Chemistry U. of Waterloo CANADA ltaylor@peptidarus.uwaterloo.ca From owner-proteins@net.bio.net Wed Feb 16 22:00:00 1994 Path: biosci!POSSUM.MURDOCH.EDU.AU!harris From: harris@POSSUM.MURDOCH.EDU.AU (leon harris) Newsgroups: bionet.molbio.proteins Subject: Sine-like bands in isoelectric focussing Date: 17 Feb 1994 14:05:03 -0000 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 18 Sender: daemon@net.bio.net Distribution: bionet Message-ID: NNTP-Posting-Host: net.bio.net Hi netters! Does anyone out there have any experience with the LKB PAGplate system for isoelectric focussing? I am having this problem where, instead of a nice straight band I am getting a curvey one (~ instead of -). What causes it? My system is refrigerated to 4oC, and I am running under the coditions suggested by LKB. It just aint right! Also does anyone have any suggestion why the bands closest to the edge (ie near the spacers) sometimes bend on native discontinuous PAGE? (I don't use the end wells now). Any suggestions would really be appreciated. Bye for now Leon Harris@possum.murdoch.edu.au From owner-proteins@net.bio.net Wed Feb 16 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!bcm!news.msfc.nasa.gov!europa.eng.gtefsd.com!howland.reston.ans.net!pipex!sunic!news.lth.se!news.lu.se!gemini.ldc.lu.se!MASKLINKEM From: masklinkem@gemini.ldc.lu.se Subject: Brookhaven in Europe? Message-ID: <1994Feb17.160612.20595@nomina.lu.se> Sender: news@nomina.lu.se (USENET News System) Nntp-Posting-Host: gemini.ldc.lu.se Reply-To: masklinkem@gemini.ldc.lu.se Organization: Lund University Sweden Date: Thu, 17 Feb 1994 16:06:12 GMT Lines: 4 Do anybody know of a european FTP site that contain the coordinates of the 3-Dfiles that otherwise are in the Brookhaven Database? Pal Szecsi University of Lund Sweden From owner-proteins@net.bio.net Thu Feb 17 22:00:00 1994 Path: biosci!daresbury!doc.ic.ac.uk!warwick!uknet!pipex!uunet!Germany.EU.net!netmbx.de!zib-berlin.de!math.fu-berlin.de!tertius.in-berlin.de!wolf_w From: wolf_w@FHI-Berlin.MPG.DE (WILFRIED WOLF) Newsgroups: bionet.molbio.proteins Subject: HELP: thermodyn data of phosphotyrosine/tyrosine Message-ID: <1994Feb18.123919.8082@rz-berlin.mpg.de> Date: 18 Feb 94 12:39:19 +0100 Organization: Fritz-Haber-Institut der MPG,Berlin Lines: 12 hello everybody out there! a friend of mine is searching for delta-H and/or delta-G of the phosphorylation of tyrosine, so any hint where to find data of formation energies of (L-para-)phosphotyrosine and tyrosine itself or thermodynamic data concerning the above mentioned reaction itself would be very much appreciated. thanx willi ps: could you please mail to me directly, since i don't often look in these newsgroups (E-Mail: WOLF_W@FHI-Berlin.MPG.DE) From owner-proteins@net.bio.net Thu Feb 17 22:00:00 1994 Path: biosci!daresbury!doc.ic.ac.uk!warwick!pipex!zaphod.crihan.fr!jussieu.fr!univ-lyon1.fr!swidir.switch.ch!scsing.switch.ch!urz.unibas.ch!bickle From: bickle@urz.unibas.ch Newsgroups: bionet.molbio.proteins Subject: Re: Floating Amm.SO4 ppts. Message-ID: <1994Feb18.085142.43580@urz.unibas.ch> Date: 18 Feb 94 08:51:42 MET References: <761426472snz@genesys.demon.co.uk> Organization: University of Basel, Switzerland Lines: 20 In article , newittja@delphi.com writes: > Duncan Clark writes: > >Does anyone know why one sometimes gets Amm.SO4 ppts of proteins that float >rather than pellet? Is there anyway to get preps that do this to pellet? In >my case the prep is E.coli, sonicated, spun down, addition of polymin P (to >ppt nucleic acids) plus 0.2M NaCl (to keep my protein in solution) and the >ppt spun down and the supernatant kept. Addition of Amm.SO4 to 70%, left O/N >at 4C then spun at 50,000g. Floating pellicle. Why? > If you are really using 70% AS the solution is simply denser than the precipitated proteins, which therefore float. Doesn't your recipe call for using 70% of *saturation*? Which is about 40 % W/V if my memory serves. -- Tom Bickle Microbiology Dept, Biozentrum, Basel University Klingelbergstrasse 70, CH-4056 Basel, Switzerland + 41 61 267 21 20 bickle@urz.unibas.ch From owner-proteins@net.bio.net Thu Feb 17 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!usenet.ins.cwru.edu!cleveland.Freenet.Edu!bl275 From: bl275@cleveland.Freenet.Edu (Dan Diaz) Newsgroups: bionet.molbio.proteins Subject: Experience with KSCN for subunit dissociation? Date: 18 Feb 1994 14:56:52 GMT Organization: Case Western Reserve University, Cleveland, OH (USA) Lines: 9 Message-ID: <2k2kvl$cdh@usenet.INS.CWRU.Edu> Reply-To: bl275@cleveland.Freenet.Edu (Dan Diaz) NNTP-Posting-Host: kanga.ins.cwru.edu i am trying to dissociate a oligomeric enzyme without unfolding the subunits. i have read that thiocynate (SCN-) is often useful in the generation of 'structured monomers'. i would appreciate hearing from those who have used SCN or some other moderate denaturant. diz From owner-proteins@net.bio.net Thu Feb 17 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!wupost!udel!pacs.sunbelt.net!lynx.unm.edu!dns1.NMSU.Edu!mwaugh Newsgroups: