From owner-proteins@net.bio.net Mon Aug 01 23:00:00 1994 Path: biosci!agate!ames!waikato!auckland.ac.nz!NewsWatcher!user From: JEB.Harrison@auckland.ac.nz (Jane Harrison) Newsgroups: bionet.molbio.proteins Subject: Baculovirus Followup-To: bionet.molbio.proteins Date: 3 Aug 1994 06:48:24 GMT Organization: Molecular Medicine Lines: 17 Distribution: world Message-ID: NNTP-Posting-Host: 130.216.55.45 Hi, Anyone out there have a way of getting bacuolovirus particles out of the supernatant? Spinning at 100 000xg for 60 min is not sucessful enough. Thanks Jane **************************************************************************** Jane Harrison 'All opinions are Mine and mine alone' Medical School "Science is like being God- University of Auckland Only I have time and buget constraints" Private Bag 92019, Auckland, New Zealand ***************************************************************************** From owner-proteins@net.bio.net Mon Aug 01 23:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!usc!crash!xipe From: xipe@crash.cts.com (Xipe Press) Subject: HIV+ and Whey Protein Organization: CTS Network Services (CTSNET), San Diego, CA Date: Tue, 2 Aug 1994 18:03:27 GMT Message-ID: X-Newsreader: TIN [version 1.2 PL2] Sender: news@crash.cts.com (news subsystem) Nntp-Posting-Host: crash.cts.com Lines: 1 From owner-proteins@net.bio.net Mon Aug 01 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!swrinde!pipex!doc.ic.ac.uk!charlie.lif.icnet.uk!pic.lif.icnet.uk!user From: p_whitehead@icrf.icnet.uk (Phil Whitehead) Newsgroups: bionet.molbio.proteins Subject: Re: West blotting Followup-To: bionet.molbio.proteins Date: Tue, 02 Aug 1994 17:03:53 +0000 Organization: Imperial Cancer Research Fund Lines: 27 Distribution: bionet Message-ID: References: <2uotjj$c8k@mserv1.dl.ac.uk> NNTP-Posting-Host: 143.65.8.48 In article <2uotjj$c8k@mserv1.dl.ac.uk>, mbsxu@s-crim1.dl.ac.uk (S. Xu) wrote: > Hi, Folks, > > I am interested in protein tyrosine phosphorylation in megakaryocyte > differentiation. Currently I have a problem doing Western blotting. I > used "Wet Electrophoric Transfer" method, but I found that the bands > with low molecular weight were not evenly transferred. BTW, I was told > that it was very tricky to study tyrosine phosphorylation by using > Western blotting, which I just touched. Any help will be appreciated. > > Thanks in advance. > > ------------------------------------------------- > Ying Hong Dept of Medicine > King's College School of Medicine > London SE5 9PJ > Email: RCHA213@MAPL.KCL.CC.AC.UK > ------------------------------------------------- What is the MW of your protein? anything below 30K will whistle straight through o.45 nitrocellulose, so, you may have to think about using 0.2 pore size instead . Phil Whitehead From owner-proteins@net.bio.net Mon Aug 01 23:00:00 1994 Path: biosci!agate!spool.mu.edu!vms.csd.mu.edu!6566FRIEDMAN From: 6566friedman@vms.csd.mu.edu Newsgroups: bionet.molbio.proteins Subject: PLASMID DRAWING SOFTWARE Date: 2 Aug 1994 15:23:38 GMT Organization: Marquette University - Computer Services Lines: 6 Message-ID: <00982579.31DDA6E0@vms.csd.mu.edu> Reply-To: 6566friedman@vms.csd.mu.edu NNTP-Posting-Host: vmsb.csd.mu.edu Can anyone recommend a good Macintosh program for drawing plasmids? Are any available in the public domain from FTP sites? Thanks, Alan Friedman 6566Friedman@VMS.CSD.MU.EDU From owner-proteins@net.bio.net Mon Aug 01 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!cs.utexas.edu!news.tamu.edu!news.utdallas.edu!wupost!golf!mizzou1.missouri.edu!DCRCEP From: DCRCEP@mizzou1.missouri.edu (Elmer M. Price) Newsgroups: bionet.molbio.proteins Subject: Test Date: Sat, 30 Jul 94 15:48:48 CDT Organization: University of Missouri, Columbia Lines: 3 Message-ID: <17003DE6FS86.DCRCEP@mizzou1.missouri.edu> NNTP-Posting-Host: mizzou1.missouri.edu This is a test. From owner-proteins@net.bio.net Mon Aug 01 23:00:00 1994 Path: biosci!VM.CC.PURDUE.EDU!ERNESTO From: ERNESTO@VM.CC.PURDUE.EDU (martin) Newsgroups: bionet.molbio.proteins Subject: Disagreement Date: 2 Aug 1994 17:58:24 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 8 Sender: daemon@net.bio.net Distribution: world Message-ID: <199408030058.RAA01171@net.bio.net> NNTP-Posting-Host: net.bio.net Phil Whitehead wrote that anything below 30 kd will pass through 0.45 nitrocell ulose paper. I donot understand why he is saying that since I have been using nitrocellulose paper with this pore size and found sometimes bands much more lower than my pr otein that is 30 kd (I know that because of non-specific cross reactions with m y antibody). So, basically I do not agree with that point. Any comments? Martin. From owner-proteins@net.bio.net Tue Aug 02 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!EU.net!sun4nl!news.nic.surfnet.nl!highway.LeidenUniv.nl!rulsfb.LeidenUniv.nl!SBTNFH From: sbtnfh@rulsfb.LeidenUniv.nl (Flip Hoedemaeker, CvF RUL/TNO, Leiden, The Netherlands) Newsgroups: bionet.molbio.proteins Subject: Re: Disagreement Date: 3 Aug 1994 08:12:30 GMT Organization: Biology, Leiden University, The Netherlands Lines: 21 Distribution: world Message-ID: <31njhe$fim@highway.LeidenUniv.nl> References: <199408030058.RAA01171@net.bio.net> Reply-To: sbtnfh@rulsfb.LeidenUniv.nl NNTP-Posting-Host: rulsfb.leidenuniv.nl In article <199408030058.RAA01171@net.bio.net>, ERNESTO@VM.CC.PURDUE.EDU (martin) writes: >Phil Whitehead wrote that anything below 30 kd will pass through 0.45 nitrocell >ulose paper. >I donot understand why he is saying that since I have been using nitrocellulose > paper with this pore size and found sometimes bands much more lower than my pr >otein that is 30 kd (I know that because of non-specific cross reactions with m >y antibody). So, basically I do not agree with that point. Any comments? > > Martin. I have not only blotted proteins as small as 6 kd on 0.45 pore size, but I also got a good N-terminal sequence from those blots! So I agree with you Martin. For anyone interested, I use Millipore Immobilon P (PVDF membrane), a semi-dry blotting device (Novablot, Pharmacia I think), and I don't blot longer than 45 mins, because if you blot longer you will lose protein. The same applies for nitrocellulose though. Yes, you get hazy bands with such small proteins, but they stain with Comassie. Flip From owner-proteins@net.bio.net Tue Aug 02 23:00:00 1994 Path: biosci!NBRF.GEORGETOWN.EDU!POSTMASTER From: POSTMASTER@NBRF.GEORGETOWN.EDU Newsgroups: bionet.molbio.proteins Subject: Re: Need comments about PIR-41 feature format Date: 3 Aug 1994 14:25:45 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 28 Sender: daemon@net.bio.net Distribution: world Message-ID: <01HFHA77ZUZE96WJ7O@NBRF.Georgetown.Edu> NNTP-Posting-Host: net.bio.net In message <940803161033.20212185@scri.fsu.edu> Victor B. Strelets (STRELETS@SCRI.FSU.EDU) said > PIR-41 was practically ready for installation on > anon.FTP when I find some strange (not described > in the extremely pure documentation for new CODATA > PIR format) frequent context in FEATURE field. > This is symbol '\' which appeares without any > visible regularity at the end of some strings > which contain feature name (text description). He soon added in another posting that he saw it was a record separator. In the document CXFSD.LIS, available from the PIR Network Request Server with the command SEND CXFSD, the use of the backslash is described as follows. The backslash is used as a general data item separator that indicates that what follows is a separate data item but of the same type. The backslash is used in the database only to indicate the concatenation of data items. I am certain that if you obtain that document and receive the PIR Technical Bulletins, you will find the answers to all your format questions. ------------------------------------------------------------------------ Dr. John S. Garavelli Database Coordinator Protein Information Resource National Biomedical Research Foundation Washington, DC 20007 POSTMAST@GUNBRF.BITNET POSTMASTER@NBRF.GEORGETOWN.EDU From owner-proteins@net.bio.net Tue Aug 02 23:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!agate!howland.reston.ans.net!EU.net!sunic!ki.se!kidec.cmb.ki.se!cib From: cib@kidec.cmb.ki.se (Carlos Ibanez) Subject: fusion protein cleavage Message-ID: Organization: CMB, Karolinska Institutet Date: Wed, 3 Aug 1994 19:46:40 GMT Lines: 7 Is anyone out there working with proteins fused with glutathione S transferase? My thrombin cleavage seems to be extremely inefficient. I would appreciate any advise you could give me. I am doing the cleavage in solution with free fusion protein (i.e. it is not bound to a matrix) Thanks in advance. Please e-mail your replies to Leopold.Ilag@mbb.ki.se From owner-proteins@net.bio.net Tue Aug 02 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!europa.eng.gtefsd.com!newsxfer.itd.umich.edu!uunet!newstf01.cr1.aol.com!search01.news.aol.com!not-for-mail From: mmkania@aol.com (MMKania) Newsgroups: bionet.molbio.proteins Subject: High level protein expression. Date: 4 Aug 1994 00:35:01 -0400 Organization: America Online, Inc. (1-800-827-6364) Lines: 9 Sender: news@search01.news.aol.com Message-ID: <31pr5l$nuc@search01.news.aol.com> NNTP-Posting-Host: search01.news.aol.com I am interested in expressing a bacterial enzyme in Drosophila cells. Does anyone have knowledge about the stablity of bacterial proteins which are expressed in eukaryotic cells? Please answer to my Email address: klingler@zi.biologie.uni-muenchen.de Thank You, Martin From owner-proteins@net.bio.net Tue Aug 02 23:00:00 1994 Path: biosci!GIBBS.OIT.UNC.EDU!vaisman From: vaisman@GIBBS.OIT.UNC.EDU (Iosif Vaisman) Newsgroups: bionet.molbio.proteins Subject: Molecular Modeling Conference 1994 Date: 3 Aug 1994 18:31:50 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 109 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Molecular Modeling Conference 1994 Fundamentals and Applications for the Pharmaceutical Industry 2-4 October 1994 Brunswick Hilton and Towers, East Brunswick, New Jersey Molecular Modeling Conference 1994 is organized by Advanstar Communications, the publishers of Pharmaceutical Technology, BioPharm, LC-GC, and Spectroscopy magazines. Conference Moderators: Alexander MacKerell, Assistant Professor, Department of Pharmaceutical Sciences, University of Maryland at Baltimore Alexander Tropsha, Assistant Professor, Director, Laboratory for Molecular Modeling, University of North Carolina at Chapel Hill Herschel J.R. Weintraub, Assistant Director, Medicinal Chemistry, R.W. Johnson Pharmaceutical Research Institute Sunday, 2 October 1994 Afternoon Session: Optional Introductory Workshop - Molecular Modeling Basics Instructors: Warren J. Hehre, Wavefunction, Inc., and University of California, Irvine Alexander Tropsha (Session Organizer), University of North Carolina, Chapel Hill Herschel J.R. Weintraub, R.W. Johnson Pharmaceutical Research Institute Monday, 3 October 1994 Plenary Lecture: Molecular Modeling - For Better, For Worse. For Richer, For Poorer. Peter Goodford, University of Oxford, UK On the Effect of Long-range Interactions on Protein Structure, Specificity, & Ligand Binding Free Energies Arnie Hagler, Biosym Technologies, Inc. Modeling Selectivity in Organic Reactions Warren J. Hehre, Wavefunction, Inc. and University of California, Irvine General Representation and Solution of the QSAR Problem Based Upon Tensor Analysis A. J. Hopfinger, University of Illinois at Chicago Rapid Prediction of Binding Energies Using Continuum Methods Barry Honig, Columbia University Pharmacophore Determination: The Critical Decision in Ligand-Based Design Richard D. Cramer, Tripos, Inc. Overview of 3D-Searching: A Powerful Technique for Computer-Assisted Molecular Design Robert S. Pearlman, University of Texas, Austin Tuesday, 4 October 1994 X-ray Crystallographic Analysis of Macromolecular Structures Wayne A. Hendrickson, Columbia University Free Energy Modeling Monte Pettitt, University of Houston Multidimensional Heteronuclear NMR of Proteins Angela M. Gronenborn, NIDDK, National Institutes of Health Models of G Protein-Linked Receptors: How Do We Get Them and What Can We Do With Them? Charles Hutchins, Abbott Laboratories Comparative Homology Modeling: What Is It Good For and How Well Does It Work? Jonathan Greer, Abbott Laboratories De Novo Predications of Quaternary Protein Structure: Applications to Coiled Coils Jeffrey Skolnick, Scripps Research Institute Computer Assisted Ligand Design I.D. Kuntz, University of California, San Francisco Retrospective and Prospective Successes of Molecular Modeling in the Pharmaceutical Industry Peter Gund, Molecular Simulations Inc. Registration Information To register or to receive a copy of the conference program brochure, please call the Molecular Modeling Conference Registrar at (800) 343-3423 or (503) 343-1200. Fees for Molecular Modeling Conference include all course materials, a copy of the conference proceedings, admission to the Technology Demonstration Room, the Optional Introductory Workshop, and refreshment breaks. Fees Early (postmarked by 19 August 1994): $545.00 Regular (postmarked after 19 August 1994): $645.00 On-Site: $695.00 For more information, contact: Molecular Modeling Conference 1994 859 Willamette Street Eugene, OR 97401-6806 Phone: (800) 343-3423 or (503) 343-1200 Fax: (503) 343-7024 From owner-proteins@net.bio.net Tue Aug 02 23:00:00 1994 Path: biosci!agate!headwall.Stanford.EDU!morrow.stanford.edu!camis.Stanford.EDU!holbrook From: holbrook@camis.Stanford.EDU (Robin Holbrook) Newsgroups: bionet.molbio.proteins Subject: NMR Summer Course Announcement Date: 4 Aug 1994 00:04:48 GMT Organization: Stanford Magnetic Resonance Laboratory Lines: 14 Sender: holbrook@camis.stanford.edu Distribution: world Message-ID: <31pbb0$m4i@morrow.stanford.edu> NNTP-Posting-Host: camis.stanford.edu May 22-30, 1995 International School of Biological Magnetic Resonance, 2nd Course: "Dynamics and the Problem of Recognition in Biological Macromolecules" - Ettore Majorana Centre for Scientific Culture, Erice, Sicily, Italy For information and/or registration contact either: holbrook@camis.stanford.edu (Robin Holbrook, Course Administrator) or the Directors: Dr. Oleg Jardetzky (Email: jardetzky@camis.stanford.edu Fax: 415/723-2253) or Dr. Jean-Francois Lefevre (Email: lefevre@bali.u-strasbg.fr Fax: +33/88 65 53 43) From owner-proteins@net.bio.net Tue Aug 02 23:00:00 1994 Path: biosci!SCRI.FSU.EDU!STRELETS From: STRELETS@SCRI.FSU.EDU ("VICTOR B. STRELETS") Newsgroups: bionet.molbio.proteins Subject: Need comments about PIR-41 feature format Date: 3 Aug 1994 13:10:41 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 23 Sender: daemon@net.bio.net Distribution: world Message-ID: <940803161033.20212185@scri.fsu.edu> NNTP-Posting-Host: net.bio.net Hi, I'm working on the creation of the compressed PIR data structure for compact storage and effective retrieval of PIR information on different computers (for DOS/Windows on IBM-compatibles and for UNIX). PIR-41 was practically ready for installation on anon.FTP when I find some strange (not described in the extremely pure documentation for new CODATA PIR format) frequent context in FEATURE field. This is symbol '\' which appeares without any visible regularity at the end of some strings which contain feature name (text description). Question to PIR stuff or well-informed netters: Does this symbol mean something informative or this is garbage object to simple kill? And by the way, is there something more detailed on the network instead of funny PIR CODATA description announced on BIONET by PIR stuff (PIRTECHx files etc.)