From owner-proteins@net.bio.net Sat Apr 01 23:00:00 1995 Path: biosci!agate!library.ucla.edu!nnrp.info.ucla.edu!usenet From: arne@hodgkin.mbi.ucla.edu (Arne Elofsson) Newsgroups: bionet.molbio.proteins Subject: Re: accessible surface area Date: 02 Apr 1995 08:45:21 -0700 Organization: University of California, Los Angeles Lines: 16 Message-ID: References: <3ko04o$ltd@mark.ucdavis.edu> <1995Mar22.194113.4776@alw.nih.gov> <3lgju8$6ci@alpha.cisi.unige.it> NNTP-Posting-Host: hodgkin.mbi.ucla.edu In-reply-to: victor@sheila.ibf.unige.it's message of 31 Mar 1995 10:01:44 GMT X-Newsreader: (ding) Gnus v0.38 The fastest algorithm I have seen is by LeGrand & Merz, J 3. LEGRAND SM; MERZ KM. RAPID APPROXIMATION TO MOLECULAR SURFACE AREA VIA THE USE OF BOOLEAN LOGIC AND LOOK-UP TABLES. JOURNAL OF COMPUTATIONAL CHEMISTRY, 1993 MAR, V14 N3:349-352. arne -- -------------------------------------------------------- From: Arne Elofsson Email: arne@hodgkin.mbi.ucla.edu WWW: http://www.doe-mbi.ucla.edu/arne/main.html From owner-proteins@net.bio.net Sat Apr 01 23:00:00 1995 Path: biosci!bloom-beacon.mit.edu!spool.mu.edu!howland.reston.ans.net!Germany.EU.net!EU.net!uknet!doc.news.pipex.net!pipex!lyra.csx.cam.ac.uk!mole.bio.cam.ac.uk!smb18 From: smb18@mole.bio.cam.ac.uk (Simon Brocklehurst (Bioc)) Newsgroups: bionet.molbio.proteins Subject: Re: Homotrimer-ONE!!! binding site? Date: 3 Apr 1995 00:04:57 GMT Organization: University of Cambridge, England Lines: 26 Message-ID: <3lne39$qbv@lyra.csx.cam.ac.uk> References: NNTP-Posting-Host: mole.bio.cam.ac.uk aflaloc@BGUMAIL.BGU.AC.IL (Claude Aflalo) writes: >On Mon, 27 Mar 1995, Ralf Morgenstern wrote: >> Our binding studies indicate that microsomal glutathione transferase binds >> only one GSH per homotrimer. Are there known examples of homotrimers (or >> homooligomers) with one binding site only. I have a hard time envisioning >> this because of symmetry reasons (or lack of imagination). Perhaps three >> overlapping sites exist of which only one can be occupied at a time. Does >> anyone know of examples that describe this situation. >> -- >> Ralf Morgenstern, IMM, Karolinska Institutet, Box 210, S-17177, SWEDEN >> Tlf. +46-8-7287574, Fax, +46-8-334467, E-mail: ralf.morgenstern@imm.ki.se >> >> The binding site could simply lie on one end of the C3 symmetry axis (if the protein has such symmetry). _________________________________________________________________________ | | ,_ o Simon M. Brocklehurst, | / //\, Oxford Centre for Molecular Sciences | \>> | Department of Biochemistry, University of Oxford, | \\, Oxford, UK. | E-mail: smb@bioch.ox.ac.uk |________________________________________________________________________ From owner-proteins@net.bio.net Sun Apr 02 23:00:00 1995 Path: biosci!agate!spool.mu.edu!uwm.edu!cs.utexas.edu!swrinde!pipex!uknet!daresbury!dlpx1!asm From: asm@dlpx1.dl.ac.uk (A.S.McAlpine) Newsgroups: bionet.molbio.proteins Subject: udp-glucose aff chromotography Date: 3 Apr 1995 15:35:38 GMT Organization: SERC Daresbury Lab, Warrington, U.K. Lines: 22 Sender: asm@dlpx1 (A.S.McAlpine) Distribution: world Message-ID: <3lp4ka$kq8@mserv1.dl.ac.uk> NNTP-Posting-Host: dlpx1.dl.ac.uk Hi, I am looking for a method of coupling UDP-glucose to an affinity resin. Does anybody have experience with purifying a protein by using immobilized UDP glucose??? Any information would be helpful. \\|// (o o) --------------------------ooO---(_)---Ooo-------------------------- Protein Crystallography ----------------------- Alan McAlpine | SRR Division Daresbury Laboratories | Daresbury | Warrington WA4 4AD | Tel: 01925 603606 England | Fax: 01925 603124 UK | E-Mail: a.s.mcalpine@dl.ac.uk ------------------------------------------------------------------- From owner-proteins@net.bio.net Sun Apr 02 23:00:00 1995 Path: biosci!bloom-beacon.mit.edu!usc!cs.utexas.edu!swrinde!pipex!oleane!jussieu.fr!u-psud.fr!zaphod.crihan.fr!BioE8.univ-rouen.fr!tberthe From: tberthe@gandalf.crihan.fr (Thierry Berthe) Newsgroups: bionet.molbio.proteins Subject: Elution of proteins from polyacrylamide gel Date: Mon, 3 Apr 1995 09:17:03 Organization: crihan, france Lines: 1 Message-ID: NNTP-Posting-Host: 193.52.150.138 X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] What is the best way to elute proteins from polyacrylamide gel ???? From owner-proteins@net.bio.net Sun Apr 02 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!agate!howland.reston.ans.net!pipex!uknet!bcc.ac.uk!mac-126.ucl-32.bcc.ac.uk!user From: zcbcl02@ucl.ac.uk (Adam Assem Rostom) Subject: MONOCLONAL ANTIBODIES Sender: news@ucl.ac.uk (Usenet News System) Message-ID: Date: Mon, 3 Apr 1995 10:50:56 GMT Organization: University College London Lines: 11 WIZARDS! I am a student doing a project on monoclonal antibodies and antibody engineering. I was wondering if any of you brain boxes know of any good papers on antibody protein engineering to start from. Thanks ADAM From owner-proteins@net.bio.net Sun Apr 02 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!pipex!oleane!jussieu.fr!univ-lyon1.fr!swidir.switch.ch!scsing.switch.ch!rzusuntk.unizh.ch!NewsWatcher!user From: maga@vetbio.unizh.ch (Giovanni Maga) Subject: D2O exchange for proteins Message-ID: Followup-To: bionet.molbio.proteins Sender: newsadm@rzu-news.unizh.ch (CNEWS ADMINISTRATION) Organization: University of Zurich Irchel- Biochemistry Date: Mon, 3 Apr 1995 21:14:38 GMT Lines: 20 Dear bionetters, we want to begin to study a small protein (30kD, pI around 5) by neutron scattering. We can purify such protein in mg amounts at homogenity, However, we need to provide it in D2O rather than that in H2O for neutron scattering analysis. We tried once by liophylizing our protein and then resuspending it in phosphate buffer at pH 6 in D2O, but we had serious troubles in getting it soluble again. Does anybody know a good method to exchange H2O buffers with D2O buffers? Is the pH important in this reaction (well, the pD in this case)? Any buffering system will work or some is preferred? I tried to get advice by reading a couple of issues of Methods in Enzymology which were dealing with nmr studies, but I didn't find an answer to my (basic, I know) questions. Any help will be greatly appreciated. Answer here or e-mail to: maga@vetbio.unizh.ch G.Maga,PhD Institute of Veterinary Biochemistry University of Zuerich-CH From owner-proteins@net.bio.net Sun Apr 02 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!pipex!sunsite.doc.ic.ac.uk!news.cc.ic.ac.uk!usenet From: "M.P.Woodland" Newsgroups: bionet.molbio.proteins Subject: Re: Elution of proteins from polyacrylamide gel Date: 3 Apr 1995 15:39:48 GMT Organization: Imperial College, London, UK Lines: 12 Message-ID: <3lp4s5$pch@oban.cc.ic.ac.uk> References: NNTP-Posting-Host: mwpc1.bc.ic.ac.uk tberthe@gandalf.crihan.fr (Thierry Berthe) wrote: > > What is the best way to elute proteins from polyacrylamide gel ???? Try the Bio-Rad 442 electroeluter. With this you can elute excised bands into 600ul.It can be used with native or SDS-PAGE, The protein bands may be stained and fixed or unfixed. In theory it is possible to electrodialyse SDS from the protein - in practise I have had less success with this aspect. m.p.woodland @ic.ac.uk From owner-proteins@net.bio.net Sun Apr 02 23:00:00 1995 Path: biosci!agate!howland.reston.ans.net!swrinde!pipex!news.maz.net!news.omnilink.net!news.gni.net!lemon!itc-biotech.com!thoenes Date: 21 Mar 1995 13:41:00 +0100 From: thoenes@itc-biotech.com (thoenes) Newsgroups: bionet.molbio.proteins Message-ID: <5iGOH762iEB@itc-biotech.com> Subject: GFP: What is Coelenterazine X-Newsreader: XP v3.02 X-Charset: ISO-8859-1 Lines: 20 ## Nachricht vom 21.03.95 weitergeleitet ## Ursprung : /bionet/molbio/methds-reagnts ## Ersteller: thoenes@itc-biotech.com Dear Bionetters, During my literture studies about the "Green Fluorescent Protein" I found a component named "coelenterazine" (Inouye and Tsuji, FEBS Letters 351 (1994) 211-214) as the organic substrate for aequorin. During the reaction it is oxidized to coelenteramide. Is there anybody out there who can tell me the chemical formula and structure as well as some details about coelenterazine? In some ealder papers one also can find "coelenterates". I never hears about this word. Is this a certain sort of organism ? Thanks for any kind of answer U. Thoenes ## CrossPoint v3.02 ## From owner-proteins@net.bio.net Sun Apr 02 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!bloom-beacon.mit.edu!gatech!howland.reston.ans.net!paladin.american.edu!newsfeed.ACO.net!edvz.sbg.ac.at!wst!floeckn From: floeckn@wst.edvz.sbg.ac.at (Floeckner Hannes) Subject: Protein Fold Identification Tool Message-ID: Lines: 33 Sender: floeckn@wst (Floeckner Hannes) Reply-To: floeckn@wst.edvz.sbg.ac.at (Floeckner Hannes) Organization: University of Salzburg / Austria Date: Mon, 3 Apr 1995 16:03:05 GMT ______________________________________________ | | | ProFIT - Protein Fold Identification Tool | |______________________________________________| Version 0.9 Beta for SGI and DEC ALPHA Workstations ProFIT (Protein Fold Identification Tool) combines an aminoacid sequence with a database of 3D structures, and has the potential to detect a fold related to the native structure of the input sequence. This is a beta-release. It is still experimental and will change during the following months. For a description of the program, the approaches used, the expected success rate and the quality of information retrieved, please consult the accompanying manuscript. ProFIT V0.9 Beta is freely available for academic users. You can down-load ProFIT V0.9 Beta by anonymous ftp from Gundi.came.sbg.ac.at (141.201.27.11) Licenses are distributed on a one machine one license basis. A license is valid for one year. Licenses are free of charge for academic users. For commercial licenses please contact: Manfred.J.Sippl Center Of Applied Molecular Engineering Jakob-Haringer Str. 1 A-5020 Salzburg / AUSTRIA E-Mail: sippl@olga.came.sbg.ac.at From owner-proteins@net.bio.net Sun Apr 02 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!usc!elroy.jpl.nasa.gov!lll-winken.llnl.gov!overload.lbl.gov!ames!waikato!waikato.ac.nz!mbdinger From: mbdinger@waikato.ac.nz Newsgroups: bionet.molbio.proteins Subject: Help with Protein Introns Message-ID: <1995Apr4.161608.37742@waikato.ac.nz> Date: 4 Apr 95 16:16:08 +1300 Organization: University of Waikato, Hamilton, New Zealand Lines: 14 Hello, I'm currently researching "Protein Introns", and am in need of information on this subject. If anybody is involved in this field could provide any information, or knows of references that I can look up, it would be greatly appreciated. Abstract searches on the libraries CD-ROMs revealed nothing. Thanks. Any replies to: mbdinger@waikato.ac.nz. From owner-proteins@net.bio.net Sun Apr 02 23:00:00 1995 Path: biosci!uwichill.edu.bb!aalleyne From: aalleyne@uwichill.edu.bb (aalleyne) Newsgroups: bionet.molbio.proteins Subject: re.glycoproteins Date: 3 Apr 1995 20:34:14 -0700 Organization: UWI, Cave Hill Lines: 12 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Hello All Netters, I have isolated a compound from culture filtrates of Collettotriichum gloeosporioides from yam Dioscorea alata cv White Lisbon. This substance has putataive toxin activity. However I have not fuly elucidated its structure. The question is this. Does anyone out there know of an enzyme that can be used to cleave the bond between a protein and a carbohydrate?? I am desperate for some advice on this problem. Angela From owner-proteins@net.bio.net Sun Apr 02 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!vixen.cso.uiuc.edu!uwm.edu!psuvax1!news.cc.swarthmore.edu!netnews.upenn.edu!cronkite.ocis.temple.edu!astro.ocis.temple.edu!driska From: driska@astro.ocis.temple.edu (Stephen P. Driska PhD) Newsgroups: bionet.molbio.proteins Subject: Re: Elution of proteins from polyacrylamide gel Date: 3 Apr 1995 22:48:57 GMT Organization: Temple University, Academic Computer Services Lines: 17 Message-ID: <3lpu0p$n60@cronkite.ocis.temple.edu> References: NNTP-Posting-Host: astro.ocis.temple.edu X-Newsreader: TIN [version 1.2 PL2] Thierry Berthe (tberthe@gandalf.crihan.fr) wrote: : What is the best way to elute proteins from polyacrylamide gel ???? The Elutrap, sold by Schleicher & Schuell in the USA works for us. I think it's a German product, so maybe it's sold by someone else in France. We use an ammonium bicarbonate/SDS/dithiothreitol buffer. (It's something that you run in a horizontal electrophoresis unit.) It works with coomassie-blue stained gels; the stain remains in the trap with the protein. If you vacuum-dry the protein solution, the coomassie-blue, dithiothreitol, and any ammonium bicarbonate that remains can be dissolved in 70% ethanol, leaving behind the protein. If you want details, let me know. Steve Driska From owner-proteins@net.bio.net Sun Apr 02 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!vixen.cso.uiuc.edu!newsrelay.iastate.edu!newsxfer.itd.umich.edu!news1.oakland.edu!cms.cc.wayne.edu!gkrause From: gkrause@cms.cc.wayne.edu (Gary Krause) Newsgroups: bionet.molbio.proteins Subject: Need Serine Kinase inhibitor Date: Mon, 3 Apr 1995 14:39:44 est Organization: Wayne State University Lines: 11 Message-ID: NNTP-Posting-Host: 146.9.12.243 X-Newsreader: Trumpet for Windows [Version 1.0 Rev B final beta #1] I am trying to isolate the serine kinase that phosphorylates eIF-2 in the brain. I am in need of a general inhibitor of serine kinases. I have tried serine with minimal success. Any suggestions would be welcome. Thanks Gary ------------------------------------------- Gary Krause (gkrause@cms.cc.wayne.edu) Department of Emergency Medicine Wayne State University, Detroit, MI USA From owner-proteins@net.bio.net Sun Apr 02 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!ns1.faseb.org!darwin.sura.net!nih-csl!loglady.ninds.nih.gov!johnk From: johnk@spasm.niddk.nih.gov (John Kuszewski) Subject: Re: D2O exchange for proteins Message-ID: <1995Apr3.190102.15564@alw.nih.gov> Sender: postman@alw.nih.gov (AMDS Postmaster) Nntp-Posting-Host: spasm.niddk.nih.gov Reply-To: johnk@spasm.niddk.nih.gov (John Kuszewski) Organization: National Insts. of Health References: Date: Mon, 3 Apr 1995 19:01:02 GMT Lines: 58 In article , maga@vetbio.unizh.ch (Giovanni Maga) writes: |> |> Dear bionetters, |> we want to begin to study a small protein (30kD, pI around |> 5) by neutron scattering. We can purify such protein in mg amounts at |> homogenity, However, we need to provide it in D2O rather than that in H2O |> for neutron scattering analysis. We tried once by liophylizing our protein |> and then resuspending it in phosphate buffer at pH 6 in D2O, but we had |> serious troubles in getting it soluble again. Does anybody know a good |> method to exchange H2O buffers with D2O buffers? Is the pH important in |> this reaction (well, the pD in this case)? Any buffering system will work |> or some is preferred? I tried to get advice by reading a couple of issues |> of Methods in Enzymology which were dealing with nmr studies, but I didn't |> find an answer to my (basic, I know) questions. I'm not positive what you're asking here, so I'll give two separate answers. If you're just looking to get the protein into D2O, then all you need to do is use a Centricon to wash in D2O to your heart's content. If you want to exchange protein protons for deuterons, then read on. As anyone who's done H/D exchange studies of protein folding could tell you, the exchange rate of amide protons is pH-dependent. The reaction is both acid and base catalyzed, with a minimum rate around pH 3-4 and maximal rates at very acid or basic pHs. The rate increases linearly with [H+] or [-OH]. If your protein doesn't tolerate extremes of pH, you could add some small amount of denaturant (say, 1-2 M gdmCl) to help move things along. Phosphate buffers, in my hands, tend to do strange things, so you can use acetate (at low pH) or glycine (at high pH). You realize, of course, that this will only replace a subset of the protein protons with deuterons. Conditions that lead to exchange of non-exchangable (ie., alpha, aliphatic, or aromatic) protons tend to do nasty things to proteins. If you want 100% of the protein protons to be deuterons, you'll have to do the expression in D2O (ie., mucho dinero). Hope this helps. -- _____________ | ___/_ | |/ / -- /\ // /-- || || / /|| || || / / || || ||/ / || John Kuszewski || |/ /| || johnk@spasm.niddk.nih.gov || / /|| || \/ / / || \/ that's MISTER protein G to you! |/__/| | /_________| From owner-proteins@net.bio.net Sun Apr 02 23:00:00 1995 Path: biosci!adam.cc.sunysb.edu!news.sprintlink.net!uunet!gumby!newsxfer.itd.umich.edu!news.itd.umich.edu!usenet From: @ Newsgroups: bionet.molbio.proteins Subject: Re: Cyanogen Bromide Digest Question Date: 3 Apr 1995 18:47:29 GMT Organization: University of Michigan Lines: 25 Message-ID: <3lpfs1$ibr@lastactionhero.rs.itd.umich.edu> References: <3lh6k0$ivj@dove.nist.gov> Reply-To: @ NNTP-Posting-Host: 50.33.med.umich.edu X-Newsreader: IBM NewsReader/2 v1.03 David Here at the facility we routinely perform this digestion doing the following: Stock soltn 10 mg/ml CNBr in 70% formic add 10 ul stock/10ug protein. Incubate @ room temp in dark overnight. Top off with 10X water and speed vac, I usually wash in 2x. If internal sequence is what your after than I would not reccomend purification by RP-HPLC, as the newly acquired formyl grps make for a broad and very messy profile. Instead, I add tricine sample buffer, with 2 ul neat 4 VP, and incubate for 1-2 hrs at room temp, load to tricine 16% gel(Novex) and blot to PVDF. Alternatively, you could reduce/alk pror to digestion, and a good protocol would be S-pyridylethylation of Cystine Residues, Applied Biosystem no.28, Hawke and Yuan. I use it exclusively with slight modification. If this is a MALDI experiment, where mass is that you need, then you may want to use TFA instead of the formic as the formylation does not work very well. Should you need any additional protocols or hard copies for any of the above feel free to contact me. Contrary to what most people say, CNBr is one of my first choices when contemplating the frgmentating of a protein. Jake Tropea tropea@brcf.med.umich.edu 313-763-6710 Protein and Carbohydrate Structure Facility Univ of Michigan From owner-proteins@net.bio.net Sun Apr 02 23:00:00 1995 Path: biosci!ns1.faseb.org!darwin.sura.net!blaze.cs.jhu.edu!jhunix1.hcf.jhu.edu!welchlink.welch.jhu.edu!dshumake From: dshumake@welchlink.welch.jhu.edu (DALE KENDRICK SHUMAKER) Newsgroups: bionet.molbio.proteins,bionet.cellbiol Subject: PKC inhibitor chelerythrine chloride Date: 3 Apr 1995 18:32:39 GMT Organization: Johns Hopkins University, Welch Medical Library Lines: 23 Distribution: world Message-ID: <3lpf07$p87@jhunix1.hcf.jhu.edu> NNTP-Posting-Host: 128.220.59.78 Xref: biosci bionet.molbio.proteins:4162 bionet.cellbiol:1985 I have been using the PKC inhibitor chelerythrine chloride recently, as well as two other PKC inhbitors which bind to the DAG regulatory site. Chelerythrine inhibits PKC by binding to the active site. Recently it came to my attention that PKC can also be activated by arachidonic acid (AA). I have the following questions: Does chelerythrine inhibit all PKC isoforms? Are there specific PKCs that can be activated only by either AA or DAG; or are PKC isoforms activated by both AA and DAG? If there are specific PKC isoforms which are only activated by AA or DAG does chelerythrine only inhibit one set of isoforms, or does chelerythrine inhibit both set of PKCs? All and any information that is known about this will be appreciated. I have not had any luck in finding out the answers to my questions through literature searches on Medline. Thank you for your help and advice. Dale From owner-proteins@net.bio.net Sun Apr 02 23:00:00 1995 Path: biosci!ns1.faseb.org!darwin.sura.net!blaze.cs.jhu.edu!jhunix1.hcf.jhu.edu!welchlink.welch.jhu.edu!dshumake From: dshumake@welchlink.welch.jhu.edu (DALE KENDRICK SHUMAKER) Newsgroups: bionet.molbio.proteins Subject: Re: Homotrimer-ONE!!! binding site? Date: 3 Apr 1995 18:17:49 GMT Organization: Johns Hopkins University, Welch Medical Library Lines: 57 Message-ID: <3lpe4d$np4@jhunix1.hcf.jhu.edu> References: <1995Mar31.202005.10900@alw.nih.gov> NNTP-Posting-Host: 128.220.59.78 In article <1995Mar31.202005.10900@alw.nih.gov>, John Kuszewski wrote: >In article , aflaloc@BGUMAIL.BGU.AC.IL (Claude Aflalo) writes: >|> On Mon, 27 Mar 1995, Ralf Morgenstern wrote: >|> >|> > Our binding studies indicate that microsomal glutathione transferase binds >|> > only one GSH per homotrimer. Are there known examples of homotrimers (or >|> > homooligomers) with one binding site only. I have a hard time envisioning >|> > this because of symmetry reasons (or lack of imagination). Perhaps three >|> > overlapping sites exist of which only one can be occupied at a time. Does >|> > anyone know of examples that describe this situation. >|> > -- >|> > Ralf Morgenstern, IMM, Karolinska Institutet, Box 210, S-17177, SWEDEN >|> > Tlf. +46-8-7287574, Fax, +46-8-334467, E-mail: ralf.morgenstern@imm.ki.se >|> > >|> > >|> Have a look at the "Alternating binding change" mechanism propose by Paul >|> D. Boyer in the 80's for the mechanism of ATP synthase (F0F1) of bacteria, >|> chloroplasts and mitochondria. The enzyme has 6 binding sites for >|> nucleotides, 3 of them act as alternating catalytic sites. The mechanism >|> is characterized by negative cooperativity in binding AND positive >|> cooperativity in catalysis. >|> >|> Claude Aflalo ########################################################### >|> Dept. of Life Sciences Phones: Office 972-7-472118 >|> Ben Gurion University of the Negev Lab 972-7-472119 >|> P.O.Box 653 Fax 972-7-276201 >|> Beer Sheva 