bionet.molbio.proteins Subject: Mac-compatable flatbed scanners and software for 2-D analysis. Message-ID: <2k2ufrINNks@dns1.NMSU.Edu> From: mwaugh@crl.nmsu.edu (Mark E. Waugh) Date: 18 Feb 1994 17:39:07 GMT Reply-To: mwaugh@crl.nmsu.edu (Mark E. Waugh) Sender: mwaugh@crl.nmsu.edu. Organization: New Mexico State University Keywords: scanner, 2-dimensional electrophoresis NNTP-Posting-Host: pp-macgouda.nmsu.edu X-Posted-From: InterNews 1.0@dns1.nmsu.edu. Lines: 16 Does anyone have any experience with Mac-compatable flatbed scanners and software for scanning autoradiographs of 2-D gels? Our lab-net includes PC's, Mac's and a remote-mounted Sun station, but the machine we want to hang the scanner off of is a Mac. We have downloaded a 2-D manipulation and analysis program from CERN (Melanie) which runs in the Unix world and is accessable to the Macs through eXodus X-windows. The problem now is finding a reasonably priced (<$2000) fairly high resolution flatbed scanner with software that will allow thresholding and will provide 256 levels of grayscale reproducibly. If anyone has any experience with Mac scanners, especially in conjunction with scanning autorads, please respond either in this news group or feel free to send me e-mail at: mwaugh@crl.nmsu.edu Any help would be greatly appreciated. From owner-proteins@net.bio.net Thu Feb 17 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!pipex!uknet!gdt!aber!spp From: spp@aber.ac.uk (Simon Peter Penson) Subject: Re: Sine-like bands in isoelectric focussing Message-ID: <1994Feb18.085921.29652@aber.ac.uk> Organization: University of Wales - Aberystwyth - Prifysgol Cymru References: Distribution: bionet Date: Fri, 18 Feb 1994 08:59:21 GMT Lines: 56 In article , leon harris wrote: > >Hi netters! > Does anyone out there have any experience with the LKB PAGplate >system for isoelectric focussing? I am having this problem where, instead >of a nice straight band I am getting a curvey one (~ instead of -). What >causes it? My system is refrigerated to 4oC, and I am running under the >coditions suggested by LKB. It just aint right! Also does anyone have any >suggestion why the bands closest to the edge (ie near the spacers) >sometimes bend on native discontinuous PAGE? (I don't use the end wells >now). Any suggestions would really be appreciated. > >Bye for now > >Leon > >Harris@possum.murdoch.edu.au > > I think you may have several possible problems. Look at a copy of the Pharmacia IEF guide. There are several sections describing problems like yours. If you can't find a copy, send me your full mail (ie real) address, and I'll send you a copy of the relevant bits. Here's a summary: Disturbances and wavy bands (in IEF gels) 1. Poor contact between electrode and electrode strip 2. Poor choice of electrode solutions 3. Uneven wetting of electrode strip 4. Excess salt I never run a sample in the outermost lane-edge effect distortion is almost inevitable. Also, try running at a lowwer temperature. Hope this helps, Simon ----------------------------------------------------------------------------- Simon Penson spp@aber.ac.uk (internet) Environmental Biology Dept., spp@uk.ac.aber (janet) AFRC Institute of Grasslands and Environmental Research, Aberystwyth, Dyfed, UK, SY23 3RH. Tel: +44 (0)970 828255 Fax: +44 (0)970 828357 From owner-proteins@net.bio.net Thu Feb 17 22:00:00 1994 Path: biosci!daresbury!doc.ic.ac.uk!bay.cc.kcl.ac.uk!bay.cc.kcl.ac.uk!udbl119 Newsgroups: bionet.molbio.proteins Subject: PP2B Inhibitor Message-ID: From: udbl119@bay.cc.kcl.ac.uk (Mandy Johnstone) Date: Fri, 18 Feb 1994 16:10:30 Organization: King's College London Nntp-Posting-Host: pgw2.rai.kcl.ac.uk X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] Lines: 9 Hi, Does anyone know of a good inhibitor for protein phosphatase 2B (calcineurin) that can be used in an in vitro kinase assay?. I recently read that cyclosporin-cyclophilin A and FK506-FKBP have been found to inhibit PP2B but I do not know how successful these immunosuppressant drugs would be in my assay system. Does anyone out there have any ideas or previos experience of these or possible other PP2B inhibitors?. Cheers, Mandy. From owner-proteins@net.bio.net Thu Feb 17 22:00:00 1994 Path: biosci!daresbury!doc.ic.ac.uk!agate!kos2mac23.berkeley.edu!user From: genecutl@mendel.berkeley.edu (gc) Newsgroups: bionet.molbio.proteins Subject: Re: Specificity of antibodies for Conformation Followup-To: bionet.molbio.proteins Date: Fri, 18 Feb 1994 15:45:42 +0200 Organization: -none- Lines: 36 Message-ID: References: <2juc7i$7b3@news.bu.edu> NNTP-Posting-Host: kos2mac23.berkeley.edu In article <2juc7i$7b3@news.bu.edu>, pfoster@bu.edu (Patricia Foster) wrote: > [ Article crossposted from bionet.molbio.methds-reagnts ] > [ Author was Patricia Foster ] > [ Posted on 17 Feb 1994 00:00:27 GMT ] > > Dear Netters, > We have to decide whether to have antibodies raised > against our already purified denatured protein, or to > try to purify it under native conditions. So, how > likely is it that polyclonal antibodies raised > against a denatured protein will immunoabsorb the > native form of the protein, or a complex of it with > other proteins, in a cell lysate? > Thanks, > Pat > -- > Patricia L. Foster > Boston University School of Medicine > Boston, MA USA > pfoster@bu.edu In the experience of my lab, it varies a lot. Some polyclonals are great for westerns and can't immunoprecipitate anything, while others are the reverse, and some can do both. All of our antibodies are raised against denatured proteins. It seems to be the luck of the draw. Inject enought rabbits and you'll get the antibodies you want. -- --gc From owner-proteins@net.bio.net Thu Feb 17 22:00:00 1994 Path: biosci!daresbury!doc.ic.ac.uk!warwick!pipex!howland.reston.ans.net!sol.ctr.columbia.edu!newsxfer.itd.umich.edu!news1.oakland.edu!condor.ic.net!usenet From: Gelman@ic.net Newsgroups: bionet.molbio.proteins Subject: Material Grants Available Date: 18 Feb 1994 21:06:37 GMT Organization: Gelman Sciences Lines: 19 Distribution: world Message-ID: <2k3alb$kiu@condor.ic.net> NNTP-Posting-Host: 152.160.5.17 X-Newsreader: We recently have learned that university material grant programs are available as a means of making donations. We want to donate filtration equipment to these material grants, especially to bio-technology groups within the university communities, in celebration of our thirty fifth anniversary. We would also consider non-profit foundations other than universities which have material grant operations. Unfortunately, our order service people don't know any of the details about the material grant programs or who to contact. You could be of help to us and to your departments. Please send your responses to Dr. James Camilleri at Gelman@ic.net. Please indicate the names of people we should contact about material grants. If your department would like to be included on a materials grant, please let us know so that we might offer free merchandise specifically to you. Thank you. Charles Gelman Chairman and Chield Executive Officer From owner-proteins@net.bio.net Thu Feb 17 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!agate!msuinfo!netnews.upenn.edu!dsinc!ub!galileo.cc.rochester.edu!news From: DSCT@BPHVAX.BIOPHYSICS.ROCHESTER.EDU (DAVID SCOTT) Subject: Re: Mac-compatable flatbed scanners and software for 2-D ana In-Reply-To: mwaugh@crl.nmsu.edu's message of 18 Feb 1994 17:39:07 GMT Message-ID: <1994Feb18.202402.21573@galileo.cc.rochester.edu> Sender: news@galileo.cc.rochester.edu Nntp-Posting-Host: bphvax.biophysics.rochester.edu Organization: DEPT. OF BIOPHYSICS, UNIVERSITY OF ROCHESTER References: <2k2ufrINNks@dns1.NMSU.Edu> Date: Fri, 18 Feb 94 20:24:02 GMT X-News-Reader: VMS NEWS v1.25 Lines: 27 In <2k2ufrINNks@dns1.NMSU.Edu> mwaugh@crl.nmsu.edu writes: > Does anyone have any experience with Mac-compatable flatbed scanners > and software for scanning autoradiographs of 2-D gels? Our lab-net > includes PC's, Mac's and a remote-mounted Sun station, but the machine > we want to hang the scanner off of is a Mac. We have downloaded a 2-D > manipulation and analysis program from CERN (Melanie) which runs in the > Unix world and is accessable to the Macs through eXodus X-windows. The > problem now is finding a reasonably priced (<$2000) fairly high > resolution flatbed scanner with software that will allow thresholding > and will provide 256 levels of grayscale reproducibly. If anyone has > any experience with Mac scanners, especially in conjunction with > scanning autorads, please respond either in this news group or feel > free to send me e-mail at: > > mwaugh@crl.nmsu.edu > > Any help would be greatly appreciated. You want to look for a program called NIH-Image, that only runs on macs. It is available for free by FTP from the NIH at zippy.nimh.nih.gov [128.231.98.32] There is a list that discusses nih-image that can be subscribed to from listserv@soils.umn.edu. It supports a variety of flat bed scanners, and may do the analysis you want. -Gavin Fischer University of Rochester Cancer Center From owner-proteins@net.bio.net Thu Feb 17 22:00:00 1994 Path: biosci!daresbury!doc.ic.ac.uk!bay.cc.kcl.ac.uk!bay.cc.kcl.ac.uk!udbl119 Newsgroups: bionet.molbio.proteins Subject: PP2B Inhibitor Message-ID: From: udbl119@bay.cc.kcl.ac.uk (Mandy Johnstone) Date: Fri, 18 Feb 1994 15:59:24 Organization: King's College London Keywords: PP2B Nntp-Posting-Host: pgw2.rai.kcl.ac.uk X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] Lines: 2 Hi, From owner-proteins@net.bio.net Thu Feb 17 22:00:00 1994 Path: biosci!JHUIGF.BITNET!GEETHA From: GEETHA@JHUIGF.BITNET Newsgroups: bionet.molbio.proteins Subject: alpha helical coiled coils in proteins Date: 18 Feb 1994 17:18:18 -0000 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 9 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <5C2B3F3EA6FF2090DC@JHUIGF.BITNET> NNTP-Posting-Host: net.bio.net Hello Netters, Can anybody help me understand the difference between coiled coils and supercoils? I believe, the alpha helical coiled coils is a result of packing interactions between parallel