? Thanks, V.Strelets From owner-proteins@net.bio.net Tue Aug 02 23:00:00 1994 Path: biosci!SCRI.FSU.EDU!STRELETS From: STRELETS@SCRI.FSU.EDU ("VICTOR B. STRELETS") Newsgroups: bionet.molbio.proteins Subject: Oops, I see.. Date: 3 Aug 1994 13:49:35 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 16 Sender: daemon@net.bio.net Distribution: world Message-ID: <940803164928.20212185@scri.fsu.edu> NNTP-Posting-Host: net.bio.net Ooooo, it was really easy. Instead of automatic thinking in "string" style it was necessary to look at symbol '\' as delimiter of particular independent feature type (except of last in FATURE block). Sorry.. But why not to describe such details somewhere in documentation? How can I feel if my automatic program well designed for previous versions of PIR/GenBank databases unexpectedly start to put in stored text some new symbols? I'm not happy to find that during random control of final data configuration.. I can remember old good times when databases were supplied by toy entry file where all possible context combinations were virtually presented and therefore explained. Where they are now? From owner-proteins@net.bio.net Tue Aug 02 23:00:00 1994 Path: biosci!MAILHOST.TCS.TULANE.EDU!rellim From: rellim@MAILHOST.TCS.TULANE.EDU (Chuck Miller) Newsgroups: bionet.molbio.proteins Subject: ftp site for Mac plasmid drawing program Date: 3 Aug 1994 07:00:43 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 25 Sender: daemon@net.bio.net Distribution: world Message-ID: <199408031359.IAA28303@mailhost.tcs.tulane.edu> NNTP-Posting-Host: net.bio.net Alan Friedman (6566friedman@vms.csd.mu.edu) asked: Can anyone recommend a good Macintosh program for drawing plasmids? Are any available in the public domain from FTP sites? Hi Allen and other cellbios, Try ftp.bio.indiana.edu and look in the molbio directory. Retrieve the MacPlasmap program. Best of luck, Chuck Miller Dr. Charles A. Miller Dept. Environmental Health Sciences (374 CBR) Tulane University Medical Center 1501 Canal St. Box SL29 New Orleans, LA 70112 Ph. 504-585-6942 Fx. 504-585-6939 rellim@mailhost.tcs.tulane.edu From owner-proteins@net.bio.net Tue Aug 02 23:00:00 1994 Path: biosci!FOX.CCE.USP.BR!szeinfel From: szeinfel@FOX.CCE.USP.BR (Rafael N Szeinfeld) Newsgroups: bionet.molbio.proteins Subject: Richard Epand's adress needed Date: 3 Aug 1994 12:39:29 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 7 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Hi all, I need to contact Richard M. Epand from the Department of Biochemistry at McMaster University Health Sciences Center, Hamilton, Ontario. Thanks for your help From owner-proteins@net.bio.net Wed Aug 03 23:00:00 1994 Path: biosci!bcm!cs.utexas.edu!news.tamu.edu!sewild1.tamu.edu!user From: YMH4375@zeus.tamu.edu (Yasha Hartberg) Newsgroups: bionet.molbio.proteins Subject: Salt Links vs. Hydrophobic Interactions? Followup-To: bionet.molbio.proteins Date: Thu, 04 Aug 1994 13:46:44 -0700 Organization: Texas A&M University Lines: 9 Message-ID: NNTP-Posting-Host: sewild1.tamu.edu I want to use site-directed mutagenesis to disrupt dimer formation of a protein I'm studying. I have a choice between either disrupting several important salt-bridges or putting in several hydrophilic residues to disrupt the hydrophobic interface. Before I design the oligos, I would like to hear some advice as to which approach seems the most likely to succeed. Any help will be greatly appreciated. Yasha Hartberg Texas A&M University From owner-proteins@net.bio.net Wed Aug 03 23:00:00 1994 Path: biosci!agate!cat.cis.Brown.EDU!tonto-slip11.cis.brown.edu!user From: Stephen_Lasky@brown.edu (Stephen R. Lasky) Newsgroups: bionet.molbio.proteins Subject: Re: Richard Epand's adress needed Date: 4 Aug 1994 13:23:18 GMT Organization: Brown University/Roger Williams Medical Center Lines: 30 Message-ID: References: NNTP-Posting-Host: tonto-slip11.cis.brown.edu In article , szeinfel@FOX.CCE.USP.BR (Rafael N Szeinfeld) wrote: > Hi all, > I need to contact Richard M. Epand from the Department of > Biochemistry at McMaster University Health Sciences Center, Hamilton, > Ontario. > Thanks for your help A GOPHER SEARCH TURNED THIS UP, HOPE THIS IS WHAT YOU NEED. -200:1: name: R F Epand -200:1: email: epandraq@McMaster.Ca -200:1: office_phone: 22237 -200:1: office_location: Health Sciences Centre Room 4h26 -200:1: department: Biochemistry -200:1: position: Research Associate -200:2: name: R M Epand -200:2: email: epand@McMaster.Ca -200:2: office_phone: 22453 -200:2: office_location: Health Sciences Centre Room 4h26 -200:2: department: Biochemistry -200:2: position: Professor *************************************************************** Stephen R. Lasky, Ph.D. Brown University/Roger Williams Medical Center e-mail: Stephen_Lasky@brown.edu LandLine: 401-456-6572 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ America may be unique in being a country which has leapt from barbarism to decadence without touching civilization: John O'Hara *************************************************************** From owner-proteins@net.bio.net Wed Aug 03 23:00:00 1994 Path: biosci!bcm!news.msfc.nasa.gov!europa.eng.gtefsd.com!newsxfer.itd.umich.edu!newsrelay.iastate.edu!news.iastate.edu!bipin From: bipin@iastate.edu (Bipin K Dalmia) Newsgroups: bionet.molbio.proteins Subject: Re: fusion protein cleavage Date: 4 Aug 1994 22:42:44 GMT Organization: Iowa State University, Ames, Iowa (USA) Lines: 28 Message-ID: <31rqt4$5ti@news.iastate.edu> References: NNTP-Posting-Host: class1.iastate.edu In article , Carlos Ibanez wrote: > Is anyone out there working with proteins fused with glutathione S > transferase? My thrombin cleavage seems to be extremely inefficient. > I would appreciate any advise you could give me. I am doing the > cleavage in solution with free fusion protein (i.e. it is not bound > to a matrix) > > Thanks in advance. Please e-mail your replies to Leopold.Ilag@mbb.ki.se what is the cleavage buffer? a 20 mM tris-hcl, pH 8.3 with 5 mM cacl2 works great. how long and at what temperature do you incubate? what is your thrombin to fusion protein ratio? what thrombin are you using? i've found that sigma's human thrombin (cat # T3010) at a 1:2000 weight ratio, overnight at room temperature works nicely (greater than 90% cleavage, estimated from sds-page). or maybe that the thrombin recognition site in your fusion protein is buried. you may try partially unfolding your fusion protein by adding 1 to 2 M urea and cleaving it in presence of urea. the urea will decrease the activity of thrombin also, so you'll have to add more of it. bip -- bipin k. dalmia the other night i was lying on my bed, looking bipin@iastate.edu up at the beautiful stars, and i said to myself, n2.bkd@isumvs.iastate.edu 'where the F*CK is my ROOF !!' -- From owner-proteins@net.bio.net Wed Aug 03 23:00:00 1994 Path: biosci!bcm!news.msfc.nasa.gov!europa.eng.gtefsd.com!newsxfer.itd.umich.edu!newsrelay.iastate.edu!news.iastate.edu!bipin From: bipin@iastate.edu (Bipin K Dalmia) Newsgroups: bionet.molbio.proteins Subject: Re: fusion protein cleavage Date: 4 Aug 1994 22:45:26 GMT Organization: Iowa State University, Ames, Iowa (USA) Lines: 12 Message-ID: <31rr26$5u2@news.iastate.edu> References: <31rqt4$5ti@news.iastate.edu> NNTP-Posting-Host: class1.iastate.edu forgot to mention this in the previos post. make sure you don't have any protease inhibitors in your fusion protein preparation (EDTA, PMSF, benzamidine, pefabloc etc.) any of these will effectively inhibit thrombin. bip -- bipin k. dalmia the other night i was lying on my bed, looking bipin@iastate.edu up at the beautiful stars, and i said to myself, n2.bkd@isumvs.iastate.edu 'where the F*CK is my ROOF !!' -- From owner-proteins@net.bio.net Wed Aug 03 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!nctuccca.edu.tw!news.cc.nctu.edu.tw!news.csie.nctu.edu.tw!mjhsieh From: mjhsieh@life.nthu.edu.tw (Meng-Juei Hsieh) Newsgroups: bionet.molbio.proteins Subject: How to do research on protein mechanism? Date: 4 Aug 1994 19:25:37 GMT Organization: Dep. Computer Sci. & Information Eng., Chiao Tung Univ., Taiwan, R.O.C Lines: 11 Message-ID: <31rfbh$s6d@news.csie.nctu.edu.tw> NNTP-Posting-Host: @life.nthu.edu.tw X-Newsreader: TIN [version 1.2 PL2] Can anyone tell how many method to do research on protein mechanism? -- /////////////////////////////////////////////////////////////////// / Francis M.J. Hsieh [A student in Life Science Dept] / / Life Science NTHU ,HsinChu ^ ^ ^ / /Email address: u801629@WinKie.Oz.nthu.edu.tw / / mjhsieh@life.nthu.edu.tw ^_^ / /////////////////////////////////////////////////////////////////// From owner-proteins@net.bio.net Wed Aug 03 23:00:00 1994 Path: biosci!agate!doc.ic.ac.uk!daresbury!trane.uninett.no!sunic!news.funet.fi!zippo.uwasa.fi!freeport.uwasa.fi!mpueyo From: mpueyo@freeport.uwasa.fi (Maria Pueyo) Newsgroups: bionet.molbio.proteins Subject: RE. West Blotting Date: 4 Aug 1994 15:46:17 GMT Organization: University of Vaasa, Finland Lines: 11 Message-ID: <31r2g9$eco@zippo.uwasa.fi> NNTP-Posting-Host: freeport.uwasa.fi My name is Maria and I'm working in a biochemist departement in Spain. I usually do blottings by diffusion. I transfer proteins of 20 Kd to 100Kd. If you are interested i can give you all explanation. My address is mpueyo@freeport.uwasa.fi I hope this will be useful for you. -- From owner-proteins@net.bio.net Wed Aug 03 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!europa.eng.gtefsd.com!newsxfer.itd.umich.edu!uunet!psinntp!sjuvm!zhltphs Nntp-Posting-Host: 149.68.2.20 Date: Thu, 4 Aug 1994 16:14:57 -0400 From: "TROMBETTA, LOUIS D" Newsgroups: bionet.molbio.proteins Subject: 2nd Ab for rat tissue - Westerns Message-ID: <04AUG94.17547876.0021@sjumusic.stjohns.edu> Sender: usenet@sjumusic.stjohns.edu Organization: St. John's University Lines: 8 Hi. We are doing western's against rat proteins. Can anyone recommend a source of labelled antibody. The labelled antibodies we are now using stain nonspecifically against liver. We are using a Biotin-Strepavidin antimouse secondary antibody. We are seeing bands without primary antibody - major bands in the 70 and 100 kD range. Any suggestion? Thanks. From owner-proteins@net.bio.net Thu Aug 04 23:00:00 1994 Path: biosci!CC.IITB.ERNET.IN!chyusia From: chyusia@CC.IITB.ERNET.IN Newsgroups: bionet.molbio.proteins Subject: query on programs like DISMAN DIANA etc Date: 5 Aug 1994 23:45:01 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 13 Sender: daemon@net.bio.net Distribution: world Message-ID: <940806120932798-MTACYBER*chyusia@cc.iitb.ernet.in> NNTP-Posting-Host: net.bio.net dear netters, i would like to know if programs like DISMAN, DIANA,DISGEO meant for distance geometry calculations work on PCs (PC 486). if so what are the hard ware requirements. also i would be very thankful if somebody would tell me where i could get programs like ECEPP/2 (hydration shell model included). this would be of a great help to me for studying the conformational features of small peptides. thanking you in advance ramkrishna graduate student email: rama@cc.iitb.ernet.in From owner-proteins@net.bio.net Thu Aug 04 23:00:00 1994 Path: biosci!bcm!news.msfc.nasa.gov!europa.eng.gtefsd.com!howland.reston.ans.net!EU.net!sunic!columba.udac.uu.se!rose.bmc.uu.se!dunten From: dunten@rose.bmc.uu.se (Pete Dunten) Newsgroups: bionet.molbio.proteins Subject: Re: How to do research on protein mechanism? Date: 5 Aug 1994 19:04:12 GMT Organization: Uppsala University Lines: 5 Distribution: world Message-ID: <31u2fc$1dbp@columba.udac.uu.se> References: <31rfbh$s6d@news.csie.nctu.edu.tw> NNTP-Posting-Host: rose.bmc.uu.se >Can anyone tell how many method to do research on >protein mechanism? There are 327 ways, and you must learn them all before you graduate. From owner-proteins@net.bio.net Thu Aug 04 23:00:00 1994 Path: biosci!ZOOL.UMD.EDU!GOODE From: GOODE@ZOOL.UMD.EDU ("Dr. Dennis Goode") Newsgroups: bionet.molbio.proteins Subject: Re: 2nd Ab for rat tissue - Westerns Date: 5 Aug 1994 10:25:52 -0700 Organization: University of Maryland Zoology Lines: 29 Sender: daemon@net.bio.net Distribution: world Message-ID: <9408051725.AA21300@umailsrv1.UMD.EDU> NNTP-Posting-Host: net.bio.net On Thu, 4 Aug 1994 16:14:57 > "TROMBETTA, LOUIS D" wrote: > Hi. > We are doing western's against rat proteins. Can anyone recommend > a source of labelled antibody. The labelled antibodies we are now using > stain nonspecifically against liver. We are using a Biotin-Strepavidin > antimouse secondary antibody. We are seeing bands without primary > antibody - major bands in the 70 and 100 kD range. Any suggestion? > Thanks. We have seen non-specific staining with the Biotin-strepavidin system also. We have obtained high specificity and sensitivity by using 10-nm gold labeled secondary antibodies (Jannsen's GoatARab or GAMouse) followed by silver enhancement with the Intense-BL silver enhancement chemicals (sold by Ammersham). Another advantage is the blots don't fade. The antibodies are somewhat more expensive, but worth it, in my opinion. Hope this helps, Dennis "It takes all the running you can do to stay in one place. If you want to get somewhere else, you must run twice as fast." -The Red Queen to Alice (Lewis Carroll) From owner-proteins@net.bio.net Thu Aug 04 23:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!agate!howland.reston.ans.net!usc!crash!xipe From: xipe@crash.cts.com (Xipe Press) Subject: HIV and whey protein Organization: CTS Network Services (CTSNET), San Diego, CA Date: Fri, 5 Aug 1994 21:26:57 GMT Message-ID: X-Newsreader: TIN [version 1.2 PL2] Sender: news@crash.cts.com (news subsystem) Nntp-Posting-Host: crash.cts.com Lines: 52 At Xipe Research, we have been actively pursuing work done examining the effects of dietary whey protein on several important areas of human health. We are most impressed with its beneficial properties for athletes, cancer patients, and those advanced in years. Specific to this group, however, are findings by a number of researchers on the effects of this protein on those with HIV or AIDS. The studies we have examined in this area primarily focus on the beneficial property of weight gain in HIV positive individuals. As you may know, one of the most debilitating effects of the HIV is a rapid loss of weight, specifically, the loss of lean muscle mass. This loss of muscle mass weakens the body not only physically, but makes the individual vulnerable to a host of new problems and illnesses. Several studies have shown that the clinical administration of undenatured whey protein as a dietary supplement leads to significant weight gain in HIV positive individuals. These studies show a gain in these patients between roughly 5 and 15 pounds. Some of the patients involved with these clinical trials have even attained ideal body weight as a result. The obvious side effect was an increased energy level, and thus, productivity, and an overall improvement in health. Other studies have focused on