84105 Israel email: aflaloc@bgumail.bgu.ac.il >|> > >Or have a look at the GroEL structure. It has 14 monomers, and each has a >binding site, but only one binding site per 14mer is active at any one time. > >-- > _____________ > | ___/_ > | |/ / > -- /\ // /-- > || || / /|| > || || / / || > || ||/ / || >John Kuszewski || |/ /| || >johnk@spasm.niddk.nih.gov || / /|| || > \/ / / || \/ >that's MISTER protein G to you! |/__/| | > /_________| I don't remember the exact protein/receptor, but there is a ligand gated ion channel which has four fold symmetry. Each subunit has a binding site but only one ligand is bound per tetramer. The talk I was at, where this was being discussed, indicated that the ligand sat down in the binding pocket formed by the tetramer, in one of four equally probable orientations. I will try and find out the researcher's name and the channel he is studying. Dale From owner-proteins@net.bio.net Sun Apr 02 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!vixen.cso.uiuc.edu!newsrelay.iastate.edu!newsxfer.itd.umich.edu!news.itd.umich.edu!usenet From: @ Newsgroups: bionet.molbio.proteins Subject: Re: C-terminal Sequencing? Date: 3 Apr 1995 17:37:37 GMT Organization: University of Michigan Lines: 22 Message-ID: <3lpbp2$ibr@lastactionhero.rs.itd.umich.edu> References: Reply-To: @ NNTP-Posting-Host: 50.33.med.umich.edu X-Newsreader: IBM NewsReader/2 v1.03 Hello Mandy Here at the Protein and Carbohydrate Structure Facility, Univ of Michigan, C-terminal sequence analysis is performed on a routine basis on a Hewlett Packard model 1009A. We routinely obtain a clear identification on at least two residues at the 0.1 to 1nmol level although results are dependent on sample preparation and properties of the protein. Some background peaks may increase by cycle #3. Sequence runs as long as 8 residues have been obtained for some proteins. Samples can be submitted either lyophilized or in solution, as PVDF is incompatible with this new chemistry. As further enhancements to the chemistry are developed, investigators will be apprised of improvements as they are implemented here at the facility, as we are working closely with the kind staff at HP. Should you have any additional questions I can be reached at the following: Jake Tropea PCSF Univ of Mich 2580 MSRB II 1150 West Medical Center Dr Ann Arbor MI 48109-0674 tropea@brcf.med.umich.edu (313)763-6710 From owner-proteins@net.bio.net Sun Apr 02 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!bcm!cs.utexas.edu!swrinde!pipex!sunic!sunic.sunet.se!news.lth.se!news.lu.se!stefanr.mbioekol.lu.se!user From: Stefan.Rosen@mbioekol.lu.se Subject: How to deglycosylate fetuin? Message-ID: Sender: news@nomina.lu.se (USENET News System) Nntp-Posting-Host: stefanr.mbioekol.lu.se Organization: Lund University Computing Center Date: Mon, 3 Apr 1995 16:46:08 GMT Lines: 14 Hello! I want to enzymatically pile off the O-linked sugars (totally) in fetuin (from fetal calf serum). Are there anyone that have own practical experience about this subject and knows which enzyme(s) that works best (company,no, extent of deglycosylation..) ?. Thank you, Stefan Rosén Department of Microbial Ecology Lund University Sweden E-mail: Stefan.Rosen@mbioekol.lu.se From owner-proteins@net.bio.net Sun Apr 02 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!torn!news.bc.net!news.mic.ucla.edu!library.ucla.edu!csulb.edu!csus.edu!news.ucdavis.edu!usenet From: dobates@ucdavis.edu (Dave Bates) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins,bionet.cellbiol Subject: CALL FOR VOTES for b.m.proteins.fluorescent Date: 3 Apr 1995 20:40:52 GMT Organization: Dept of Human Physiology, UC Davis Lines: 103 Sender: -Not-Authenticated-[1543] Message-ID: <3lpmgk$sou@mark.ucdavis.edu> NNTP-Posting-Host: norm.hph.ucdavis.edu X-Posted-From: InterNews 1.0.1@norm.hph.ucdavis.edu Xdisclaimer: No attempt was made to authenticate the sender's name. Xref: biosci bionet.molbio.methds-reagnts:26758 bionet.molbio.proteins:4171 bionet.cellbiol:1993 This has been posted in bionet.general. I'm crossposting it to the relevent nesgroups so you can vote for it. Please. Thank you. Voting is now open on the following proposal to create the mailing list & newsgroup FLUORESCENT-PROTEINS/ The charter was not modified as a result of the discussion. *** NOTE *** We are often running several votes for other newsgroups, so please be certain to follow the voting directions *carefully*! If you just send in a message saying "YES" or "NO" it will not be counted if it is not clear which proposal you are responding to. ---------------------------------------------------------------------- Proposal to establish: FLUORESCENT-PROTEINS/bionet.molbio.proteins.fluorescent Proposed USENET name: bionet.molbio.proteins.fluorescent Proposed mailing list name: FLUORESCENT-PROTEINS Proposed e-mail addresses: fluorpro@net.bio.net fluorpro@daresbury.ac.uk Discussion Leaders: Paul Kitts e-mail: paulki@clontech.com Steve Kain e-mail: stevek@clontech.com Bioluminescence, the production of visible light by living organisms, is a fascinating biological phenomenon. In addition, bioluminescent organisms have provided biologists with several powerful research tools: Luciferases which are used as highly sensitive quantitative reporter genes, Aequorin which is used to report on intracellular levels of Ca++ ions, the pycobiliproteins, and most recently Green fluorescent protein (GFP) which is a novel reporter that