or antiparallel or relatively staggered axes. It must be something analogous to the twisted beta sheets then. What are those supercoils? I might be wrong. Pl.help me. --Geetha. From owner-proteins@net.bio.net Thu Feb 17 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!torn!watserv2.uwaterloo.ca!peptidarus.UWaterloo.ca!ltaylor From: ltaylor@peptidarus.UWaterloo.ca (Lorne Taylor) Subject: REPOST: PD Distance Geometry Software?? Message-ID: Sender: news@watserv2.uwaterloo.ca Organization: University of Waterloo Date: Fri, 18 Feb 1994 15:18:52 GMT Lines: 11 Greetings Sorry if this is a FAQ - Is there a public domain, net retrievable distance geometry package (suitable for protein NMR)?? Cheers Lorne ltaylor@peptidarus.uwaterloo.ca From owner-proteins@net.bio.net Thu Feb 17 22:00:00 1994 Path: biosci!headwall.Stanford.EDU!agate!library.ucla.edu!csulb.edu!nic-nac.CSU.net!usc!cs.utexas.edu!uunet!noc.near.net!das-news.harvard.edu!husc-news.harvard.edu!husc.harvard.edu!husc10!robison1 Newsgroups: bionet.molbio.proteins Subject: Re: Swiss protein sequence Message-ID: From: robison1@husc10.harvard.edu (Keith Robison) Date: 16 Feb 94 05:02:58 GMT References: <2joiap$dm8@gap.cco.caltech.edu> Organization: Harvard University, Cambridge, Massachusetts NNTP-Posting-Host: husc10.harvard.edu Lines: 27 wqc@hood.caltech.edu (Wangqin Chen) writes: >How do I access to the SWISS:PROT protein sequence data base? If anybody >knows, please help! Thank. Okay, here's 1 sample for each of 4 popular access methods E-mail: retrieve@ncbi.nlm.nih.gov FTP: ncbi.nlm.nih.gov /repository gopher: ftp.bio.indiana.edu WWW: http://expasy.hcuge.ge (Note: I don't have the paths fully specified for the last 3, but what I specified gets you within sight of the goal) Enjoy! Keith Robison Harvard University Department of Cellular and Developmental Biology Department of Genetics / HHMI krobison@nucleus.harvard.edu From owner-proteins@net.bio.net Fri Feb 18 22:00:00 1994 Path: biosci!daresbury!doc.ic.ac.uk!uknet!pipex!howland.reston.ans.net!noc.near.net!bigboote.WPI.EDU!wpi.WPI.EDU!kitten From: kitten@wpi.edu (Kitten) Newsgroups: bionet.molbio.proteins Subject: prions Date: 18 Feb 1994 19:28:25 GMT Organization: Worcester Polytechnic Institute Lines: 14 Message-ID: <2k34sp$ojl@bigboote.WPI.EDU> NNTP-Posting-Host: wpi.wpi.edu X-Newsreader: TIN [version 1.2 PL1] I think this is the right newsgroup for this... I was just wondering if anybody had any information on prions. They are pure protein viruses, no nucleic acids. I don't even need all facts I would love to read theories on their reproduction etc... you can either post or email me at kitten@wpi.wpi.edu thanx... jen From owner-proteins@net.bio.net Sat Feb 19 22:00:00 1994 Path: biosci!daresbury!doc.ic.ac.uk!warwick!pipex!howland.reston.ans.net!europa.eng.gtefsd.com!gatech!usenet.ins.cwru.edu!magnus.acs.ohio-state.edu!mwadhwa From: mwadhwa@magnus.acs.ohio-state.edu (Manpreet S Wadhwa) Newsgroups: bionet.molbio.proteins,bionet.molbio.methds-reagnts Subject: stability of polylysine Date: 20 Feb 1994 08:13:17 GMT Organization: The Ohio State University Lines: 21 Message-ID: <2k762t$1lo@charm.magnus.acs.ohio-state.edu> NNTP-Posting-Host: bottom.magnus.acs.ohio-state.edu Xref: biosci bionet.molbio.proteins:1398 bionet.molbio.methds-reagnts:11689 hi netters, i have been working with polylysine in some of my experiments. I generally dissolve the polylysine (hydrobromide) in deionized water and keep aliquots of the concentrated stock solution (50mg/ml) in the freezer. My working solution is 4mg/ml and this i keep either in the freezer, or sometimes refrigerated at 4 degrees (when in regular use). The label on the sigma bottle for polylysine says "Dessicate" and "Store at less than 0 degree C". I've been wondering lately if the dessication requirement is only to avoid hygroscopicity (which would change the weight of the powder), or is there a stability issue involved? Provided that there are no proteases swimming around, is there any problem in storing the polylysine in the way i am? (probably shouldn't be, given the stability of amide bonds...) please email me any comments, or post here. thanks, /manpreet s. wadhwa From owner-proteins@net.bio.net Sat Feb 19 22:00:00 1994 Path: biosci!FOX.NSTN.NS.CA!ewright From: ewright@FOX.NSTN.NS.CA ("Edwin Wright") Newsgroups: bionet.molbio.proteins Subject: Longest Protein Sequences Date: 21 Feb 1994 01:04:13 -0000 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 20 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <75890.ewright@fox.nstn.ns.ca> NNTP-Posting-Host: net.bio.net Does anyone have references for the following: (a) Longest viral protein sequenced to date (b) Shortest viral protein sequenced to date (c) Longest non-viral protein sequenced to date (d) Shortest non-viral protein sequenced to date (e) Consensus data for (a), (b), (c), and (d) Edwin Wright 85 Spinnaker Drive, A-602 Halifax, Nova Scotia CANADA B3N 3E3 Telephone: (902) 477-5037 Email: ewright@fox.nstn.ns.ca =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= From