whey protein's enhancement of immune system function. Specifically, whey protein has been shown to enhance the antibody-producing ability of B-cell lymphocytes, stimulate Helper T-cell lymphocytes, and, in a secondary effect, increase the activity of Killer T-cells. All of this has been related to the nature of whey protein, which is high in glutamylcysteine groups. Cysteine is crucial to the body's synthesis of glutathione, the real agent in all of this. And as very few food proteins contain glutamylcysteine groups, we have not only been researching whey protein more thoroughly for proof of its benefits, we have been looking for other dietary sources of these agents. We are interested in discussion and information regarding all aspects of whey protein, including other works that confirm the findings we have uncovered, projects involving the protein currently in progress, other benefits of the protein that we have overlooked, and where, if at all, we may find a commercially produced undenatured whey protein. We have seen a great number of dietary whey proteins, mostly marketed towards high-stress athletes, but none that we have encountered so far have been appropriate, as the process by which the product was produced leaves the protein denatured, and useless for the purposes of our inquiries. We have placed similar postings in other related newsgroups to generate as much information exchange and discussion as possible. It is our intent, as well, to contact appropriate mailing lists, but due to the massive amount of mail we will receive through the mail servers, we will attempt to contain ourselves to the public newsgroups. Anybody with information we may be interested in, however seemingly unimportant, or with questions as to just what the heck we're talking about, feel free to respond. Thanks Xipe Research xipe@crash.cts.com From owner-proteins@net.bio.net Thu Aug 04 23:00:00 1994 Path: biosci!agate!spool.mu.edu!torn!utnut!utcsri!yonge.cs.toronto.edu!relay.cs.toronto.edu!neuron.ai.toronto.edu!ai.toronto.edu!steeg Newsgroups: bionet.molbio.proteins From: steeg@cs.toronto.edu ("Evan W. Steeg") Subject: Re: How to do research on protein mechanism? Message-ID: <94Aug5.105246edt.1046@neuron.ai.toronto.edu> Originator: root@yonge.cs Nntp-Posting-Host: yonge.cs Organization: CS Lab, University of Toronto References: <31rfbh$s6d@news.csie.nctu.edu.tw> Date: 5 Aug 94 14:53:27 GMT Lines: 22 In article <31rfbh$s6d@news.csie.nctu.edu.tw> mjhsieh@life.nthu.edu.tw (Meng-Juei Hsieh) writes: > >Can anyone tell how many method to do research on >protein mechanism? > 1. Apply for grants. 2. Get grad students to do lots of work. 3. Have your post-doc explain the results to you. -- Evan -- Evan W. Steeg steeg@ai.toronto.edu Dept of Computer Science University of Toronto, Tel: (416) 978-5182 Toronto, Canada M5S 1A4 FAX: (416) 978-1455 From owner-proteins@net.bio.net Thu Aug 04 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!EU.net!sunic!columba.udac.uu.se!rose.bmc.uu.se!dunten From: dunten@rose.bmc.uu.se (Pete Dunten) Newsgroups: bionet.molbio.proteins Subject: Re: Salt Links vs. Hydrophobic Interactions? Date: 5 Aug 1994 14:45:26 GMT Organization: Uppsala University Lines: 16 Distribution: world Message-ID: <31tja6$1das@columba.udac.uu.se> References: <31stq3$sb8@lyra.csx.cam.ac.uk> NNTP-Posting-Host: rose.bmc.uu.se :: I want to use site-directed mutagenesis to disrupt dimer formation of a :: protein I'm studying. I have a choice between either disrupting several :: important salt-bridges or putting in several hydrophilic residues to :: disrupt the hydrophobic interface. Before I design the oligos, I would :: like to hear some advice as to which approach seems the most likely to :: succeed. Any help will be greatly appreciated. : Presumably the salt bridge is buried, since its in the interface. :Removing that will leave a buried charge, which will be massively unstable ( :I can dig out a ref or a ballpark figure if you like). I think this would be :your best bet. Sounds good but life isn't always so predictable! See the work on attempts to disrupt the insulin hexamer, which included introducing a negative charge in each monomer at the center of the hexamer. J Cryst Growth Vol 122 (1992) 144-151. From owner-proteins@net.bio.net Thu Aug 04 23:00:00 1994 Path: biosci!agate!spool.mu.edu!vms.csd.mu.edu!6566FRIEDMAN From: 6566friedman@vms.csd.mu.edu Newsgroups: bionet.molbio.proteins Subject: Help Converting Enzymekinetics Date: 5 Aug 1994 14:29:34 GMT Organization: Marquette University - Computer Services Lines: 12 Message-ID: <009827CD.24ACC0E0@vms.csd.mu.edu> Reply-To: 6566friedman@vms.csd.mu.edu NNTP-Posting-Host: vmsb.csd.mu.edu I recently obtained a copy of Enzymekinetics from the Indiana FTP site. After Bin Hexing and unstuffing the program, I obtained what appeared to be a functional hypercard stack. However when ever I attempted to enter data, a window popped up saying that I needed to "convert"the stack before new data could be entered. Since this software has no menu bar, I have now idea how to convert the data; any suggestions? Alan Friedman Dept. Biology Marquette University Milwaukee, WI 6566FRIEDMAN@VMS.CSD.MU.EDU From owner-proteins@net.bio.net Thu Aug 04 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!pipex!lyra.csx.cam.ac.uk!bjd12 From: bjd12@cus.cam.ac.uk (Ben Davis) Newsgroups: bionet.molbio.proteins Subject: Re: Salt Links vs. Hydrophobic Interactions? Date: 5 Aug 1994 08:38:27 GMT Organization: University of Cambridge, England Lines: 36 Message-ID: <31stq3$sb8@lyra.csx.cam.ac.uk> References: NNTP-Posting-Host: grus.cus.cam.ac.uk X-Newsreader: TIN [version 1.2 PL2] Yasha Hartberg (YMH4375@zeus.tamu.edu) wrote: : I want to use site-directed mutagenesis to disrupt dimer formation of a : protein I'm studying. I have a choice between either disrupting several : important salt-bridges or putting in several hydrophilic residues to : disrupt the hydrophobic interface. Before I design the oligos, I would : like to hear some advice as to which approach seems the most likely to : succeed. Any help will be greatly appreciated. Presumably the salt bridge is buried, since its in the interface. Removing that will leave a buried charge, which will be massively unstable ( I can dig out a ref or a ballpark figure if you like). I think this would be your best bet. Just asked around - the consensus of opinion is yes, mutate out the salt bridge. Removal of a buried salt bridge destabilises by ~4 kcal/mol. To minimise disruption of the monomer structure, try mutating the acidic residue to its amide counterpart (eg Glu --> Gln, Asp --> Asn). This should work fine, and not cause too many side effects. If the salt bridge isn't buried, life is going to be harder. You'll have to disrupt the hydrophobic interface, and your running the risk of either still forming dimers, or disrupting the monomer stucture. Hope this helps. Ben : Yasha Hartberg : Texas A&M University -- ______________________________________________________________________________ Ben Davis, MRC Protein Function and Design, Cambridge, UK ______________________________________________________________________________ "They can make me do it, but they can't make me do it with dignity." From owner-proteins@net.bio.net Thu Aug 04 23:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!agate!howland.reston.ans.net!EU.net!sun4nl!sci.kun.nl!ronsmul From: ronsmul@sci.kun.nl (Ronald Smulders) Subject: CBB staining mechanism Message-ID: Sender: news@sci.kun.nl (News owner) Nntp-Posting-Host: wn2.sci.kun.nl Organization: University of Nijmegen, The Netherlands Date: Fri, 5 Aug 1994 07:12:21 GMT Lines: 12 Dear protein experts, As you all know, Coomassie brilliant blue is a very popular dye for protein staining. However, in our lab nobody seems to know how CBB interacts with a protein. I assume this interaction is non-covalent, but I would like to know whether CBB 'recognizes' certain residues or just binds to peptide bonds or something. Thanks is advance for your reply. Ronald (ronsmul@sci.kun.nl) From owner-proteins@net.bio.net Thu Aug 04 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!spool.mu.edu!bloom-beacon.mit.edu!senator-bedfellow.mit.edu!aaRS!lluis From: lluis@aaRS (Lluis Ribas) Newsgroups: bionet.molbio.proteins Subject: Re: Salt Links vs. Hydrophobic Interactions? Date: 5 Aug 1994 16:49:01 GMT Organization: Massachvsetts Institvte of Technology Lines: 10 Message-ID: <31tqht$n4h@senator-bedfellow.MIT.EDU> References: NNTP-Posting-Host: aars.mit.edu X-Newsreader: TIN [version 1.2 PL2] Yasha Hartberg (YMH4375@zeus.tamu.edu) wrote: : I want to use site-directed mutagenesis to disrupt dimer formation of a : protein I'm studying. I have a choice between either disrupting several : important salt-bridges or putting in several hydrophilic residues to : disrupt the hydrophobic interface. Before I design the oligos, I would : like to hear some advice as to which approach seems the most likely to : succeed. Any help will be greatly appreciated. : Yasha Hartberg : Texas A&M University From owner-proteins@net.bio.net Fri Aug 05 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!newsserver.jvnc.net!yale.edu!news.ycc.yale.edu!commons-107-node.net.yale.edu!user From: smith@minerva.cis.yale.edu (Albert Smith) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Protocol for Fluorescently Labeling Antibody Wanted Followup-To: bionet.molbio.methds-reagnts Date: Sat, 06 Aug 1994 15:58:19 +0100 Organization: A large organization with structure Lines: 32 Message-ID: Reply-To: smith@minerva.cis.yale.edu NNTP-Posting-Host: 130.132.107.14 Xref: biosci bionet.molbio.methds-reagnts:17014 bionet.molbio.proteins:2417 I want to directly conjugate some rabbit polyclonal antisera with rhodamine and/or fluorescein. I have gotten the appropriate succinimidyl esters from Molecular Probes and would like some insight into methods that I can use to label my antisera. In a first attempt I tried to combine fluorescent labeling with affinity purification. I generated a protocol similar to one used for affinity purification/biotinylation of antibodies described in BioTechniques 13:546-8. But I didn't end up with labeled antibody. I know that my affinity purification protocol alone works, so the problem is with the fluorescent dye coupling. Can anyone provide an appropriate protocol for fluorescently labeling an antibody. I would be interested in protocols that combine affinity purification with labeling as well as protocols for solely labeling the antisera. As an aside, would a succinimidyl ester be reactive with nitrocellulose? The biotinylation protocol mentioned above used a combination of nitrocellulose and a succinimidyl ester and ended up with biotinylated antibody. For that reason I attempted my labeling with a rhodamine succimidyl ester and protein immobilized on nitrocellulose. The end product of my conjugation was a very red piece of nitrocellulose, which makes me wonder if I had labeled the filter better than the protein. Does this theory hold any water? I am not a chemist by any stretch of the imagination, so I ask this question out of complete naivete. Thanks for any information, -bert -- smith@minerva.cis.yale.edu From owner-proteins@net.bio.net Sat Aug 06 23:00:00 1994 Newsgroups: bionet.molbio.proteins,bionet.molec-model Path: biosci!daresbury!trane.uninett.no!eunet.no!nuug!EU.net!uunet!munnari.oz.au!mel.dit.csiro.au!dmp.csiro.au!lachlan From: lachlan@dmp.csiro.au (Lachlan Cranswick) Subject: Announcing Scientist Friendly MS-Windows Internet Kit Message-ID: <1994Aug7.031046.24953@dmp.csiro.au> Organization: CSIRO Division of Mineral Products, Melbourne, AUSTRALIA Date: Sun, 7 Aug 1994 03:10:46 GMT Lines: 548 Xref: biosci bionet.molbio.proteins:2418 bionet.molec-model:64 ANNOUNCING SCIENTIST FRIENDLY MS WINDOWS INTERNET KIT ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ (One stop Winsock Shop) QUICK INSTALL :- ---- Obtaining the files. anonymous ftp to :- sol.dmp.csiro.au:/pub/internet/all/internet.exe (192.68.132.2) sol.dmp.csiro.au:/pub/internet/floppy/internt1.exe sol.dmp.csiro.au:/pub/internet/floppy/internt2.exe US mirror (expires in October) :- ftp://ftp.alumni.caltech.edu/pub/dank/internet-kit/internet.exe (ftp.alumni.caltech.edu:/pub/dank/internet-kit) --- Installing the files :- (the -d option is very important as this creates subdirectories and puts files in their correct place). internet c:\ -d or internt1 c:\ -d internt2 c:\ -d ----- 1. INTRODUCTION 2. GETTING THE SOFTWARE VIA ANONYMOUS FTP 3. ASSUMED KNOWLEDGE FOR INSTALLATION 4. WHAT SOFTWARE IS IN THIS KIT 5. BEFORE STARTING 6. NETWORK HARDWARE AND PC/SOFTWARE REQUIREMENTS 7. TO INSTALL OR NOT TO INSTALL THE TRUMPET WINSOCK? 8. LOADING ALL THE SOFTWARE ONTO YOUR PC 9. INSTALLING TRUMPET WINSOCK THE WINSOCK INSTALLATION BEGINS 10. INSTALLING THE INTERNET SOFTWARE ADDING THE WINDOWS PROGRAM ICONS HGOPHER WS_FTP TRMPTEL Wintrumpet Newsreader WinQVT 11. FINISHED! ========================================= INTRODUCTION This might help people who are trying to find MS-Windows internet software without having to browse several different sites and go through several different installation procedures. This is version 1.00 of this one-stop-shop so please be on the lookout for any errors. Also, suggestions of new software and/or viewers for MOSAIC and GOPHER and how to install them would be appreciated. ------- GETTING THE SOFTWARE VIA ANONYMOUS FTP This Scientist friendly MS Windows internet kit available by anonymous ftp from :- sol.dmp.csiro.au:/pub/ms-win-internet-kit/all/internet.exe (2181885) (192.68.132.2) (All the files in one pkzip self extracting exe file). US mirror (expires in October) :- ftp://ftp.alumni.caltech.edu/pub/dank/internet-kit/internet.exe (ftp.alumni.caltech.edu:/pub/dank/internet-kit) or sol.dmp.csiro.au:/pub/ms-win-internet-kit/floppy/internt1.exe (1200132) sol.dmp.csiro.au:/pub/ms-win-internet-kit/floppy/internt2.exe (994559) (Files to fit onto 1.2 Meg (and thus 1.44 Meg) floppy disks). All the software packages given in this kit is either public domain software or shareware. Some of this software is deliberately not the most recent versions. But it has been found to work reliably and be fairly easy for a "casual" MS-Windows user to install. Please email if there are any errors in the installation. ----- User friendly internet software is also available for Mac users at :- sol.dmp.csiro.au:/pub/mac And (friendly?) UNIX software also at :- sol.dmp.csiro.au:/pub/unix ----------------------------- ASSUMED KNOWLEDGE FOR INSTALLAION Some basic knowledge of the internet is required as well as editing with Windows Notepad. It also assumes you know how to use anonymous ftp to get the files. If not - consult you local PC/internet guru who should be able to help you. -------------------------------------------------- WHAT SOFTWARE IS IN THIS KIT This kit includes :- Trumpet winsock - shareware TCP-IP software (one of the best!) WINQVT - mainly telnet (but has other services - i.e., POP mail, NEWS, FTP) (you can print telnet screens to printer with ALT F2) NCSA Mosaic WWW client - old 16 bit version with picture viewer pre-installed. (easy to load). A new fast 32 bit version is available but requires Win32s to be installed prior to use for Win 3.1 and 3.11. HGOPHER - gopher client