can be used to monitor gene expression and protein localization inside living cells and organisms. This newsgroup is intended to provide a forum for discussion of bioluminescence, to promote further development of reporter proteins obtained from bioluminescent organisms, and to facilitate their application to interesting biological questions. We anticipate that the participants in this newsgroup will be: - scientists studying the biology and biochemistry of bioluminescence. - scientists developing new or improved fluorescent proteins, luciferases or aequorin-like proteins. - scientists using these reporter proteins in their research. - scientists developing instruments for the imaging or quantitation of the light emitted by these reporters. The newsgroup is intended to provide : - a forum for the discussion of ideas, problems and recent developments concerning light emitting reporter proteins. - a place for sharing unpublished results, both successes and failures. - a source of help on methodological and technical problems. - opportunities for collaborative efforts between labs. The newsgroup will run unmoderated but we are willing to act as coordinators. Paul Kitts & Steve Kain CLONTECH Laboratories Palo Alto, California. ---------------------------------------------------------------------- Voting is now open on the proposal for FLUORESCENT-PROTEINS/bionet.molbio.proteins.fluorescent and will run through 24:00 hrs Pacific Time on 2 May 1995. Please send your vote to either of the following addresses: Address Location Network ------- -------- ------- biovote@daresbury.ac.uk U.K. JANET biovote@net.bio.net U.S.A. Internet/BITNET PLEASE BE SURE TO FOLLOW THE FORMAT BELOW - WE OFTEN RUN MORE THAN ONE VOTE AT A TIME SO A SIMPLE "YES" OR "NO" MESSAGE WITHOUT THE NEWSGROUP NAME MAY BE AMBIGUOUS. Your vote should contain a single line: YES on FLUORPRO if you favor allowing the creation of this newsgroup or NO on FLUORPRO ------------------------------------------------------------------------ - Dave Bates PhD Tel: 916 752-7081 Postdoctoral Researcher Fax: 916 758-2554 Dept of Human Physiology email: dobates@ucdavis.edu University of California at Davis drink: anything Davis, California 95616, USA No I don't have a sense of humour now buy me a beer ------------------------------------------------------------------------ ---- From owner-proteins@net.bio.net Sun Apr 02 23:00:00 1995 Path: biosci!agate!howland.reston.ans.net!pipex!news.pavilion.co.uk!line06.kemp-du.pavilion.co.uk!user From: jpwscyto@pavilion.co.uk (Jon Waterman-Smith) Newsgroups: bionet.molbio.proteins Subject: LAUNCH OF FREE MAGS/WWW PAGES FOR BIONET... Date: 3 Apr 1995 15:53:15 GMT Organization: Pavilion Internet plc, Brighton, England Lines: 29 Message-ID: NNTP-Posting-Host: line06.kemp-du.pavilion.co.uk Within the next 6 weeks a new series of magazines will be launched, which will have a hard copy available to interested parties, as well as a major publication series available on the web. The magazines will deal with many subject areas, and will have hypertext points imprinted into features, so the reader can access the WWW, and simply go to the relevant pages, following the same hypertext links as shown in the magazines. The future....... Our aim is to promote science, and scientific issues mainly in the field of bioscience. Both the magazines and the WWW pages will eventually have articles and links to publications, posters, and as many products and suppliers as we can get interested in publishing with us. So, if you know of anyone who would be interested in providing articles, publishing information to assist other scientists, or you work for a company who wants to advertise , and set up pages on the WWW, we aim to become the one stop point to leap off into the web, and other internet areas. Send me an email, and I'll keep you up to date on our progress. Also let me know if you want to register for the first issue of the first magazine to be published early May '95. Diagnostics, Biotechnology and Life-sciences will be the areas first targeted for publication. TO ALL INTERESTED ADVERTISERS - the deadline for the first issue will be in 3-4 weeks time. Please contact me urgently for more information - your advert will also secure you web space and hypertext links!!!!! I look forward to hearing from you all... Best wishes, Jon Waterman-Smith E-mail address..... jpwscyto@pavilion.co.uk From owner-proteins@net.bio.net Sun Apr 02 23:00:00 1995 Path: biosci!FREENET.UFL.EDU!afn08460 From: afn08460@FREENET.UFL.EDU ("Brian A. Hollander") Newsgroups: bionet.molbio.proteins Subject: Re: Need Serine Kinase inhibitor Date: 3 Apr 1995 21:53:19 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 27 Sender: daemon@net.bio.net Distribution: world Message-ID: References: NNTP-Posting-Host: net.bio.net I'm not surprised that serine did not work as the recognition sites for kinases are usually multiple amino acids. You might try staurosporine, which inhibits a broad range of ser/thr kinases including PKC and proline-dependent kinases. PKA can be inhibited by a synthetic peptide called wiptide (I think). If none of those inhibit, you may check for casein kinases. Casein kinase I can be inhibited by the selective inhibitor CKI-7 (from Sekagaku; sp?), while CKII can be inhibited by heparin. Casein kinases are found at pretty high levels in the brain too. Hope this helps. BAH On Mon, 3 Apr 1995, Gary Krause wrote: > I am trying