owner-proteins@net.bio.net Sat Feb 19 22:00:00 1994 Path: biosci!daresbury!doc.ic.ac.uk!uknet!bnr.co.uk!corpgate!news.utdallas.edu!wupost!howland.reston.ans.net!sol.ctr.columbia.edu!news.columbia.edu!bonjour.cc.columbia.edu!ps44 From: ps44@bonjour.cc.columbia.edu (Pavel Sova) Newsgroups: bionet.molbio.proteins Subject: Re: Trouble binding GST fusion protein to glutathione column Date: 21 Feb 1994 01:08:12 GMT Organization: Columbia University Lines: 49 Message-ID: <2k91hs$h8@apakabar.cc.columbia.edu> References: <1994Feb18.014008.4188@midway.uchicago.edu> NNTP-Posting-Host: bonjour.cc.columbia.edu Keywords: GST fusion proteins In article <1994Feb18.014008.4188@midway.uchicago.edu>, Bob Litt wrote: >I am having trouble binding a GST fusion protein to a glutathione >column. I am attempting to purify fusion proteins with Ypt1, a yeast >GTP binding protein. I have succesfully purified the wild-type >protein and two different mutants of this protein using fusions to >GST. My third mutant (which is, of course, the most interesting) >expresses at high levels in E. coli (checked by Western) at similar >levels to the wild-type Ypt1 fusion. The protein is soluble (not in >inclusion bodies). This troublesome GST fusion does not bind well to >the glutathione column. This troublesome fusion protein binds 20 >times less than the analogous wild-type fusion protein. My yields are >abysmal. I need more protein. I have tried: > ---stuff deleted--- >I am at a loss. Has anyone ever had similar problems with GST >fusions, or heard of similar problems? Yes, I've just encountered the same problem. My fusion protein of GST with vif (of HIV) doesn't bind to glutathione-sepharose, although, under the same conditions and in parallel, I get strong binding (->band on SDS-PAGE) of plain GST. My thought was that addition of another piece of protein to GST distorts its structure and binding capacity to glutathione. But your system is somewhat different - how similar are those mutant alleles? So far, I figured to try to transform different strain of bacteria (JM109 instead of DH5alfa) and perhaps to play a little with incubation temperature (should affect solubility and maybe conformation?) and detergents (those may affect conformation, either - although you've tried it). > >Any and all help is greatly appreciatted. > >Thanks in advance, >Bob Litt > ER | Bob Litt (rjl4@midway.uchicago.edu) Sorry I couldn't help you. I will also appreciate any help of general public. Thanks in advance, Pavel Sova = ps44@columbia.edu| Molecular Virology Laboratory| Columbia University| New York From owner-proteins@net.bio.net Sat Feb 19 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!daresbury!doc.ic.ac.uk!uknet!pipex!howland.reston.ans.net!sol.ctr.columbia.edu!news.kei.com!newsstand.cit.cornell.edu!cornell!uw-beaver!netnews.nwnet.net!serval!wsuaix!pxu From: pxu@wsuaix (Pin Xu) Subject: remove polysaccharide from protein prep? Message-ID: <1994Feb20.055500.16795@serval.net.wsu.edu> Sender: news@serval.net.wsu.edu (USENET News System) Organization: Washington State University X-Newsreader: Tin 1.1 PL4 Date: Sun, 20 Feb 94 05:55:00 GMT Lines: 13 Greating, Could anyone tell me how to separate protein from soluble polysaccharide? Recently I tried to purify a protein from 100,000 g supernatant of pollen homogenate by ammonium sulfate precipitation. However, it seems that protein can be co-precipitated by polysaccharide (or liopsaccharide?). Please send your kind reply to me via e-mail: pxu@wsuaix.csc.wsu.edu Thanks, p. xu From owner-proteins@net.bio.net Sun Feb 20 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!pipex!uknet!bcc.ac.uk!bsm.bioc.ucl.ac.uk!martin From: martin@bsm.bioc.ucl.ac.uk (Andrew Martin) Subject: Info on sequence entries Message-ID: <1994Feb21.141520.83266@ucl.ac.uk> Date: Mon, 21 Feb 1994 14:15:20 GMT Organization: Bloomsbury Computing Consortium Lines: 18 I wish to extract the sequences of a few hundred antibodies from one of the sequence databases, but also need to know the antigen to which each binds (if known). Does anyone know if any of the sequence databases include this level of information, or will I simply have to take the reference from each entry and spend a week in the library looking them up? All suggestions welcome! Andrew -- **************************************************************************** Dr. Andrew C.R. Martin, University College London & SciTech Software INTERNET: martin@bsm.bioc.ucl.ac.uk -OR- amartin@scitec.adsp.sub.org JANET: martin@uk.ac.ucl.bioc.bsm UUCP: {uunet|rutgers}!cbmehq!cbmuk!scitec!amartin **************************************************************************** From owner-proteins@net.bio.net Sun Feb 20 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!sol.ctr.columbia.edu!usenet.ucs.indiana.edu!news From: Bill Nowatzke Subject: Re: Floating Amm.SO4 ppts. Message-ID: X-Xxdate: Mon, 21 Feb 94 19:22:23 GMT Sender: news@usenet.ucs.indiana.edu (USENET News System) Nntp-Posting-Host: nowatzke-willia.tuliptree.indiana.edu Organization: IU