software WINTRUMPET - presently the best user-friendly Usenet newsreader around (Also has decent POP Mail) (my opinion only). WS_FTP - Very user friendly FTP program (point and click). TRMPTEL - Simple no-frills VT100 telnet program. -------- BEFORE STARTING (you must consult your system manager). You need to know :- Your network's "Domain Name" Your logon name - for telnet. Your Email address. The IP address and name of your PC The IP address and name of your Name Server. The IP address and name of your Gateway. The IP address and name of your News Server (Your system manager will have to edit the news server's nntp_access file to allow your PC to get news from the news server) The IP address and name of your Mail (SMTP/POP) Server. --- NETWORK HARDWARE AND INSTALLATION REQUIREMENTS This assumes that your PC has a correctly installed ethernet card and a link into the internet. SLIP is also possible via a PC serial port but your internet vendor would have to give you installation instructions. If you do not have Winsock already running, it assumes packet drivers are already installed and correctly configured. While it can be changed with user modification, this installation also assumes that :- MS-Windows is installed in the c:\windows directory. That you have at least 6 Meg free hard-disk on the C:\ drive. ---- TO INSTALL OR NOT TO INSTALL THE TRUMPET WINSOCK? A very brief (and possibly inaccurate) explanation of WINSOCK. The winsock protocol (Winsockets) is the "standard" TCP-IP protocol for MS-Windows to allow running of internet and network software that is Winsock complient. If you already have winsock(tcp-ip) installed on your system, ignore the following winsock installation procedures. If you are using another network protocol such as Windows for Workgroups NDIS drivers, PCNFS, etc, consult your system manager on how to get Winsock on your system. (i.e., ignore the following winsock installation procedures). For Windows for Workgroups 3.11 - a Microsoft 32 bit beta (bit flakey) winsock is available from :- sol.dmp.csiro.au:/pub/ms-winsock/MTCPB3.EXE (192.68.132.2) (self extracting exe file). (i.e., ignore the following winsock installation procedures). Note that some network software supports winsock by default. This includes Frontier Technology's Super TCP for Windows. (i.e., ignore the following winsock installation procedures). If you have Windows NT - buy the TCP-IP pack from Microsoft which gives you winsock by default. (i.e., ignore the following winsock installation procedures). If you have no network protocol installed or just have packet drivers installed - try applying the winsock installation procedures which follow. ------- LOADING ALL THE SOFTWARE ONTO YOUR PC. Assuming you have already ftp'd the software over :- Go into the directory or drive the file is located and type (this creates subdirectories) :- (the -d option is very important as this creates subdirectories and puts files in their correct place). internet c:\ -d For floppy disk files. internt1 c:\ -d internt2 c:\ -d This will load files into your c:\windows directory. mosaic.ini hgopher.ini internet.grp vt220.fon wnnetdll.sym wnqvtnet.sym Into your c:\windows\system directory vt100.fon wnnetdll.sym wnqvtnet.sym And will also create the following directory structure with files installed in their correct place :- c:\tmp c:\temp c:\internet c:\internet\annotate c:\internet\autoexec c:\internet\ethernet c:\internet\ethernet\ne2000 c:\internet\ethernet\ne2000\pktdrv c:\internet\ethernet\smc c:\internet\ethernet\smc\diagnose c:\internet\ethernet\smc\ezsetup c:\internet\ethernet\smc\pkt_drv c:\internet\gopher c:\internet\mail c:\internet\mosaic c:\internet\mosaic\lview c:\internet\mosaic\wham c:\internet\mosaic\wplany c:\internet\news c:\internet\qvtnet c:\internet\vt100 c:\internet\windows c:\internet\windows\system c:\internet\winsock c:\internet\wintrump c:\internet\ws_ftp ------------------ INSTALLING TRUMPET WINSOCK This is for a PC with network/ethernet card correctly installed and configured with packet drivers installed with packet vector pointing to 0x60. If you do not know what these are, consult your PC networking guru. If you do not have a packet driver for your ethernet card - a large list of packet drivers are at :- sol.dmp.csiro.au:/pub/ms-winsock/pkt_drv Packet drivers and sample autoexec.bat modifications are given with the internet kit for two common network cards - ne2000 and WD/SMC Elite plus. DO NOT DO ANY OF THIS IF YOU HAVE ANOTHER NETWORK PROTOCOL INSTALLED - THIS WILL CAUSE SERIOUS TROUBLE. AGAIN - BEFORE STARTING (you must consult your system manager). You need to know :- Your network's "Domain Name" Your logon name - for telnet. Your Email address. The IP address and name of your PC The IP address and name of your Name Server. The IP address and name of your Gateway. The IP address and name of your News Server (Your system manager will have to edit the nntp_access file to allow your PC to get news from the news server) The IP address and name of your Mail Server. ---------------- THE WINSOCK INSTALLATION BEGINS. IGNORE THIS IF YOU HAVE ANY OTHER NETWORK SOFTWARE RUNNING). Assuming that you have your packet driver correctly installed. --- Use the Windows notepad to edit c:\autoexec.bat Directly after the packet driver line add:- c:\internet\winsock\winpkt 0x60 Save the file and exit. --- EDIT (using notepad) C:\INTERNET\WINSOCK\TRUMPWSK.INI Where indicated :- Add your PC's IP address, gateway IP address, dns (domain name server) IP address, and the domain name. Save and exit the file. ---- Copy the c:\internet\windows\protocol file to c:\windows Copy the c:\internet\windows\services file to c:\windows Copy the c:\internet\windows\hosts file to c:\windows ---- Use Notepad to edit the c:\windows\hosts file. Add the IP and Name of your PC. Add the IP and Name of the DNS (domain name server) Add the IP and Name of the Gateway Save and exit the file. --- Go into the STARTUP FOLDER. Add a new ICON by File, New - and Browse to the c:\internet\winsock\ directory and select the tcpman.exe file. Click on the Run Minimized button and press OK. Now exit Windows and re-boot your computer. When you run Windows again - you will have Winsock/TCP-IP running and can use a variety of internet software - CONGRATULATIONS!!!! ========================= ========================= INSTALLING THE INTERNET SOFTWARE. ---- ADDING THE WINDOWS PROGRAM ICONS In Windows. Add a new folder (Program Group) with the name "Internet Kit" and Group File of - internet.grp. You will now have a new folder called "Internet Kit" with all the icons correctly installed. Do not run any of them yet as they still have to be configured. ------- HGOPHER Gopher will run quite OK. If you wish to change the homepage, go into Options, Gopher Setup and enter the home page you wish to see on startup. ------- WS_FTP This will work Instantly. The only host there is to anonymously log into sol.dmp.csiro.au. Add other hosts as you desire. ------- TRMPTEL (telnet program) This will work without configuration. Just type in the host you wish to log into. If you wish to log into a host all the time, use File Properties to add the host in the Command Line. ------- MOSAIC WWW (World Wide Web) Client. The LVIEW gif, jpg, etc picture viewer is already correctly installed. Use notepad to edit the c:\windows\mosaic.ini file. Add your email address on the second line. Mosaic will now work properly with your real email address should you use this option You can now go wild on the WWW. There are other options you can modify - but this will start you off quite happily. ------- Wintrumpet Newsreader. When you run WinTrumpet, you will be prompted for information such as your :- News Host Name Mail Host Name Your Email Address Full Name Organisation (Your signature file is at c:\internet\sig.txt, use notepad to edit this file). The rest is optional and depends if you want to use WinTrumpet as your POP mail program). You will then be prompted for newsgroups to add. Select a couple immediately to test out your NNTP connection (i.e., sci.chem.electrochem). ------- WINQVT. Use notepad to edit the c:\internet\qvtnet\qvtnet.ini file. Search for ??? and replace as required. It occurs in this order :- PC host name. PC IP Address. Router/Gateway Domain Name Name Server (Domain Name Server) The rest is optional depending of whether you want to Use QVTNET also as a mail program, ftp client and/or server, newsreader and talk to a line printer. ------ FINISHED In theory, you have now completed the installation. Good luck! :-) ------- Standard disclaimers apply - user is 100% responsible for any hassles, problems or loss of data. (It might not be lawyer proof but it will let me sleep nights!) :-) ------------------------- -- Lachlan Cranswick - CSIRO _--_|\ lachlan@dmp.CSIRO.AU Division of Mineral Products / \ tel +61 3 647 0367 PO Box 124, Port Melbourne \_.--._/ fax +61 3 646 3223 3207 AUSTRALIA v From owner-proteins@net.bio.net Sat Aug 06 23:00:00 1994 Path: biosci!daresbury!not-for-mail From: Newsgroups: bionet.molbio.proteins Subject: PNGaseF Date: 7 Aug 1994 23:09:48 +0100 Lines: 12 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <323m3c$1ih@mserv1.dl.ac.uk> Original-To: proteins@dl.ac.uk Dear netters, I'm looking for a good protocol for deglycosilating a glycoprotein with PNGase F. The one in Current Protocols for M.B. did not work in my hands and is also a little bit imprecise.For istance it says: add 1 e protein, w ithout specifiyng in what should it be diluted. Pleas post to me by E-Mail or to the Newsgro ill compile a summary. Thanks. Giorgio Spagl Sorry, Giorgio Spagnol SPAGNOL@PLA UTO.CSATA.IT From owner-proteins@net.bio.net Sat Aug 06 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!news.cac.psu.edu!news.tc.cornell.edu!travelers.mail.cornell.edu!newsstand.cit.cornell.edu!NewsWatcher!user From: cmf5@cornell.edu (Casey M. Finnerty) Newsgroups: bionet.molbio.proteins Subject: Re: PLASMID DRAWING SOFTWARE Followup-To: bionet.molbio.proteins Date: Sun, 07 Aug 1994 17:24:59 -0500 Organization: Boyce Thompson Institute at Cornell University Lines: 18 Sender: cmf5@cornell.edu (Verified) Message-ID: References: <00982579.31DDA6E0@vms.csd.mu.edu> NNTP-Posting-Host: 132.236.156.75 In article <00982579.31DDA6E0@vms.csd.mu.edu>, 6566friedman@vms.csd.mu.edu wrote: > Can anyone recommend a good Macintosh program for drawing plasmids? Are > any available in the public domain from FTP sites? > > Thanks, > Alan Friedman > 6566Friedman@VMS.CSD.MU.EDU MacPlasmap is quite good. You can get it direct from the author (Jingdong Liu) at Jliu@bioscience.utah.edu or I think it's available in the IUBio and/or EMBL archives. I believe the program is free with the caveat that if you like it the author requests you send him 25 bucks. -- Casey M. Finnerty Boyce Thompson Institute at Cornell University cmf5@cornell.edu From owner-proteins@net.bio.net Sun Aug 07 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!pipex!lyra.csx.cam.ac.uk!mole.bio.cam.ac.uk!rgs From: rgs@mole.bio.cam.ac.uk (Robert Solomon (Bioc)) Newsgroups: bionet.molbio.proteins Subject: Re: Salt Links vs. Hydrophobic Interactions? Date: 8 Aug 1994 08:43:27 GMT Organization: U. of Cambridge, England Lines: 31 Message-ID: <324r7f$kv0@lyra.csx.cam.ac.uk> References: NNTP-Posting-Host: mole.bio.cam.ac.uk In article YMH4375@zeus.tamu.edu (Yasha Hartberg) writes: >I want to use site-directed mutagenesis to disrupt dimer formation of a >protein I'm studying. I have a choice between either disrupting several >important salt-bridges or putting in several hydrophilic residues to >disrupt the hydrophobic interface. Before I design the oligos, I would >like to hear some advice as to which approach seems the most likely to >succeed. Any help will be greatly appreciated. > >Yasha Hartberg >Texas A&M University Yasha, I would advise disrupting the hydrophobic interface, if there is a substantial surface area involved, replacing the residues with a mix of charged and polar residues. If you just remove the salt bridges, you will leave this surface intact, and it will probably cause no dimer disruption at all, or non-specific aggregation. Just a thought floating through the ether, Rob Solomon Std disclaimer: The opinions expressed are those of the author - my employer will deny my existence if pushed on the matter. <.sig deleted due to loss of sense of humour> From owner-proteins@net.bio.net Sun Aug 07 23:00:00 1994 Path: biosci!rutgers!gatech!news-feed-1.peachnet.edu!news.duke.edu!solaris.cc.vt.edu!swiss.ans.net!malgudi.oar.net!witch!abr!jstiehr Newsgroups: bionet.molbio.proteins Message-ID: <56@abr.win.net> Reply-To: jstiehr@abr.win.net (James Stiehr) From: jstiehr@abr.win.net (James Stiehr) Date: Mon, 08 Aug 1994 15:49:46 GMT Subject: Sequence/hydrophilicity Software Lines: 17 I am looking for comments, sugestions, experiences, etc. about software (DOS or Windows) for amino acid sequence aligment for multiple proteins as well as hydrophilicity plotting for peptide selection of antigenic epitopes. I know of PC/Gene and Lasergene. Both companies are sending info, but both are very expensive (PC/Gene = ~$3500, Megline & Protean modules of Lasergene = ~$2500). Does anyone have experience with either of these or with any other suppliers which can do the same thing? Are there any freeware programs for these application available on the Net? Any opinions would be welcome. Also is there a way to search Genebank or other databases for proteins which share a peptide sequence on the Net? If so, how would a newbie like me go about doing this? Thanks in advance for the feedback. James Stiehr \\ // Affinity BioReagents \ / Internet:jstiehr@abr.win.net || 908-369-3799, 908-369-8894 (FAX) || From owner-proteins@net.bio.net Sun Aug 07 23:00:00 1994 Newsgroups: bionet.structural-nmr,bionet.molbio.proteins,bionet.molec-model Path: biosci!agate!msuinfo!harbinger.cc.monash.edu.au!bunyip.cc.uq.oz.au!munnari.oz.au!mel.dit.csiro.au!dmp.csiro.au!lachlan From: lachlan@dmp.csiro.au (Lachlan Cranswick) Subject: SCA - "CRYSTAL XIX" - Ballarat Australia - April 1995 Message-ID: <1994Aug8.084900.15720@dmp.csiro.au> Organization: CSIRO Division of Mineral Products, Melbourne, AUSTRALIA Date: Mon, 8 Aug 1994 08:49:00 GMT Lines: 157 Xref: biosci bionet.structural-nmr:192 bionet.molbio.proteins:2422 bionet.molec-model:65 CRYSTAL XIX First Announcement of the Nineteenth Meeting of the Society of Crystallographers in Australia 18 - 21 April, 1995 University of Ballarat, Victoria INTRODUCTION The Organising Committee invites all crystallographers to attend the Nineteenth Meeting of the Society of Crystallographers in Australia. The conference will be held at the University of Ballarat in country Victoria, from the evening of Tuesday the 18th of April ('Easter Tuesday') to lunchtime on Friday the 21st of April, 1995. THE VENUE Ballarat University is set on 110 hectares of natural bushland about 10 km south east of the city of Ballarat. Very pleasant on-campus accommodation is provided by the Mount Helen Residence, capable of accommodating 514 people in single bed/study rooms at about $23 per night (room only). Common room and games room, kitchen areas, laundries and outdoor barbecue facilities are available in the residential complex. Numerous motels and other forms of accommodation are available within a few km of the campus. The University has a full range of lecture and seminar facilities. It also has full AARNet access,and e-mail and Internet access can be easily arranged on request for all participants. Excellent recreational facilities are available including fully equipped gymnasia, jogging and walking tracks and an Arboretum. All meals are provided by the University's catering service and fully licensed bar facilities serve the various function rooms. TRANSPORT Ballarat University is one and a half hours drive from the centre of Melbourne. There are up to six shuttle bus services per day from Tullamarine airport to Ballarat. Cost, about $15 each way. Numbers permitting, these buses will detour to the University campus. The committee will help coordinate private car transport from Melbourne. Train and private bus services also run from the centre of Melbourne to Ballarat. TENTATIVE COSTS The Conference Committee has obtained a favourable arrangement with the University to keep costs to a minimum. The University has its own catering system for all meals at reasonable prices. Drinks are at normal bar prices. We anticipate that the all inclusive cost of accommodation plus meals from Tuesday evening to Friday lunch will be less than $250 per person. Tentative registration costs are $100 for normal registration and $50 for students. SCIENTIFIC PROGRAM The Conference Lecture will be presented by the 1987 Fellow, Dr. Michael Hart. In addition to the usual topic areas (protein crystallography, inorganic and mineral structures etc) we welcome contributions in areas such as Applications of synchrotron radiation Structure studies using less traditional methods such as XANES, EXAFS, MASNMR, DAFS Structure determinations using TEM/SAED and ND Crystallographic teaching New developments in crystallographic hardware/software 1995 is the centenary of the discovery of X-rays and the Australian Academy of Science is sponsoring a conference celebrating this event in the week following Crystal XIX. We envisage that a special lecture will be devoted to commemorating this event in the Australian context. Further details will be given in the second circular. SOCIAL PROGRAM This will include a mixer (with possibly a wine tasting) on the Tuesday evening and the conference dinner on the Thursday evening. The Conference Lecture will be presented on Wednesday evening, allowing a free afternoon on Wednesday for various activities which could include visits to Sovereign Hill, to the Pyrenees Wineries and a tour of Ballarat and its surrounds. We will be seeking delegates preferences at the time of registration. ABSTRACTS/REGISTRATION Registration forms and a call for abstracts will be sent out at the end of October and the deadline for the submission of both will be 1st February 1995. CONTACT PERSON Further information can be obtained by contacting Lachlan Cranswick at CSIRO Division of Mineral Products, PO Box 124, Port Melbourne, Victoria 3207. Phone (613) 647-0211, Fax (613) 646-3223, e-mail: lachlan@dmp.csiro.au ORGANISING COMMITTEE Lachlan Cranswick (see above) Ian Grey CSIRO Division of Mineral Products, Port Melbourne Phone (613)-647-0211, Fax (613)-646-3223 e-mail: iang@dmp.csiro.au Mike Lawrence Biomolecular Research Institute, Parkville Phone (613)-342-4200, Fax (613)-342-4301 e-mail: mike@rox.mel.dbe.csiro.au Andrew Stevenson CSIRO Division of Materials Science and Technology, Clayton Phone (613)-542-2917, Fax (613)-544-1128 e-mail: stevens@rivett.mst.csiro.au Jonathan White Melbourne University, Chemistry Department, Parkville Phone (613)-344-4621, Fax (613)-347-5180 e-mail: jonathan_white@muwayf.unimelb.edu.au We thank Nikki Scarlett and Neil Manning for their help with the preparation of the first circular. -- Lachlan Cranswick - CSIRO _--_|\ lachlan@dmp.CSIRO.AU Division of Mineral Products / \ tel +61 3 647 0367 PO Box 124, Port Melbourne \_.--._/ fax +61 3 646 3223 3207 AUSTRALIA v From owner-proteins@net.bio.net Mon Aug 08 23:00:00 1994 Newsgroups: bionet.molbio.proteins From: jm@ambident.demon.co.uk (Jonathan Montagu) Path: biosci!daresbury!doc.ic.ac.uk!warwick!pipex!demon!ambident.demon.co.uk!jm Subject: Protein folding and AI Lines: 8 X-Newsreader: Archimedes ReadNews Date: Tue, 9 Aug 1994 17:55:50 +0000 Message-ID: Sender: usenet@demon.co.uk Can anybody suggest some references on the theoretical prediction of protein conformations using artificial intelligence (eg genetic algorithms)? Is the 'Brookland Protein Database' on the Internet? Many thanks, Jonathan Montagu ----------------------------------------------------------------------------- E-mail: jm@ambident.demon.co.uk ----------------------------------------------------------------------------- From owner-proteins@net.bio.net Mon Aug 08 23:00:00 1994 Path: biosci!ARSERRC.GOV!CTHOMPSON From: CTHOMPSON@ARSERRC.GOV Newsgroups: bionet.molbio.proteins Subject: Research Date: 9 Aug 1994 13:28:16 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 5 Sender: daemon@net.bio.net Distribution: world Message-ID: <01HFPMYRXHD4000AU1@arserrc.gov> NNTP-Posting-Host: net.bio.net Does anyoneone have any ideas on what would be a good graduate school to do collagen rsearch with? Thanks, Craig Thompson CTHOMPSON@arserrc.gov From owner-proteins@net.bio.net Mon Aug 08 23:00:00 1994 Path: biosci!ARSERRC.GOV!CTHOMPSON From: CTHOMPSON@ARSERRC.GOV Newsgroups: bionet.molbio.proteins Subject: telopeptides Date: 9 Aug 1994 13:19:12 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 5 Sender: daemon@net.bio.net Distribution: world Message-ID: <01HFPMOQXTZM000AU1@arserrc.gov> NNTP-Posting-Host: net.bio.net Does anyone know where or what the sequence is for the C-telopeptide and the N-telopeptide for type(I) collagen? Thanks, Craig Thompson CTHOMPSON@arserrc.gov From owner-proteins@net.bio.net Mon Aug 08 23:00:00 1994 Path: biosci!FOX.CCE.USP.BR!szeinfel From: szeinfel@FOX.CCE.USP.BR (Rafael N Szeinfeld) Newsgroups: bionet.molbio.proteins Subject: Re: Protein folding and AI Date: 9 Aug 1994 12:43:50 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 28 Sender: daemon@net.bio.net Distribution: world Message-ID: References: NNTP-Posting-Host: net.bio.net See Protein Engineering 7(4):475-485 (1994). Hope this help. Rafael. ###################################### # Rafael Iosef Najmanovich Szeinfeld # # Department of Biochemistry # # University of Sao Paulo, Brasil. # # E-mail : szeinfel@fox.cce.usp.br # # szeinfel@snfma1.if.usp.br # # szeinfel@iris.iq.usp.br # # 1762545@cat.cce.usp.br # ###################################### On Tue, 9 Aug 1994, Jonathan Montagu wrote: > Can anybody suggest some references on the theoretical prediction of > protein conformations using artificial intelligence (eg > genetic algorithms)? Is the 'Brookland Protein Database' on the > Internet? Many thanks, > > Jonathan Montagu > ----------------------------------------------------------------------------- > E-mail: jm@ambident.demon.co.uk > ----------------------------------------------------------------------------- > > From owner-proteins@net.bio.net Mon Aug 08 23:00:00 1994 Path: biosci!daresbury!trane.uninett.no!nac.no!eunet.no!nuug!EU.net!howland.reston.ans.net!europa.eng.gtefsd.com!library.ucla.edu!news.mic.ucla.edu!hodgkin.mbi.ucla.edu!arne From: arne@hodgkin.mbi.ucla.edu (Arne Elofsson) Newsgroups: bionet.molbio.proteins Subject: Re: Protein folding and AI Date: 9 Aug 1994 19:41:23 GMT Organization: OrgFreeware Lines: 28 Distribution: world Message-ID: <328m53$16l@news.mic.ucla.edu> References: Reply-To: arne@hodgkin.mbi.ucla.edu (Arne Elofsson) NNTP-Posting-Host: hodgkin.mbi.ucla.edu >Can anybody suggest some references on the theoretical prediction of >protein conformations using artificial intelligence (eg >genetic algorithms)? Is the 'Brookland Protein Database' on the >Internet? Many thanks, GA:s have been used by: Dandekar & Argus, Sun, LeGrand & Merz, Bowie & Eisenberg all during the last year for protein folding simulations. It works OK Neural Nets have been used: XX & Sander and XX & Karplus for secondary structure prediction. Others have used NN:s for predicting other stuff (including long-range contacts, don't ask me how they did that) It works as good as any other method. arne -- ****************************************************************************************** ** Arne Elofsson (arne@hodgkin.mbi.ucla.edu) ** ** Molecular Biology Institute, UCLA ** ** 405 Hilgard Avenue, Los Angeles ** ** 90024-1570 California, USA ** ** tel: +1-(310)-825-1402 Fax: +1-(310)-206-3914 ** ** Home: +1-(310)-268-8006 SWE: +46-8-306129 ** ****************************************************************************************** From owner-proteins@net.bio.net Mon Aug 08 23:00:00 1994 Path: biosci!rutgers!gatech!europa.eng.gtefsd.com!ulowell!umassd.edu!news.umass.edu!nic.umass.edu!usenet From: JMANGOR@UCSVAX.UCS.UMASS.EDU (Jodie Mangor) Newsgroups: bionet.molbio.proteins Subject: muscle protein extracts Date: 9 Aug 1994 19:15:58 GMT Organization: University of Massachusetts at Amherst Lines: 15 Distribution: world Message-ID: <328kle$8gs@nic.umass.edu> NNTP-Posting-Host: phobos.ucs.umass.edu X-News-Reader: VMS NEWS v1.25 I am in need of protocols for preparing ACTIVE total protein extracts from insect muscle (Manduca in particular). I may also need to prepare ACTIVE extracts from the nuclei and cytoplasm separately, but I don't know that yet. If anyone has any protocols, please forward them to CASCONE@marlin.bio.umass.edu -- please do not send the info to this address -- thanks a lot!!!! P. Cascone Jodie Mangor ###########* *##* *##* *##* *##* Biology Department *: : : * * : : *: : : * * : : University of Massachusetts * : : * * : : * * : : * * : : * Amherst, MA 01003 * * : : * : : * * : : * : : * JMANGOR@BIO.UMASS.EDU *##* *##* *##* *##* From owner-proteins@net.bio.net Mon Aug 08 23:00:00 1994 Path: biosci!bloom-beacon.mit.edu!senator-bedfellow.mit.edu!aaRS!lluis From: lluis@aaRS (Lluis Ribas) Newsgroups: bionet.molbio.proteins Subject: Re: Protein folding and AI Date: 9 Aug 1994 23:41:11 GMT Organization: Massachvsetts Institvte of Technology Lines: 25 Distribution: world Message-ID: <32946n$kfb@senator-bedfellow.MIT.EDU> References: <328m53$16l@news.mic.ucla.edu> NNTP-Posting-Host: aars.mit.edu X-Newsreader: TIN [version 1.2 PL2] Arne Elofsson (arne@hodgkin.mbi.ucla.edu) wrote: : Neural Nets have been used: : XX & Sander and XX & Karplus for secondary structure prediction. : Others have used NN:s for predicting other stuff (including long-range : contacts, don't ask me how they did that) : It works as good as any other method. : arne I have no experience with Karplus's methods, but I have used extensively most of the available secondary structure prediction algorythms an I have to disagree with your statement. While I could hardly see any difference with the methods of Chou&Fasman, GOR (Garnier, Osguthorpe & Robson), McLachlan, etc. etc., PhD (the Rost&Sander method using neural networks) works definetely better. If you have good alignments for your problem sequence the predictions are astonishingly good, particularly for beta-strands, which are a traditional weak point of the older methods based on pure statistics. Of course, still far from what we'd like it to be, but certanly better. best regards, LLuis From owner-proteins@net.bio.net Mon Aug 08 23:00:00 1994 Path: biosci!daresbury!not-for-mail From: Newsgroups: bionet.molbio.proteins Subject: TRUE OR FALSE? Date: 9 Aug 1994 15:50:37 +0100 Lines: 7 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <32853t$621@mserv1.dl.ac.uk> Original-To: proteins@dl.ac.uk, immuno@dl.ac.uk I intend to purify a protein with an immunoaffinity column. Current Protocols in MB states that the ratio Ab/Activated sepharose in the columnn should be 2mg/ml. To me seems such an exaggerated quantity of antibody that I suspect a Typing mistake,but I'm not so sure of myself. Is anybody out there willing to offer his expertize? Cordially, yours Giorgio Spagnol Spagnol@plauto.csata.it From owner-proteins@net.bio.net Mon Aug 08 23:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!bcm!news.msfc.nasa.gov!europa.eng.gtefsd.com!darwin.sura.net!gatekeeper.es.dupont.com!esds01.es.dupont.com!GREENERA@ldoc03.ldc.dupont.com From: greenera@wmvx.lvs.dupont.com Subject: Re: Postdoctoral position Message-ID: <1994Aug9.211013.22416@es.dupont.com> Sender: news@es.dupont.com (USENET News System) Nntp-Posting-Host: ldoc03.lvs.dupont.com Reply-To: greenera@wmvx.lvs.dupont.com Organization: DuPont (Opinions are those of the writer only) References: <31af6s$a78@obelix.cica.es> Date: Tue, 9 Aug 1994 21:10:13 GMT Lines: 16 end ggg=======`y In article <31af6s$a78@obelix.cica.es>, pmateo@ugr.es(Pedro L. Mateo) writes: > From owner-proteins@net.bio.net Tue Aug 09 23:00:00 1994 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!scripps.edu!manuel From: manuel@scripps.edu (Manuel Baca) Newsgroups: bionet.molbio.proteins Subject: what unit is "pkat" Date: 10 Aug 1994 18:11:24 GMT Organization: The Scripps Research Institute, La Jolla, California, USA Lines: 9 Distribution: world Message-ID: <32b58c$8b6@riscsm.scripps.edu> NNTP-Posting-Host: terrapin.scripps.