Chemistry X-Useragent: Nuntius v1.1.1d7 References: <761426472snz@genesys.demon.co.uk> <1994Feb18.085142.43580@urz.unibas.ch> Date: Tue, 22 Feb 1994 00:21:40 GMT Lines: 6 I have seen this same problem- getting a flocculant mess even after 20kXg spin. I deanted as much as I could without too much protein loss and respun at 40k X g. The reulting pellet was firm enough to separate from the amm sulfate sup, but I am wondering are there cautious limits to consider when collecting protein ppt? I wasn't able to resuspend all of the ppt (fine particles) upon dialyzing. From owner-proteins@net.bio.net Sun Feb 20 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!pipex!uknet!EU.net!Germany.EU.net!netmbx.de!zib-berlin.de!news.th-darmstadt.de!brewhq!atkins!wolfgang From: wolfgang@atkins.swb.de (Wolfgang Sachs) Newsgroups: bionet.molbio.proteins,bionet.general,bionet.software Subject: looking for pdb description of proteases Message-ID: <5837.2102942253@atkins.swb.de> Date: Mon, 21 Feb 1994 21:53:24 GMT Reply-To: wolfgang@atkins.swb.de (Wolfgang Sachs) Organization: Institute for Artificial Insanity Followup-To: bionet.molbio.proteins Content-Type: text/plain Content-Transfer-Encoding: 8bit Lines: 51 Xref: biosci bionet.molbio.proteins:1405 bionet.general:7881 bionet.software:7309 Hi bionet'ers, can anyone send me descriptions of 3d structures of proteases in the pdb file format? I have to give a talk about the "structure - function relationship" and I want to display the 3d structures of some enzymes to show the catalytic site, so I can talk about the mechanism more vivid. I want to structure my talk according to the classes of proteases: Serine proteases: Chymotrypsin A Trypsin (EC 3.4.21.4) Thrombin Factor X Plasmin Elastase Cysteine proteases: Papain (EC 3.4.22.2) Calpain Cathepsins B, H, L and S Aspartic proteases: Pepsin Cathepsin D Chymosin (EC 3.4.23.4) Penicillopepsin (EC 3.4.23.6) Renin (EC 3.4.99.19) Metallo-proteases: Thermolysin (EC 3.4.24.4) Bovine Carboxypeptidase A (EC 3.4.17.1) Does anybody have any hints...yes I'm actually doing literature-search ;-) Since I have to do the talk next week and have no account at the university I cannot download, ftp or even search via gophers and WWW. And since I don't know the exact names I even can't question the archie-servers :-| So I depend on you kindness...if you have apropriate material and are willing to help a fellow student..please send it to me... Thank you very much for the work _I_ cause _you_ to do... Wolfgang Sachs -- Wolfgang Sachs, Neckarstr. 25, D-64390 Erzhausen, Germany Voice: +49 6150 82882 Domain: Wolfgang@atkins.swb.{de | sub.org} From owner-proteins@net.bio.net Sun Feb 20 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: parmentf@ERE.UMontreal.CA (Parmentier Fabrice) Newsgroups: bionet.molbio.proteins Subject: problems with inclusions bodies Date: 21 Feb 1994 16:58:43 -0000 Lines: 11 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2kap83$mun@mserv1.dl.ac.uk> Original-To: methods@dl.ac.uk Hi netters, Recently I did a construction using the GST gene fusion system with a hydrophobic peptide of 40 amino acids. This fusion protein was well expressed in E.Coli but was unfortunately associated with the pellet fraction and probably localized in inclusion bodies. I have tried to alter the growth conditions of the bacteria but in each case the fusion protein still associated with the pellet fraction. I have tried to solubilize the inclusion bodies of the pellet fraction with: CHAPS 1%, Triton X-100,1%, urea or Gn-HCl and every time the fusion protein is still present in the pellet fraction. I could try a more drastic treatment of this fraction. However, I will need to purify the fusion protein with glutathione Sepharose 4B beads. Any suggestions or comments are appreciated Isabelle Lemire parmentf@ere.umontreal.ca From owner-proteins@net.bio.net Sun Feb 20 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!sol.ctr.columbia.edu!newsxfer.itd.umich.edu!destroyer!caen!batcomputer!ghost.dsi.unimi.it!univ-lyon1.fr!swidir.switch.ch!scsing.switch.ch!rzusuntk.unizh.ch!newsadm From: newsadm@rzu-news.unizh.ch (CNEWS ADMINISTRATION) Subject: re: prions Message-ID: <1994Feb21.161245.15443@rzu-news.unizh.ch> Organization: University of Zurich, Switzerland References: <2k34sp$ojl@bigboote.WPI.EDU> Date: Mon, 21 Feb 1994 16:12:45 GMT Lines: 16 In article <2k34sp$ojl@bigboote.WPI.EDU> kitten@wpi.edu (Kitten) writes: > >I think this is the right newsgroup for this... > >I was just wondering if anybody had any information on prions. >They are pure protein viruses, no nucleic acids. I don't even need all facts I >would love to read theories on their reproduction etc... > >you can either post or email me at > >kitten@wpi.wpi.edu > >thanx... > >jen From owner-proteins@net.bio.net Sun Feb 20 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!pipex!doc.ic.ac.uk!bay.cc.kcl.ac.uk!bay.cc.kcl.ac.uk!R.Burge Newsgroups: bionet.molbio.proteins Subject: Re: prions Message-ID: From: R.Burge@bay.cc.kcl.ac.uk (Richard Burge) Date: Mon, 