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit Can anyone tell me what the units "pkat" are? I have seen it used in an enzyme kinetics paper as the Vmax units. Any enlightenment would be much appreciated. Many thanks, Manuel Baca Scripps Research Institute La Jolla CA manuel@scripps.edu From owner-proteins@net.bio.net Tue Aug 09 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!swiss.ans.net!malgudi.oar.net!ucbeh!allendn From: allendn@ucbeh.san.uc.edu Newsgroups: bionet.molbio.proteins Subject: Reverse Phase HPLC at neutral pH Message-ID: <1994Aug10.121324.3175@ucbeh> Date: 10 Aug 94 12:13:24 EST Distribution: world Organization: University of Cincinnati Lines: 7 I am attempting to purify a protein from bovine pituitaries. I have been using primarily gel filtration and reverse phase HPLC. I have also done some pilot experiments with cation-exchange HPLC, isoelectric focusing, and gel electrophoresis. Most of the reverse phase HPLC has used trifloroacetic acid or heptaflorobutyric acid. I am starting to try reverse phase HPLC at less acidic or neutral pH using ammonium acetate or triethylamine. Has anyone done this, and do you have any helpful advice. Thanks. From owner-proteins@net.bio.net Tue Aug 09 23:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!agate!overload.lbl.gov!ames!elroy.jpl.nasa.gov!swrinde!pipex!bt!uknet!bcc.ac.uk!bsm.bioc.ucl.ac.uk!mcdonald From: mcdonald@bsm.bioc.ucl.ac.uk (Ian McDonald) Subject: HBPLUSv3.0 Hydrogen Bond Calculator Message-ID: <1994Aug10.203608.29082@ucl.ac.uk> Date: Wed, 10 Aug 1994 20:36:08 GMT Organization: University College London Lines: 194 HBPLUS v 3.0 ************ Hydrogen Bond Calculation Program ================================= Introduction ============ HBPLUS is a hydrogen bond calculation program that has been developed in-house over the course of over four years and has been used in countries as diverse as the USA, Taiwan and South Africa. It . . o Calculates the Geometries of all Hydrogen Bonds o Optionally Lists of Neighbour Interactions o Calculates Hydrogen Positions o Deals with Hydrogens that can Occupy More Than one Position o Optionaly includes amino-aromatic H-bonds. o Supports Full Customisation, eg of o H-bond criteria o Donor and Acceptor Atoms types o Analyses H-bonding Near Asn, Gln and His Side-Chains and Suggests Optimal Conformation o Supports .hbplusrc Files o Outputs PDB File Including Extrapolated Polar Hydrogen Positions Technical Side ============== It is written in ANSI C and has been checked on several UNIX systems as well as VAX/VMS. It unfortunately requires too much memory to run on an IBM PC or an Apple Macintosh. If you would like a copy of HBPLUS, simply download the confidentiality agreement, sign it, snailmail it to me and I'll email you a password that you can use to un-crypt the ftp-able hbplus.tar.Z.cr file, after loading it to disk as a binary file. The agreement essentially promises not to give anyone outside your lab copies of HBPLUS, and to reference us if you use HBPLUS results in a paper - HBPLUS is free to academic users. HBPLUS compiles using a makefile. This release also includes ACCESS to determine accessible surface areas, and CLEAN to tidy Brookhaven files. These programs are written by other people and also covered by the confidentiality agreement. ftp: bsm.bioc.ucl.ac.uk (128.40.46.11) /pub/hbplus WWW: Copyrights ========== HBPLUS is (c) I.K. McDonald, D. Naylor, D. Jones and J.M. Thornton 1994 ACCESS is (c) S. Hubbard 1992-4 CLEAN is (c) D.K. Smith, R. Laskowski and G. Hutchinson 1992-4 Reference ========= I.K. McDonald and J.M. Thornton (1994), "Satisfying Hydrogen Bonding Potential in Proteins", JMB 238:777-793. mcdonald@bsm.bioc.ucl.ac.uk Confidentiality Agreement ========================= Correspondence to: Ian McDonald (PG) Biological Structure and Modelling Unit Department of Biochemistry and Molecular Biology University College London Gower Street LONDON WC1E 6BT UK / EC Email mcdonald@uk.ac.ucl.bsm HBPLUS - Hydrogen Bond Calculation ---------------------------------- CONFIDENTIALITY AGREEMENT ------------------------- In regard to the HBPLUS program, specified in Appendix 1 herewith (the Software) supplied to us, the copyright and other intellectual property rights to which belong to the authors, we __________________________________________________________________ undertake to the authors that we shall be bound by the following terms and conditions:- 1. We will receive the Software and any related documentation in confidence and will not use the same except for the purpose of the department's own research. The Software will be used only by such of our officers or employees to whom it must reasonably be communicated to enable us to undertake our research and who agree to be bound by the same confidence. The department shall procure and enforce such agreement from its staff for the benefit of the authors. 2. The publication of research using the Software must reference "McDonald IK & Thornton JM (1994), 'Satisfying Hydrogen Bonding Potential in Proteins', Journal of Molecular Biology 238:777-793" 3. Research shall take place solely at the department's premises at __________________________________________________________________ 4. All forms of the Software will be kept in a reasonably secure place to prevent unauthorised access. 5. Each copy of the Software or, if not practicable then, any package associated therewith shall be suitably marked (and such marking maintained) with the following copyright notice: "Copyright 1991-3 Ian McDonald, Dorica Naylor, David Jones, S Hubbard, R A Laskowski and Janet M Thornton All Rights Reserved". 6. The Software may be modified but any changes made shall be made available to the authors. 7. The Software shall be used exclusively for academic teaching and research. The Software will not be used for any commercial research or research associated with an industrial company. 8. The confidentiality obligation in paragraph one shall not apply: (i) to information and data known to the department at the time of receipt hereunder (as evidenced by its written records); (ii) to information and data which was at the time of receipt in the public domain or thereafter becomes so through no wrongful act of the department; (iii) to information and data which the department receives from a third party not in breach of any obligation of confidentiality owed to the authors. Please sign this Undertaking and return a copy of it to indicate that you have read, understood and accepted the above terms. For and on behalf of _____________________________ _________________________________________________ .................................................. Dated ............................................ Address __________________________________________ _________________________________________________ _________________________________________________ Country _________________ Postcode ______________ Telephone ________________________________________ Electronic Mail Address to which HBPLUS shall be sent _____________________________________________ _________________________________________________ APPENDIX 1 - DETAILS OF THE HBPLUS PROGRAM PROVIDED --------------------------------------------------- Files to be included -------------------- 1. hbplus.h } 2. hbp_gen.c } Source program files, 3. hbp_inpdb.c } ,formerly hbplus.c 4. hbp_findh.c } 5. hbp_hhb.c } 6. hbp_main.c } 7. hbplus.man } Documentation 8. accall.f } Supplemental, (c) S. Hubbard 9. vdw.radii } "ACCESS" / "NACCESS" 10. clean.f } Supplemental, (c) DK Smith et al. 11. brkcln.par } "BRKCLN" 12. chkqnh } C Shell Script 13. Makefile } Unix Makefile From owner-proteins@net.bio.net Tue Aug 09 23:00:00 1994 Path: biosci!agate!spool.mu.edu!sdd.hp.com!cs.utexas.edu!news.unt.edu!hermes.oc.com!convex!convex!news.duke.edu!solaris.cc.vt.edu!swiss.ans.net!europa.eng.gtefsd.com!newsxfer.itd.umich.edu!uunet!EU.net!sunic!news.funet.fi!news.csc.fi!news.helsinki.fi!not-for-mail From: pemakine@cc.Helsinki.FI (Pekka Makinen) Newsgroups: bionet.molbio.proteins Subject: Re: what unit is "pkat" Date: 10 Aug 1994 22:13:03 +0300 Organization: University of Helsinki Lines: 11 Message-ID: <32b8rv$37l@serifos.Helsinki.FI> References: <32b58c$8b6@riscsm.scripps.edu> Reply-To: Pekka.Makinen@Helsinki.FI NNTP-Posting-Host: serifos.helsinki.fi Mime-Version: 1.0 Content-Type: text/plain; charset=ISO-8859-1 Content-Transfer-Encoding: 8bit manuel@scripps.edu (Manuel Baca) writes in bionet.molbio.proteins: ]Can anyone tell me what the units "pkat" are? I have seen it used in an ]enzyme kinetics paper as the Vmax units. pkat = picokatals Katal is unit of enzyme activity defined as 1 kat = 1 mol/s. -- _____________________________________________________ Be realistic! Pekka.Makinen@Helsinki.FI pemakine@kruuna.helsinki.fi Demand the impossible. From owner-proteins@net.bio.net Tue Aug 09 23:00:00 1994 Path: biosci!rutgers!koriel!cs.utexas.edu!howland.reston.ans.net!europa.eng.gtefsd.com!library.ucla.edu!news.mic.ucla.edu!unixg.ubc.ca!quartz.ucs.ualberta.ca!news From: rndy@wimsey.mmid.ualberta.ca (Randy Read) Newsgroups: bionet.molbio.proteins Subject: Re: Protein folding and AI Date: 10 Aug 1994 18:26:17 GMT Organization: Computer and Network Services, U of Alberta, Edmonton, Canada Lines: 12 Distribution: world Message-ID: <32b649$du5@quartz.ucs.ualberta.ca> References: <328m53$16l@news.mic.ucla.edu> Reply-To: rndy@wimsey.mmid.ualberta.ca NNTP-Posting-Host: wimsey.mmid.ualberta.ca In article <328m53$16l@news.mic.ucla.edu> arne@hodgkin.mbi.ucla.edu (Arne Elofsson) writes: > > GA:s have been used by: > Dandekar & Argus, Sun, LeGrand & Merz, Bowie & Eisenberg > all during the last year for protein folding simulations. Also Unger & Moult, "Genetic algorithms for protein folding simulations", Journal of Molecular Biology. 231(1):75-81, 1993. Randy Read rndy@mycroft.mmid.ualberta.ca From owner-proteins@net.bio.net Tue Aug 09 23:00:00 1994 Path: biosci!agate!spool.mu.edu!vms.csd.mu.edu!6566FRIEDMAN From: 6566friedman@vms.csd.mu.edu Newsgroups: bionet.molbio.proteins Subject: Solved Problem w/ Enzymekinetics Date: 10 Aug 1994 14:44:31 GMT Organization: Marquette University - Computer Services Lines: 14 Message-ID: <00982BBD.0FC67F60@vms.csd.mu.edu> Reply-To: 6566friedman@vms.csd.mu.edu NNTP-Posting-Host: vmsb.csd.mu.edu I finally solved the problem I was having with Enzymekinetics, a hypercard stack that plots enzyme kinetic data as V vs [S] or 1/V vs 1/[S]. Initially I used Hypercard version 2.1 and got error messages upon entering my data. However, when I used an old version of hypercard, version 1.2.1 (1987), the program ran just fine. The software does exactly what it claims to do, but will only plot a single data set on a plot. Therefore, data containing various inhibitor concentrations can not be plotted together. Nevertheless, I think this program is a usefull teaching tool for introductory biochemisty courses. Alan Friedman Dept Biology Marquette University 6566FRIEDMAN@VMS.CSD.MU.EDU From owner-proteins@net.bio.net Tue Aug 09 23:00:00 1994 Path: biosci!agate!headwall.Stanford.EDU!hsdndev!NewsWatcher!user From: rdoe@waste_time.edu (Robert Doe) Newsgroups: bionet.molbio.proteins Subject: Immunoaffinity Followup-To: bionet.molbio.proteins Date: 10 Aug 1994 11:20:56 GMT Organization: Not enough to do ? Lines: 6 Distribution: world Message-ID: NNTP-Posting-Host: 134.174.88.62 It's not a typo, 2mg mAb per ml of hydrated, activated Sepharose is OK. Personally, I use 5-10 mg mAb per ml. Smaller columns per given quantity of mAb yield material that is more pure, after elution. If your mAb is limiting, I've used a coupling density of as low as 0.5 mg/ml gel. Buy the CNBr activated Sepharose from Sigma, not Pharmacia. From owner-proteins@net.bio.net Tue Aug 09 23:00:00 1994 Path: biosci!agate!spool.mu.edu!vms.csd.mu.edu!6566FRIEDMAN From: 6566friedman@vms.csd.mu.edu Newsgroups: bionet.molbio.proteins Subject: Review of MacPlasmap Date: 10 Aug 1994 15:29:04 GMT Organization: Marquette University - Computer Services Lines: 32 Message-ID: <00982BC3.48BECA60@vms.csd.mu.edu> Reply-To: 6566friedman@vms.csd.mu.edu NNTP-Posting-Host: vmsb.csd.mu.edu In a recent posting, I asked about Macintosh plasmid drawing software recommendations. Several of you suggested MacPlasmap which is available from as macplasmap.hqx, a binary, compressed file in the directory /molbio/mac. The program comes complete with a detailed manual and a small library of the most commonly used plasmids, including pBR322, pUC19, pBluscript SK, M13mp18. MacPlasmap uses the typical Macintosh graphical interface and is easy to use. The user has the option of modifying an existing plasmid map or starting a new map from scratch. Various tools, including restriction site, polylinker, fragment insert and fragment delete allow you to add and delete all of the usual map features into a circular or linear map. Genes, ORIs, selectable markers etc. are indicated by arcs along the circular map and can be arrowed and stippled in various patterns. The only significant complaint that I have concerns the gene dialog box; the width of the gene is determined by clicking and dragging a bar; a numerical adjustment is not provided, making it difficult to accurately adjust the width of various inserts on the same map and even more difficult between different maps. This criticism not withstanding, I highly recommend MacPlasmap Version 1.82, written by Jingdong Liu at the University of Utah. It is provided as $25 shareware; a great value. It is easy to use, keeps track of the map position of various features, includes a note tool for your record keeping and provides publication ready output to a Laserwritter. Alan Friedman Dept Biology Marquette University 6566FRIEDMAN@VMS.CSD.MU.EDU From owner-proteins@net.bio.net Tue Aug 09 23:00:00 1994 Path: biosci!bcm!news.msfc.nasa.gov!europa.eng.gtefsd.com!howland.reston.ans.net!EU.net!sun4nl!news.nic.surfnet.nl!highway.LeidenUniv.nl!rulsfb.LeidenUniv.nl!SBTNFH From: sbtnfh@rulsfb.LeidenUniv.nl (Flip Hoedemaeker, CvF RUL/TNO, Leiden, The Netherlands) Newsgroups: bionet.molbio.proteins Subject: Re: Protein folding and AI Date: 10 Aug 1994 09:32:27 GMT Organization: Biology, Leiden University, The Netherlands Lines: 16 Message-ID: <32a6rb$p51@highway.LeidenUniv.nl> References: Reply-To: sbtnfh@rulsfb.LeidenUniv.nl NNTP-Posting-Host: rulsfb.leidenuniv.nl In article , jm@ambident.demon.co.uk (Jonathan Montagu) writes: > Is the 'Brookland Protein Database' on the >Internet? Many thanks, > >Jonathan Montagu >----------------------------------------------------------------------------- >E-mail: jm@ambident.demon.co.uk >----------------------------------------------------------------------------- You'll probably mean the Brookhaven Protein Databank. Yes, i'ts availible. FTP to pdb.pdb.bnl.gov or use Gopher. Flip From owner-proteins@net.bio.net Wed Aug 10 23:00:00 1994 Path: biosci!AMBIDENT.DEMON.CO.UK!jm From: jm@AMBIDENT.DEMON.CO.UK (Jonathan Montagu) Newsgroups: bionet.molbio.proteins Subject: Re: Protein folding and AI Date: 11 Aug 1994 13:40:45 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 9 Sender: daemon@net.bio.net Distribution: world Message-ID: References: NNTP-Posting-Host: net.bio.net Rafael Many thanks for the reference. This, and another list will provide lots of papers. Jonathan Montagu ----------------------------------------------------------------------------- E-mail: jm@ambident.demon.co.uk ----------------------------------------------------------------------------- From owner-proteins@net.bio.net Wed Aug 10 23:00:00 1994 Path: biosci!daresbury!dlpx1!asm From: asm@dlpx1.dl.ac.uk (A.S.McAlpine) Newsgroups: bionet.molbio.proteins Subject: Re: TRUE OR FALSE? Date: 11 Aug 1994 17:55:24 GMT Organization: SERC Daresbury Lab, Warrington, U.K. Lines: 12 Sender: asm@dlpx1 (A.S.McAlpine) Distribution: bionet Message-ID: <32domc$62f@mserv1.dl.ac.uk> References: <32853t$621@mserv1.dl.ac.uk> NNTP-Posting-Host: dlpx1.dl.ac.uk IIn article <32853t$621@mserv1.dl.ac.uk>, writes: |> I intend to purify a protein with an immunoaffinity column. Current Protocols |> in MB states that the ratio Ab/Activated sepharose in the columnn should be |> 2mg/ml. To me seems such an exaggerated quantity of antibody that I suspect a |> Typing mistake,but I'm not so sure of myself. Is anybody out there willing to |> offer his expertize? Cordially, yours |> Giorgio Spagnol |> Spagnol@plauto.csata.it I have previously used cyanogen bromide activated sepharose from SIGMA (I think?) and they also advise that 2mg/ml protein will be bound. I managed to get around 2,.4mg/ml bound so the figure you quote is not unreasonable ASM From owner-proteins@net.bio.net Wed Aug 10 23:00:00 1994 Path: biosci!agate!headwall.Stanford.EDU!b226-mac.stanford.edu!user From: mmd@cmgm.stanford.edu (mdavis) Newsgroups: bionet.molbio.proteins Subject: How to Generate Streptavidin Tetramer Coordinates? Date: Thu, 11 Aug 1994 18:04:47 +0900 Organization: hhmi Lines: 8 Distribution: world Message-ID: NNTP-Posting-Host: b226-mac.stanford.edu Keywords: Streptavidin structure I'd like to look at the streptavidin tetramer structure on the computer screen, but the pdb file (1stp) has only the coordinates of the monomer. I don't know if the appropriate symmetry operators are in the pdb file to generate the other three monomers. Can anyone help me out with this? John Altman altman@cmgm.stanford.edu