21 Feb 1994 14:20:28 References: <2k34sp$ojl@bigboote.WPI.EDU> Organization: King's College London Nntp-Posting-Host: pgw2.rai.kcl.ac.uk X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] Lines: 20 In article <2k34sp$ojl@bigboote.WPI.EDU> kitten@wpi.edu (Kitten) writes: >I was just wondering if anybody had any information on prions. >They are pure protein viruses, no nucleic acids. I don't even need all facts I >would love to read theories on their reproduction etc... Check out the following paper for a general review. It should be a pointer to some of the key papers: Weissmann, Charles - Molecular Biology of Prion Diseases. Trends in Cell Biology Jan 94 v4 no.1, pp10-14. I hope this helps, __________________________________________________ Richard Burge | e-mail: King's College London | R.Burge@bay.cc.kcl.ac.uk ______________________|___________________________ From owner-proteins@net.bio.net Sun Feb 20 22:00:00 1994 Path: biosci!daresbury!doc.ic.ac.uk!agate!library.ucla.edu!europa.eng.gtefsd.com!howland.reston.ans.net!vixen.cso.uiuc.edu!sdd.hp.com!saimiri.primate.wisc.edu!news.doit.wisc.edu!sweetprotein.ahabs.wisc.edu!user From: ming@ahabs.wisc.edu (Ding Ming) Newsgroups: bionet.molbio.proteins Subject: Re: Floating Amm.SO4 ppts. Followup-To: bionet.molbio.proteins Date: Mon, 21 Feb 1994 22:25:25 -0600 Organization: University of Wisconsing-Madison Lines: 27 Message-ID: References: <761426472snz@genesys.demon.co.uk> <1994Feb18.085142.43580@urz.unibas.ch> NNTP-Posting-Host: sweetprotein.ahabs.wisc.edu In article , Bill Nowatzke wrote: > I have seen this same problem- getting a flocculant mess even after 20kXg > spin. I deanted as much as I could without too much protein loss and > respun at 40k X g. The reulting pellet was firm enough to separate from > the amm sulfate sup, but I am wondering are there cautious limits to > consider when collecting protein ppt? I wasn't able to resuspend all of > the ppt (fine particles) upon dialyzing. I have seen this problem too and the solution I figured out finally was to let it stay under 4 degree o/n and the fine fluff will coagulate together. The higher saturation of amm sulfate, the longer staying under cold!! The additional common problem is that protein ppt. may form foam floating on the surface of the sup if you add in solid ammsulfate and this kind of foam won't dissipate ever you spin it in high speed. The solution is that you either to vaccum it a little bit before the spin or add a drop of octanol. It is interesting to learn that you cannot dissolve the fine fluffy particles and maybe the protein ppt is not soluble in your dialyzing buffer(water?). Did you try to dissolve it in salt solution? ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Ding Ming Telephone: (608)265-3544 University of Wisconsin-Madison Fax: (608)262-7420 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ From owner-proteins@net.bio.net Mon Feb 21 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!newsserver.jvnc.net!cyanamid!igate.cyanamid.com!sandy From: sandy@nmr1.pt.cyanamid.COM (Sandy Silverman) Subject: Re: homology, identity.... In-Reply-To: moreaut@univ-tours.fr's message of Tue, 22 Feb 94 15:39:46 GMT Message-ID: Sender: news@cyanamid.uucp Organization: American Cyanamid Company References: <2kd9j2$e4q@news.univ-rennes1.fr> Date: Tue, 22 Feb 1994 17:31:03 GMT Lines: 6 I think it is best to reserve the word "homology" for evolutionary relation- ships and similarity/identity % for molecular look-alikes. -- Sanford Silverman >Opinions expressed here are my own< American Cyanamid sandy@pt.cyanamid.com, silvermans@pt.cyanamid.com "Yeast is Best" From owner-proteins@net.bio.net Mon Feb 21 22:00:00 1994 Path: biosci!s.u-tokyo!news.tisn.ad.jp!news.u-tokyo.ac.jp!sinetnews!daffy!uwvax!uwm.edu!vixen.cso.uiuc.edu!howland.reston.ans.net!pipex!zaphod.crihan.fr!news.univ-rennes1.fr!newsmaster From: moreaut@univ-tours.fr Newsgroups: bionet.molbio.proteins Subject: homology, identity.... Date: Tue, 22 Feb 94 15:39:46 GMT Organization: UNIVERSITE FRANCOIS RABELAIS - TOURS - FRANCE Lines: 29 Message-ID: <2kd9j2$e4q@news.univ-rennes1.fr> NNTP-Posting-Host: rablai.univ-tours.fr dear colleagues, I would like to have your point of view on the problem of sequence homology, sequence similarity and percent identity. When I align two related proteins with Gap or Bestfit in the GCG package, the % identity and % similarity vary between about 35 to 50 % identity. This percentage obviously depends of the gap parameters chosen for this alignment. OK I would like to know what is the best thing to do to quantify the alignment is it better to give a range for the % identity or is it better to give % identity for different sections of the 2 proteins ? Same questions for homology and/or similarity ? I should be happy to get clear definitions on homology, similarity and identity percentages Thanks in advance for any suggestions ------------------------------------------------------------------------ Thierry MOREAU URA CNRS 1334 Laboratoire d'Enzymologie et Chimie