From owner-proteins@net.bio.net Wed Aug 10 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!pipex!lyra.csx.cam.ac.uk!mole.bio.cam.ac.uk!rgs From: rgs@mole.bio.cam.ac.uk (Robert Solomon (Bioc)) Newsgroups: bionet.molbio.proteins Subject: Re: Salt Links vs. Hydrophobic Interactions? Date: 11 Aug 1994 11:13:05 GMT Organization: U. of Cambridge, England Lines: 21 Message-ID: <32d141$fa0@lyra.csx.cam.ac.uk> References: NNTP-Posting-Host: mole.bio.cam.ac.uk Yasha, I was going to follow up my earlier posting with a more detailed reply through email, but your address, as below, does not work - drop me a line. RSol In article YMH4375@zeus.tamu.edu (Yasha Hartberg) writes: >I want to use site-directed mutagenesis to disrupt dimer formation of a >protein I'm studying. I have a choice between either disrupting several >important salt-bridges or putting in several hydrophilic residues to >disrupt the hydrophobic interface. Before I design the oligos, I would >like to hear some advice as to which approach seems the most likely to >succeed. Any help will be greatly appreciated. > >Yasha Hartberg >Texas A&M University From owner-proteins@net.bio.net Wed Aug 10 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!vixen.cso.uiuc.edu!news.uoregon.edu!gaia.ucs.orst.edu!ava.bcc.orst.edu!navarrer From: navarrer@ava.bcc.orst.edu (Roy Navarre) Newsgroups: bionet.molbio.proteins Subject: Protein accumulating at stacking gel interface Date: 12 Aug 1994 05:40:26 GMT Organization: Oregon State University, Corvallis, Oregon Lines: 10 Message-ID: <32f20a$1q5@gaia.ucs.orst.edu> NNTP-Posting-Host: ava.bcc.orst.edu We are radiolabelling proteins in plants and then extracting them in an aqueous extraction buffer. The samples are never dried. We are finding a large amount of radioactivity at the stacking gel separating gel interface. Ie whatever this is, enters the stacking gel, but not the separating gel. (SDS-PAGE) Any suggestions as to how to get this material to enter the gel? Thanks a lot Roy From owner-proteins@net.bio.net Thu Aug 11 23:00:00 1994 Path: biosci!agate!msuinfo!harbinger.cc.monash.edu.au!bunyip.cc.uq.oz.au!sueslab.biosci.uq.oz.au!cochran From: duncan cochran Newsgroups: bionet.molbio.proteins Subject: WHAT IF Date: 13 Aug 1994 05:42:17 GMT Organization: University of Queensland Lines: 4 Distribution: world Message-ID: <32hmfp$sip@dingo.cc.uq.oz.au> NNTP-Posting-Host: sueslab.biosci.uq.oz.au X-UserAgent: Version 1.1.3 X-XXMessage-ID: X-XXDate: Sat, 13 Aug 94 15:47:34 GMT Does anyone use WHAT IF by Gert Vriend at EMBL? Can I ask you a few questions for example: where do you get it ? Please reply to cochran@biosci.uq.oz.au. Thanks Duncan From owner-proteins@net.bio.net Thu Aug 11 23:00:00 1994 Path: biosci!CS.Arizona.EDU!math.arizona.edu!news.Arizona.EDU!chuck.biosci.arizona.edu!user From: Dress@biosci.arizona.edu (Virginia Dress) Newsgroups: bionet.molbio.proteins Subject: Re: Protein accumulating at stacking gel interface Followup-To: bionet.molbio.proteins Date: 12 Aug 1994 23:09:20 GMT Organization: University of Arizona Lines: 26 Distribution: world Message-ID: References: <32f20a$1q5@gaia.ucs.orst.edu> NNTP-Posting-Host: chuck.biosci.arizona.edu In article <32f20a$1q5@gaia.ucs.orst.edu>, navarrer@ava.bcc.orst.edu (Roy Navarre) wrote: > > We are radiolabelling proteins in plants and then extracting > them in an aqueous extraction buffer. The samples are never dried. > We are finding a large amount of radioactivity at the stacking gel > separating gel interface. Ie whatever this is, enters the stacking > gel, but not the separating gel. (SDS-PAGE) > Any suggestions as to how to get this material to enter the gel? > > Thanks a lot > > Roy I've got 2 thoughts Roy (may be a record): 1) Adding urea to sample to help denature the protein 2) Gradient gels, of course you haven't said what % running gel you are using. If you are already using 4% stack and a 6% gel, this may not help very much. Good luck Ginnie From owner-proteins@net.bio.net Thu Aug 11 23:00:00 1994 Path: biosci!rutgers!gatech!newsxfer.itd.umich.edu!newsrelay.iastate.edu!vixen.cso.uiuc.edu!koniskyj3.life.uiuc.edu!user From: edbeaty@uxa.cso.uiuc.edu (ed beaty) Newsgroups: bionet.molbio.proteins Subject: Re: Protein accumulating at stacking gel interface Followup-To: bionet.molbio.proteins Date: 12 Aug 1994 18:09:41 GMT Organization: University of Illinois at Urbana Lines: 24 Distribution: world Message-ID: References: <32f20a$1q5@gaia.ucs.orst.edu> NNTP-Posting-Host: koniskyj3.life.uiuc.edu In article <32f20a$1q5@gaia.ucs.orst.edu>, navarrer@ava.bcc.orst.edu (Roy Navarre) wrote: > > We are radiolabelling proteins in plants and then extracting > them in an aqueous extraction buffer. The samples are never dried. > We are finding a large amount of radioactivity at the stacking gel > separating gel interface. Ie whatever this is, enters the stacking > gel, but not the separating gel. (SDS-PAGE) > Any suggestions as to how to get this material to enter the gel? > > Thanks a lot > > Roy The same thing happens when I silver stain gels; there's a distinct dark band at the interface between stacking and separating gels. I don't know a way around this, except to try running the gel without using a stacking gel. If your sample volume is fairly small, you should still get fairly tight bands. Of course, the proteins that didn't make it into the separating gel will probably be stuck at the bottom of the loading well... Just an idea. Ed Beaty edbeaty@uxa.cso.uiuc.edu From owner-proteins@net.bio.net Thu Aug 11 23:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!swrinde!news.uh.edu!mac-923.sr2-building.uh.edu!user From: rravi@uh.edu (Ravi Ramachandran) Newsgroups: bionet.molbio.proteins Subject: Peptide mapping Followup-To: bionet.molbio.proteins Date: Fri, 12 Aug 1994 11:58:14 -0700 Organization: University of Houston Lines: 8 Message-ID: NNTP-Posting-Host: mac-923.sr2-building.uh.edu Hi!, I am interested in doing peptide mapping, and as I have never done this before, I would be extremely grateful if anyone out there could suggest any protocols to use. Either post to this newsgroup or send me email at "rravi@uh.edu". Thanx en avance! :) ravi From owner-proteins@net.bio.net Thu Aug 11 23:00:00 1994 Path: biosci!BANGOR.AC.UK!BSS169 From: BSS169@BANGOR.AC.UK Newsgroups: bionet.molbio.proteins Subject: Re: True or False Date: 12 Aug 1994 07:57:31 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 21 Sender: daemon@net.bio.net Distribution: world Message-ID: <199408121457.HAA25859@net.bio.net> NNTP-Posting-Host: net.bio.net In article <32domc$62f@mserv1.dl.ac.uk> writes:"I intend to purify a protein with an immunoaffinity column. Current protocols in MB states that the ratio ab/activated sepharose in the column should be 2 mg/ml. To me seems such an exaggerated quantity of antibody that I suspect a typing mistake, but I am not so sure of myself. ... " My experience is: 2 mg affinity ligands (AL) per ml CNBr activated sepharose 4B is a minimum amount for coupling, 5-10 mg /ml is even better. After coupling your antibody to the beads/matrix, you will have to block excess active groups on the beads by other amino group containing chemicals (OC), such as ethanolamine, amino acids, Tris, etc. but not proteins! More AL in the coupling, higher proportion of AL to OC on the matrix, which would give you a better result when purifying your protein (an antigen). Other factors, e.g. matrix type, coupling buffer, ratio of buffer:gel, pH and temperature also affect coupling. Best wishes. D.H.W. From owner-proteins@net.bio.net Thu Aug 11 23:00:00 1994 Path: biosci!BANGOR.AC.UK!BSS169 From: BSS169@BANGOR.AC.UK Newsgroups: bionet.molbio.proteins Subject: (none) Date: 12 Aug 1994 07:33:06 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 15 Sender: daemon@net.bio.net Distribution: world Message-ID: <199408121433.HAA24570@net.bio.net> NNTP-Posting-Host: net.bio.net In article ID: <32f20a$1q5@gaia.ucs.orst.edu> writes: "We are radiolabelling proteins in plants and then extracting them in an aqueous extraction buffer. The samples are never dried. We are finding a large amount of radioactivity at the stacking gel separating gel interface. Ie whatever this is, enters the stacking gel, but not the separating gel. (SDS-PAGE) Any suggestions as to how to get this material to enter the gel?" Probably it was caused by problems in sample preparation. Add some Triton X-100 in extraction buffer, centrifuge at >10000 xg, and filter with glass-fibro not cellulose filter. Also check the pH and SDS concentration in the seperating and stacking gels. Best wishes. D.H.W From owner-proteins@net.bio.net Thu Aug 11 23:00:00 1994 Path: biosci!bloom-beacon.mit.edu!senator-bedfellow.mit.edu!cloutier From: cloutier@ATHENA.MIT.EDU (Normand J Cloutier) Newsgroups: bionet.molbio.proteins Subject: Re: Protein folding and AI Date: 12 Aug 1994 14:24:43 GMT Organization: Massachusetts Institute of Technology Lines: 19 Sender: cloutier@mit.edu Message-ID: <32g0nb$3ph@senator-bedfellow.MIT.EDU> NNTP-Posting-Host: bolognese.mit.edu In article jm@AMBIDENT.DEMON.CO.UK (Jonathan Montagu) writes: >Rafael > >Many thanks for the reference. This, and another list will provide lots >of papers. > >Jonathan Montagu I must have missed the posting with this apparently useful reference. Can someone please repost this. Thanks Normand Cloutier (cloutier@mit.edu) From owner-proteins@net.bio.net Thu Aug 11 23:00:00 1994 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!network.ucsd.edu!mskidmor.extern.ucsd.edu!user From: mskidmore@ucsd.edu (Michael Skidmore) Newsgroups: bionet.molbio.proteins Subject: Multiphor II Reswelling Cassette Followup-To: bionet.molbio.proteins Date: Thu, 11 Aug 1994 21:31:45 -0700 Organization: UCSD, Dept of Pathology Lines: 13 Message-ID: NNTP-Posting-Host: mskidmor.extern.ucsd.edu We just got a Pharmacia Mutiphor II and I am trying to run an Immobililine Dry Plate gel. I can't seem to set up the reswelling cassette with just half a gel. It leaks right where the plastic support is cut. The book says it's okay to just cut a gel in half and since they run around $115/ 3 gels, I would really like to do this. I tried using some really strong alligator clips but it still doesn't work. -- Michael SKidmore (619)534-1894 UCSD, Dept of Pathology mskidmore@ucsd.edu La Jolla CA 92093-0612 "Standard orbit, Mr. Sulu" From owner-proteins@net.bio.net Thu Aug 11 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!EU.net!sun4nl!news.nic.surfnet.nl!highway.LeidenUniv.nl!rulsfb.LeidenUniv.nl!SBTNFH From: sbtnfh@rulsfb.LeidenUniv.nl (Flip Hoedemaeker, CvF RUL/TNO, Leiden, The Netherlands) Newsgroups: bionet.molbio.proteins Subject: re:running gel Date: 12 Aug 1994 09:59:15 GMT Organization: Biology, Leiden University, The Netherlands Lines: 14 Message-ID: <32fh5j$cb1@highway.LeidenUniv.nl> Reply-To: sbtnfh@rulsfb.LeidenUniv.nl NNTP-Posting-Host: rulsfb.leidenuniv.nl In article <32f20a$1q5@gaia.ucs.orst.edu>, navarrer@ava.bcc.orst.edu (Roy Navarre) writes: >Any suggestions as to how to get this material to enter the gel? > >Thanks a lot > >Roy If your radiolabeled proteins are aroud 100 kD and op top of that also heavily glycosylated, they will not go into your running gel whatever you do! Maybe you should do some gelfiltration analysis first. Flip From owner-proteins@net.bio.net Thu Aug 11 23:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!swrinde!pipex!lyra.csx.cam.ac.uk!doc.ic.ac.uk!uknet!liv!news From: agmclen@liv.ac.uk Subject: SDS removal Message-ID: Sender: news@liverpool.ac.uk (News System) Nntp-Posting-Host: 138.253.30.13 Organization: Liverpool University, Biochemistry Dept. Date: Wed, 10 Aug 1994 07:54:26 GMT Lines: 5 I am sure this must be an FAQ, but does anyone have an opinion on the best way to remove unbound and bound SDS from proteins recovered from a preparative SDS polyacrylamide gel that is mild enough so that significant renaturation and recovery of enzyme activity can ultimately be achieved? Thanks From owner-proteins@net.bio.net Thu Aug 11 23:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!bcm!cs.utexas.edu!sdd.hp.com!saimiri.primate.wisc.edu!news.doit.wisc.edu!psl.wisc.edu!psl.wisc.edu!MCMAHAN From: mcmahan@psl.wisc.edu Subject: protein-protein interaction Message-ID: <1994Aug12.213555.11273@pslu1.psl.wisc.edu> Sender: news@pslu1.psl.wisc.edu (USENET News System) Reply-To: mcmahan@psl.wisc.edu Organization: University of Wisconsin - Physical Science Lab Date: Fri, 12 Aug 94 21:35:55 GMT Lines: 8 I'm thinking about doing some binding studies with peptides. I was wondering, if the computer-predicted structure of the region in question is a alpha helix, how many turns should be maintained? Can the binding still occur if the full length protein predicts a 4 turn helix but the peptide is predicted to be only 2 turns? Please email your reponses to mcmahan@oncology.wisc.edu. Thank you. Scott McMahan From owner-proteins@net.bio.net Fri Aug 12 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!vixen.cso.uiuc.edu!newsfeed.ksu.ksu.edu!moe.ksu.ksu.edu!crcnis1.unl.edu!unlinfo.unl.edu!markwell From: markwell@unlinfo.unl.edu (john markwell) Newsgroups: bionet.molbio.proteins Subject: Re: Protein accumulating at stacking gel interface Date: 12 Aug 1994 12:39:26 GMT Organization: University of Nebraska--Lincoln Lines: 36 Distribution: world Message-ID: <32fqhu$j7a@crcnis1.unl.edu> References: <32f20a$1q5@gaia.ucs.orst.edu> NNTP-Posting-Host: unlinfo.unl.edu navarrer@ava.bcc.orst.edu (Roy Navarre) writes: >We are radiolabelling proteins in plants and then extracting >them in an aqueous extraction buffer. The samples are never dried. >We are finding a large amount of radioactivity at the stacking gel >separating gel interface. Ie whatever this is, enters the stacking >gel, but not the separating gel. (SDS-PAGE) >Any suggestions as to how to get this material to enter the gel? >Thanks a lot >Roy Roy, This is fairly commonly observed with chloroplast preparations. What we (and other groups) have found is that there are three things which seem to help. 1. Include a fairly high concentration of dithiothreitol in the denaturing solution rather than 2-mercaptoethanol. I just put some DTT on the end of a spatula and add it to the sample in a microfuge tube with the denaturing solution. 2. Don't boil the samples. We heat at about 80 C for 30 to 60 min. 3. Including urea (at least 4 M) in the gel. I hope this helps solve your problem. John -- John Markwell Phone: 402-472-2924 Dept. Biochemistry FAX: 402-472-7842 University of Nebraska Internet: markwell@unl.edu Lincoln, NE 68583-0718 From owner-proteins@net.bio.net Fri Aug 12 23:00:00 1994 Path: biosci!agate!cat.cis.Brown.EDU!rodday.biomed.brown.edu!Suzanne_Rodday From: Suzanne_Rodday@brown.edu (Suzanne Rodday ) Newsgroups: bionet.molbio.proteins Subject: Re: Removing SDS Date: Sat, 13 Aug 1994 10:26:57 Organization: Brown University Lines: 6 Message-ID: NNTP-Posting-Host: rodday.biomed.brown.edu You might try Extracti-Gel D Columns for removing detergents. They are made by Pierce (800-874-3723 USA; 31.1860.19277 Europe). They work great for removing Tx-100 and other detergents! Suzanne Rodday Brown University From owner-proteins@net.bio.net Fri Aug 12 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!pipex!sunic!columba.udac.uu.se!rigel.bmc.uu.se!gerard From: gerard@rigel.bmc.uu.se (Gerard Kleijwegt) Newsgroups: bionet.molbio.proteins Subject: Re: WHAT IF Date: 13 Aug 1994 15:31:22 GMT Organization: Uppsala University Lines: 13 Distribution: world Message-ID: <32ip0a$1chl@columba.udac.uu.se> References: <32hmfp$sip@dingo.cc.uq.oz.au> NNTP-Posting-Host: rigel.bmc.uu.se In article <32hmfp$sip@dingo.cc.uq.oz.au>, duncan cochran writes: |> Does anyone use WHAT IF by Gert Vriend at EMBL? Can I ask you a few |> questions for example: where do you get it ? Please reply to |> cochran@biosci.uq.oz.au. |> Thanks Duncan Gert Vriend's E-mail address is: Gert.Vriend@EMBL-Heidelberg.DE I'm sure he'll tell you all about it ! --gerard From owner-proteins@net.bio.net Fri Aug 12 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!EU.net!Germany.EU.net!netmbx.de!zib-berlin.de!informatik.tu-muenchen.de!lrz-muenchen.de!ipp-garching.mpg.de!genmac7.biochem.mpg.de!user From: spang@vms.biochem.mpg.de (Anne Spang) Newsgroups: bionet.molbio.proteins Subject: Re: SDS removal Date: 13 Aug 1994 12:46:35 GMT Organization: Rechenzentrum der Max-Planck-Gesellschaft in Garching Lines: 13 Message-ID: References: NNTP-Posting-Host: genmac7.biochem.mpg.de I have had some good experiences with dialysis or using Centricon Concentrators to change the buffers and to remove SDS. Hope it helps! Anne In article , agmclen@liv.ac.uk wrote: > I am sure this must be an FAQ, but does anyone have an opinion on the best way > to remove unbound and bound SDS from proteins recovered from a preparative SDS > polyacrylamide gel that is mild enough so that significant renaturation and > recovery of enzyme activity can ultimately be achieved? > Thanks From owner-proteins@net.bio.net Fri Aug 12 23:00:00 1994 Path: biosci!agate!ames!lll-winken.llnl.gov!fnnews.fnal.gov!nntp-server.caltech.edu!seqvax.caltech.edu!jaime From: jaime@seqvax.caltech.edu (Jaime Arenas) Newsgroups: bionet.molbio.proteins Subject: Re: Protein accumulating at stacking gel interface Followup-To: bionet.molbio.proteins Date: 13 Aug 1994 18:22 PST Organization: Division of Biolgy, CALTECH Lines: 39 Distribution: usa Message-ID: <13AUG199418220952@seqvax.caltech.edu> References: <32f20a$1q5@gaia.ucs.orst.edu> NNTP-Posting-Host: seqvax.bio.caltech.edu News-Software: VAX/VMS VNEWS 1.41 In article , mskidmore@ucsd.edu (Michael Skidmore) writes... >In article <32f20a$1q5@gaia.ucs.orst.edu>, navarrer@ava.bcc.orst.edu (Roy >Navarre) wrote: > >> We are radiolabelling proteins in plants and then extracting >> them in an aqueous extraction buffer. The samples are never dried. >> We are finding a large amount of radioactivity at the stacking gel >> separating gel interface. Ie whatever this is, enters the stacking >> gel, but not the separating gel. (SDS-PAGE) >> Any suggestions as to how to get this material to enter the gel? > >Tried using a gel with no stacking at all? We get nearly the same separtion >using Novex precast 12% tris-glycine gels that we get with the regular >Lammeli gel. Difference is that they don't have a pH difference in the >stacking gel. There is just a portion of low% acrylamide that focuses, sort >of like a short gradient gel. You don't have to use one of their rigs >either. We run them in BRL Mini V8 rigs. >- >Michael SKidmore (619)534-1894 >UCSD, Dept of Pathology mskidmore@ucsd.edu >La Jolla CA 92093-0612 > >"Standard orbit, Mr. Sulu" The idea suggested above may just work. There is many reasons why the proteins will get stuck in the interface. A common reason for people that uses butanol to overlay their separating when polymerizing is that it is very hard to get all of it out before you pour the stacking gel. Apparently the butanol resi- dues cause some proteins to precipitate and thats the end of the story. To avoid that I always overlay my gels with water. It works just as well Good luck Jaime Arenas Division of Biology CALTECH From owner-proteins@net.bio.net Fri Aug 12 23:00:00 1994 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!network.ucsd.edu!mskidmor.extern.ucsd.edu!user From: mskidmore@ucsd.edu (Michael Skidmore) Newsgroups: bionet.molbio.proteins Subject: Re: Protein accumulating at stacking gel interface Followup-To: bionet.molbio.proteins Date: Sat, 13 Aug 1994 11:39:02 -0700 Organization: UCSD, Dept of Pathology Lines: 22 Message-ID: References: <32f20a$1q5@gaia.ucs.orst.edu> NNTP-Posting-Host: mskidmor.extern.ucsd.edu In article <32f20a$1q5@gaia.ucs.orst.edu>, navarrer@ava.bcc.orst.edu (Roy Navarre) wrote: > We are radiolabelling proteins in plants and then extracting > them in an aqueous extraction buffer. The samples are never dried. > We are finding a large amount of radioactivity at the stacking gel > separating gel interface. Ie whatever this is, enters the stacking > gel, but not the separating gel. (SDS-PAGE) > Any suggestions as to how to get this material to enter the gel? Tried using a gel with no stacking at all? We get nearly the same separtion using Novex precast 12% tris-glycine gels that we get with the regular Lammeli gel. Difference is that they don't have a pH difference in the stacking gel. There is just a portion of low% acrylamide that focuses, sort of like a short gradient gel. You don't have to use one of their rigs either. We run them in BRL Mini V8 rigs. - Michael SKidmore (619)534-1894 UCSD, Dept of Pathology mskidmore@ucsd.edu La Jolla CA 92093-0612 "Standard orbit, Mr. Sulu" From owner-proteins@net.bio.net Sat Aug 13 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!wupost!golf!mizzou1.missouri.edu!DCRCEP From: DCRCEP@mizzou1.missouri.edu (Elmer M. Price) Newsgroups: bionet.molbio.proteins Subject: Re: Protein accumulating at stacking gel interface Date: Sat, 13 Aug 94 20:02:23 CDT Organization: University of Missouri, Columbia Lines: 37 Message-ID: <17011119D6S86.DCRCEP@mizzou1.missouri.edu> References: <32f20a$1q5@gaia.ucs.orst.edu> NNTP-Posting-Host: mizzou1.missouri.edu In article <32f20a$1q5@gaia.ucs.orst.edu> navarrer@ava.bcc.orst.edu (Roy Navarre) writes: > >We are radiolabelling proteins in plants and then extracting >them in an aqueous extraction buffer. The samples are never dried. >We are finding a large amount of radioactivity at the stacking gel >separating gel interface. Ie whatever this is, enters the stacking >gel, but not the separating gel. (SDS-PAGE) >Any suggestions as to how to get this material to enter the gel? > >Thanks a lot > >Roy Hi Roy Just my two cents worth Use a SDS-PAGE sample buffer with 6M urea, 2% SDS and 150mM DTT (plus the usual 60 some odd mM Tris, pH 6.8) Do not heat AT ALL, just sit on the bench for 30-60 minutes with occasional vortexing. Hope this helps Elmer From owner-proteins@net.bio.net Sun Aug 14 23:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!agate!howland.reston.ans.net!EU.net!sunic!news.lth.se!news.lu.se!q700.plantbio.lu.se!Lars.S From: RoundUp Subject: Re: Multiphor II Reswelling Cassette Message-ID: <1994Aug15.103818.22844@nomina.lu.se> X-Xxmessage-Id: X-Xxdate: Mon, 15 Aug 1994 10:32:57 GMT Sender: news@nomina.lu.se (USENET News System) Nntp-Posting-Host: q700.plantbio.lu.se Organization: Plant biochemistry U of Lund X-Newsreader: Nuntius Version 1.2 References: Date: Mon, 15 Aug 1994 10:38:18 GMT Lines: 22 In article Michael Skidmore, mskidmore@ucsd.edu writes: >We just got a Pharmacia Mutiphor II and I am trying to run an Immobililine >Dry Plate gel. I can't seem to set up the reswelling cassette with just >half a gel. It leaks right where the plastic support is cut. The book says >it's okay to just cut a gel in half and since they run around $115/ 3 gels, >I would really like to do this. I tried using some really strong alligator >clips but it still doesn't work. I use the imobilone strips for IEF and use a standard glass test tube, siliconized, for reswelling and it works OK. I would suggest you try a siliconized glass tray instead of the cassette. Just be sure to add enough reswelling solution to cover the gel and cover the ray with some wrapping. Lars.S@Plantbio.lu.se, Plant biochemistry, Lund University _________________________________________________________________ From owner-proteins@net.bio.net Mon Aug 15 23:00:00 1994 Path: biosci!CV3.CHEM.PURDUE.EDU!GREGC From: GREGC@CV3.CHEM.PURDUE.EDU Newsgroups: bionet.molbio.proteins Subject: Re: SDS removal Date: 16 Aug 1994 19:34:45 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 6 Sender: daemon@net.bio.net Distribution: world Message-ID: <940816213438.2021a260@CV3.CHEM.PURDUE.EDU> NNTP-Posting-Host: net.bio.net Hi, Regarding SDS removal from protein solutions... Try Pierce "Extracti- Gel" columns. They are size-exclusion gels with an affinity for some detergents, but are limited to perhaps 50 mg or so. Good luck! Greg From owner-proteins@net.bio.net Mon Aug 15 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!gatech!newsxfer.itd.umich.edu!zip.eecs.umich.edu!panix!cmcl2!is.NYU.EDU!beavis From: beavis@is.nyu.edu (beavis) Newsgroups: bionet.molbio.proteins Subject: Re: SDS removal Date: 16 Aug 1994 12:27:25 GMT Organization: New York University Lines: 43 Distribution: world Message-ID: <32qbbd$lkg@cmcl2.NYU.EDU> References: <199408160931.CAA27574@net.bio.net> NNTP-Posting-Host: is.nyu.edu X-Newsreader: TIN [version 1.2 PL2] BSS169@BANGOR.AC.UK wrote: : 1> : Message ID: says: : ************************************************************************** : "I am sure this must be an FAQ, but does anyone have an opinion on the best : way to remove unbound and bound SDS from proteins recovered from a : preparative SDS polyacrylamide gel that is mild enough so that significant : renaturation and recovery of enzyme activity can ultimately be achieved?" : ************************************************************************** etc ... : 6. Ion-pair extraction, by mixtures of organic chemicals, see ref. : W.H.Koigsberg et al. Methods in Enzymol. Vol.91, p.254. (claims : complete removal of SDS). I have had some experience removing SDS for mass spectrometry analysis of proteins. MS is very sensitive to SDS complexed with proteins, which supresses protein ionization. I have found that the method refered to above, which I have dubbed "the Konigsberg procedure" works very well with small amounts of protein. It involves mixing the following solvent: 5:5:5:85 water:acetic acid:triethylamine:acetone (v/v). Add an excess of the solvent to a dried sample of the protein and swirl it a bit on a vortex mixer. Centrifuge the tube and remove the solvent. If there is no obvious solid in the tube, cool the tube for a while and re-centrifuge. Wash the pellet with acetone several times and dry. I have had good success with picomole-sized samples in 1 ml Eppendorfs. I don't know if the protein was still active (although I doubt it), but it did give good MS signals. Acetone & TCA precipitation and dialysis do not dissociate protein:SDS complexes and only remove the free SDS present in a sample. Ion exchange chromatography can work very well, but reverse-phase HPLC can be a bit tricky (rHPLC doesn't much care for the presence of a lot of SDS). Just my few cents Ron Beavis Skirball Institute of Biomolecular Medicine New York University beavis@is.nyu.edu From owner-proteins@net.bio.net Mon Aug 15 23:00:00 1994 Path: biosci!BANGOR.AC.UK!BSS169 From: BSS169@BANGOR.AC.UK Newsgroups: bionet.molbio.proteins Subject: Re: SDS removal Date: 16 Aug 1994 02:31:39 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 45 Sender: daemon@net.bio.net Distribution: world Message-ID: <199408160931.CAA27574@net.bio.net> NNTP-Posting-Host: net.bio.net 1> Message ID: says: ************************************************************************** "I am sure this must be an FAQ, but does anyone have an opinion on the best way to remove unbound and bound SDS from proteins recovered from a preparative SDS polyacrylamide gel that is mild enough so that significant renaturation and recovery of enzyme activity can ultimately be achieved?" ************************************************************************** There are some methods to remove unbound and bound SDS: 1. Dialysis. 2. Precipitation, by TCA or aceton, but you may have to re-introduce some other detergents to re-solubilize the proteins. 3. Ultrafiltration, by membranes with certain cut-off pore size, but protein recovery is low. Try Amicon or Millipore. 4. Ion-exchange chromatography, by AG 1-X4, AG 1-X2 resins, try Bio Rad. 5. Ion-retardation chromatography, by AG 11 A8 resin, try Bio Rad. See ref. S.N.Vinogradov et al. (1983)? Methods in Enzymology, Vol. 91, p.258. 6. Ion-pair extraction, by mixtures of organic chemicals, see ref. W.H.Koigsberg et al. Methods in Enzymol. Vol.91, p.254. (claims complete removal of SDS). 7. Methods based on electrophoresis. Run high percentage (e.g.30%) non-SDS PAGE in big i.d. column, with frequent changes of anode buffer which collects SDS. After PAGE, resolubilize or electro-elute proteins from the gel. To remove unbound SDS from solution is quite easy, but to remove bound SDS from proteins completely is NOT. Significance of renaturation of proteins and recovery of enzyme activity varies from protein to protein, enzyme to enzyme, function to function (even by same protein/enzyme). Some are very sensitive to SDS, some are not, e.g. endoproteinase glu-C (from Staphyloccus aureus V8) is fully active in SDS (2 mg/ml) at 25 C, and mostly active in SDS (5 mg/ml) plus 6M urea at 0 C. Best wishes. Dan H. Wu School of Biol. Sci. Univ. College of N. Wales, Bangor, U.K. From owner-proteins@net.bio.net Mon Aug 15 23:00:00 1994 Path: biosci!agate!dog.ee.lbl.gov!news.cs.utah.edu!u.cc.utah.edu!ls-31.biology.utah.edu!user From: scottb@lsiris2.biology.utah.edu (Scott beeser) Newsgroups: bionet.molbio.proteins Subject: Oxford University press phone number ? Followup-To: bionet.molbio.proteins Date: 16 Aug 1994 20:19:23 GMT Organization: university of utah Lines: 10 Distribution: world Message-ID: NNTP-Posting-Host: ls-31.biology.utah.edu Hi, Sorry if this is mispalced. I bought a book from Oxford university press about a month ago at a conference and they said they would send it to me. It has been more than a month since i paid for the book and i haven't recieved it yet. I was wondering if anybody has their phone number so i can check out what the deal is. Email is probably prefered. scott beeser department of biology university of utah From owner-proteins@net.bio.net Mon Aug 15 23:00:00 1994 Path: biosci!daresbury!bioftp.unibas.ch!rc1.vub.ac.be!dec5!nismail From: nismail@dec5 (Naima Ismail) Newsgroups: bionet.molbio.proteins Subject: dephosphorilation Date: 16 Aug 1994 20:03:11 GMT Organization: Brussels Free Universities VUB/ULB Lines: 21 Message-ID: <32r61v$deo@rc1.vub.ac.be> NNTP-Posting-Host: dbmdec5.ulb.ac.be. X-Newsreader: TIN [version 1.2 PL0] hello everybody I ve been purifying a protein that is supposed to be phosphorilated on serine residues. I am getting the protein but to make sure it s the good one I'de like first to dephosphorilate it and see if I get the apropriate MW ( the MW of the phospho and dephosphorilated forms are known. Can anybody tell me what s the best way to do this . Many thanx B B A B B B B A C C known). Can anybody tell me what s the best way to do this From owner-proteins@net.bio.net Mon Aug 15 23:00:00 1994 Path: biosci!agate!ames!elroy.jpl.nasa.gov