des Proteines TOURS France Email: moreaut@univ-tours.fr ------------------------------------------------------------------------ From owner-proteins@net.bio.net Mon Feb 21 22:00:00 1994 Path: biosci!daresbury!doc.ic.ac.uk!uknet!pipex!howland.reston.ans.net!nctuccca.edu.tw!news!news.csie.nctu.edu.tw!mjhsieh From: mjhsieh@life.nthu.edu.tw (Meng-Juei Hsieh) Newsgroups: bionet.molbio.proteins Subject: Inhibition of Topoisomerase II Date: 22 Feb 1994 15:06:02 GMT Organization: Dep. Computer Sci. & Information Eng., Chiao Tung Univ., Taiwan, R.O.C Lines: 16 Message-ID: <2kd70q$k9s@news.csie.nctu.edu.tw> NNTP-Posting-Host: @life.nthu.edu.tw X-Newsreader: TIN [version 1.2 PL2] Hello there, Is there anyone doing research on this topic , " inhibitor of topoisomerase II " ? I am a newbie in this field. It would be great if you tell me some infomation about this topic.... :) -- /////////////////////////////////////////////////////////////////// / Francis M.J. Hsieh Á©sèû / / Life Science NTHU ,HsinChu ²M¤j¥Í©R¬ì¾Ç¨t¤E¤­¯Å / /Email address: u801629@WinKie.Oz.nthu.edu.tw / / mjhsieh@life.nthu.edu.tw / /////////////////////////////////////////////////////////////////// From owner-proteins@net.bio.net Mon Feb 21 22:00:00 1994 Newsgroups: bionet.general,bionet.molbio.proteins,bionet.software Path: biosci!daresbury!doc.ic.ac.uk!warwick!pipex!uknet!comlab.ox.ac.uk!gjb From: gjb@bioch.ox.ac.uk (Geoff Barton) Subject: Protein Prediction Preprint Message-ID: <1994Feb16.131627.3051@geoff.bioch.ox.ac.uk> Originator: gjb@geoff.biop Keywords: Tyrosine phosphatase Factor XIIIa Organization: Department of Biochemistry, University of Oxford Date: Wed, 16 Feb 1994 13:16:27 GMT Lines: 52 Xref: biosci bionet.general:7893 bionet.molbio.proteins:1409 bionet.software:7319 PROTEIN PREDICTION PREPRINT AVAILABLE BY FTP PROTEIN TYROSINE PHOSPHATASE BLOOD CLOTTING FACTOR XIIIa 16 Feb 1994 We are making available by ftp a preprint of our paper entitled: "Secondary Structure Prediction from Multiple Sequence Data: Blood Clotting Factor XIII and Yersinia Protein-Tyrosine Phosphatase" by Craig D. Livingstone and Geoffrey J. Barton This paper will appear in due course in the International Journal of Peptide and Protein Research. However, this will take some time to happen and we are anxious that our predictions are available to the community prior to publication of crystal structures for members of either protein family. We were unable to include the full details of the predictions and alignments in the short IJPPR account. We are making these availble on our ftp server geoff.biop.ox.ac.uk. Please see the README file in /preprints/ptp_factor13. Be warned that some of the files are quite large - for those who access using a Unix system, you can download a compressed tar file that contains the preprint and all data and is called Everything.tar.Z. All other files should be transferred as text. Most of the files are in PostScript and should be printable on any PostScript printer. Geoffrey J. Barton and Craig D. Livingstone Laboratory of Molecular Biophysics University of Oxford Rex Richards Building South Parks Road Oxford OX1 3QU U.K. -- -------------------------------------------------------------------------------- Internet: gjb@bioch.ox.ac.uk (Janet: gjb@uk.ac.ox.bioch) From owner-proteins@net.bio.net Mon Feb 21 22:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!daresbury!doc.ic.ac.uk!agate!howland.reston.ans.net!wupost!decwrl!decwrl!olivea!koriel!lll-winken.llnl.gov!fastrac.llnl.gov!osi-east2.es.net!pnl-oracle!netnews.nwnet.net!serval!wsuaix.csc.wsu.edu!pxu From: pxu@wsuaix.csc.wsu.edu (Pin Xu) Subject: Re: Floating Amm.SO4 ppts. Message-ID: <1994Feb22.053248.9525@serval.net.wsu.edu> Sender: news@serval.net.wsu.edu (USENET News System) Organization: Washington State University X-Newsreader: Tin 1.1 PL4 References: <761426472snz@genesys.demon.co.uk> Date: Tue, 22 Feb 94 05:32:48 GMT Lines: 25 Duncan@genesys.demon.co.uk (Duncan Clark) writes: : Hi Netters, : : Does anyone know why one sometimes gets Amm.SO4 ppts of proteins that float : rather than pellet? Is there anyway to get preps that do this to pellet? In : my case the prep is E.coli, sonicated, spun down, addition of polymin P (to : ppt nucleic acids) plus 0.2M NaCl (to keep my protein in solution) and the : ppt spun down and the supernatant kept. Addition of Amm.SO4 to 70%, left O/N : at 4C then spun at 50,000g. Floating pellicle. Why? Is your "protein" gel-like? Does it swell in water? I tried to purify proteins from pollen and I also got floating gel-like stuff-- most of this are (lipo-)polysaccharides I think. : : Thanks : : Duncan : -- : ----------------------------------------------------------------------------- : Duncan Clark | Internet: duncan@genesys.demon.co.uk : GeneSys Ltd. | Compuserve: 100015.1406@compuserve.com : ----------------------------------------------------------------------------- From owner-proteins@net.bio.net Mon Feb 21 22:00:00 1994 Path: biosci!daresbury!trane.uninett.no!nntp.uio.no!usenet From: rushing@titan.ksc.nasa.gov (