to isolate the serine kinase that phosphorylates eIF-2 in the > brain. I am in need of a general inhibitor of serine kinases. I have tried > serine with minimal success. Any suggestions would be welcome. Thanks > > Gary > > > ------------------------------------------- > Gary Krause (gkrause@cms.cc.wayne.edu) > Department of Emergency Medicine > Wayne State University, Detroit, MI USA > > From owner-proteins@net.bio.net Sun Apr 02 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!vixen.cso.uiuc.edu!newsrelay.iastate.edu!news.iastate.edu!baker From: baker@iastate.edu (Wayne R. Baker) Newsgroups: bionet.molbio.proteins Subject: Re: D2O exchange for proteins Date: 3 Apr 1995 17:29:22 GMT Organization: Biochemistry & Biophysics, Iowa State University Lines: 40 Message-ID: <3lpb9i$av5@news.iastate.edu> References: NNTP-Posting-Host: pv0a05.vincent.iastate.edu In bionet.molbio.proteins, Giovanni Maga wrote : :Dear bionetters, :we want to begin to study a small protein (30kD, pI around :5) by neutron scattering. We can purify such protein in mg amounts at :homogenity, However, we need to provide it in D2O rather than that in H2O :for neutron scattering analysis. We tried once by liophylizing our protein :and then resuspending it in phosphate buffer at pH 6 in D2O, but we had :serious troubles in getting it soluble again. Does anybody know a good :method to exchange H2O buffers with D2O buffers? Is the pH important in :this reaction (well, the pD in this case)? Any buffering system will work :or some is preferred? I tried to get advice by reading a couple of issues :of Methods in Enzymology which were dealing with nmr studies, but I didn't :find an answer to my (basic, I know) questions. :Any help will be greatly appreciated. Answer here or e-mail to: The exchange rate is dependent on pH. Different proton types will have different rates; see p24+ in "NMR of Proteins and Nucleic Acids" by Kurt Wutrich for a discussion of the pH-dependence. While you may select a pH where *most* of the amide protons will exchange, ones involved in strong hydrogen bonds will not exchange under native conditions. The extent of the exchange is used to measure the H-bonding characteristics of the proton which can give you information about secondary structure. There is a neutron diffraction study of RNase A which may address many of your questions. See Wlodawer, A., & Sjolin, L. (1982) Proc. Natl. Acad. Sci. USA vol 79 p1418 and Wlodawer, A., & Sjolin, L. (1983) Biochemistry vol 22 p2720. They exchanged in deuterated buffer for six months. Finally, increased temperature or denaturants will help in completion of exchange, if your protein can handle it. However, IMHO, the information to be gained from incomplete exchange may be just as valuable. Wayne Baker (baker@iastate.edu) Always do right! 4288 Molecular Biology Building This will gratify some people Iowa State University and astonish the rest. Ames IA 50011 --Mark Twain (515) 294 0781 From owner-proteins@net.bio.net Mon Apr 03 23:00:00 1995 Path: biosci!daresbury!hgmp.mrc.ac.uk!news From: (Pete Jenner) Newsgroups: bionet.molbio.proteins Subject: Protein introns (inteins) Date: 4 Apr 1995 15:12:07 GMT Organization: NIMR Lines: 3 Message-ID: <3lrnk7$rhh@mercury.hgmp.mrc.ac.uk> Reply-To: p-jenner@nimr.mrc.ac.uk NNTP-Posting-Host: nimp326.nimr.mrc.ac.uk X-Newsreader: WinVN 0.92.2 I saw an article from mbdinger@waikato about Help with Protein Introns, but couldn't retreive it. We have been working on inteins for some time, so if you would like some information, please repost. From owner-proteins@net.bio.net Mon Apr 03 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!Germany.EU.net!zib-berlin.de!fu-berlin.de!idomeneo.ukbf.fu-berlin.DE!not-for-mail From: heicappell@ukbf.fu-berlin.de (Ruediger Heicappell) Newsgroups: bionet.molbio.proteins Subject: Adhesion molecules Date: 4 Apr 1995 15:10:27 GMT Organization: Freie Unversitaet Berlin Lines: 6 Message-ID: <3lrnh3$pbn@fu-berlin.de> NNTP-Posting-Host: idomeneo.ukbf.fu-berlin.de (160.45.185.6) X-Access: 16 17 19 X-Newsreader: WinVN 0.93.11 I need information on commercially available antibodies with specificity for alpha, beta and gamma catenin and plakoglobin. Information on companies that sell these antibodies would be highly appreciated. Ruediger Heicappell (heicappell@ukbf.fu-berlin.del) From owner-proteins@net.bio.net Mon Apr 03 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!agate!library.ucla.edu!csulb.edu!csus.edu!news.ucdavis.edu!okra.ece.ucdavis.edu!varma From: varma@okra.ece.ucdavis.edu (Hemant Varma) Newsgroups: bionet.molbio.proteins,bionet.software Subject: E-mail address needed Date: 4 Apr 1995 17:09:21 GMT Organization: University of California, Davis Lines: 25 Message-ID: <3lrug1$r6s@mark.ucdavis.edu> NNTP-Posting-Host: okra.ece.ucdavis.edu X-Newsreader: TIN [version 1.2 PL2] Xref: biosci bionet.molbio.proteins:4179 bionet.software:11697 i Hello, I was wondering if anybody knows an e-mail address of the following authors in Brazil: A.C. Gaudio and Y. Takahata of the following paper: Calculation of molecular surface area with numerical factors Computers & Chemistry Vol. 16, No.4, 277-284 I am basically looking for the program that they wrote to compute the accessible surface areas. Any help would be appreciated. Critiques on their method are welcome as well! Thanks, Hemant From owner-proteins@net.bio.net Mon Apr 03 23:00:00 1995 Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Path: biosci!daresbury!trane.uninett.no!Norway.EU.net!EU.net!howland.reston.ans.net!vixen.cso.uiuc.edu!uchinews!woodlawn!scb1 From: scb1@woodlawn.uchicago.edu (Samuel C. Blackman) Subject: Critique of Expression Cloning in Xenopus (Need Info!) X-Nntp-Posting-Host: midway.uchicago.edu Message-ID: Sender: news@midway.uchicago.edu (News Administrator) Reply-To: scb1@midway.uchicago.edu Organization: University of Chicago -- Academic Information Technologies Date: Tue, 4 Apr 1995 14:57:41 GMT Lines: 22 Xref: biosci bionet.molbio.methds-reagnts:26765 bionet.molbio.proteins:4175 I was wondering if anyone could point me to (or provide me with) any criticisms or critiques of expression cloning using Xenopus oocytes. I've read Dascal's thorough review in CRC Critical Reviews in Biochemistry (22(4):317-387,1987), but it's 8 years old. I'd appreciate it if someone could provide me with any up-to-date info or cites (for example, concerns about the accuracy of post-translational modifications in expressed proteins vs. native proteins, changes in 2nd messgr. couplings ins expressed vs. native, etc.). In addition, if anyone knows of a citation for a more recent review of this technique, I'd appreciate it. Thanks for the help!! -- Sam -- Samuel C. Blackman ! InterNet : scb1@midway.uchicago.edu MD/PhD 2/7 (Pharmacology) ! Disclaimer : Who cares what I say, I'm a student! 5513 S. Cornell Ave. #1 ! Quote : I'm not a doctor, but I play one on TV. Chicago, IL 60637-1914 ! Phone : 312/752-1082 (h) Fax: 312/996-1225 From owner-proteins@net.bio.net Mon Apr 03 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!agate!library.ucla.edu!csulb.edu!csus.edu!news.ucdavis.edu!okra.ece.ucdavis.edu!varma From: varma@okra.ece.ucdavis.edu (Hemant Varma) Newsgroups: bionet.molbio.proteins,bionet.software Subject: GEPOL Date: 4 Apr 1995 16:06:40 GMT Organization: University of California, Davis Lines: 18 Message-ID: <3lrqqg$i2t@mark.ucdavis.edu> NNTP-Posting-Host: okra.ece.ucdavis.edu X-Newsreader: TIN [version 1.2 PL2] Xref: biosci bionet.molbio.proteins:4177 bionet.software:11695 hello everyone, Here is one more request. I am looking for a program called GEPOL. It has been developed by E. Silla et al. (J. of Mol. Garphics, 1990, Vol 8 (168-172). They give a e-mail address, but that does not work from here.. Any information will be greatly appreciated. I will soon post a summary of all the available programs that compute accessible surface areas..Thanks to all those who responded and were kind enough to share the information.. Hemant varma@cs.ucdavis.edu From owner-proteins@net.bio.net Mon Apr 03 23:00:00 1995 Path: biosci!BOTANY.UFL.EDU!harmon From: harmon@BOTANY.UFL.EDU Newsgroups: bionet.molbio.proteins Subject: Re: GFP: What is Coelenterazine Date: 4 Apr 1995 05:53:22 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 29 Sender: daemon@net.bio.net Distribution: world Message-ID: <199504041253.IAA05239@cutter.clas.ufl.edu> NNTP-Posting-Host: net.bio.net U. Thoenes asked: >During my literture studies about the "Green Fluorescent Protein" I found >a component named "coelenterazine" (Inouye and Tsuji, FEBS Letters 351 >(1994) 211-214) as the organic substrate for aequorin. During the reaction >it is oxidized to coelenteramide. >Is there anybody out there who can tell me the chemical formula and >structure as well as some details about coelenterazine? > >In some ealder papers one also can find "coelenterates". I never hears >about this word. Is this a certain sort of organism ? > Coelenterazine is the substrate of aequorin from Aequoria victoria and of the luciferase from Renilla reniformis. Aequoria is a jelly fish and Renilla is a colonial marine animal also called sea pansy. Both organisms used to be called coelenterates, but now are called cnidarians. Coelenterazine was originally named luciferin, a generic name for a substrate of a luciferase, a light producing enzyme. Coelenterazine is 2-(p-hydroxybenzyl)-6-(p-hydroxyphenyl)- 3,7-dihydroimidazo[1,2-a]pyrazin-3-one. Its structure and chemistry were first described by Hori et al. 1973, Biochemistry, 12:4463. A review is: M.J. Cormier, 1981, Renilla and Aequorea Bioluminescence in "Bioluminescence and Chemiluminescence" published by Academic Press, Inc. Alice C. Harmon University of Florida From owner-proteins@net.bio.net Mon Apr 03 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!vixen.cso.uiuc.edu!newsrelay.iastate.edu!gw1.phibred.com!nicholssepc!NICHOLSSE From: NICHOLSSE@phibred.com (Scott E. Nichols) Subject: Hyaluronic acid biosynthesis Message-ID: Lines: 8 Sender: news@phibred.com (USENET News System) Organization: Pioneer Hi-Bred International X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] Date: Tue, 4 Apr 1995 19:17:24 GMT I am looking for a recent review on the enzymology of hyaluronic acid biosynthesis in both procaryotic and eucaryotic organisms. If you are aware of one, will you please post the citation? Thank you Scott E. Nichols nicholsse@phibred.com Pioneer Hi-Bred International From owner-proteins@net.bio.net Mon Apr 03 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!ns1.faseb.org!darwin.sura.net!nih-csl!helix.nih.gov!krzychwl From: "Krzysztot W. Lazowski" Subject: fluorescent labeling of oligonucleotide Content-Type: TEXT/PLAIN; charset=US-ASCII Message-ID: Sender: postman@alw.nih.gov (AMDS Postmaster) Nntp-Posting-Host: helix.nih.gov Organization: National Institutes of Health Mime-Version: 1.0 Date: Tue, 4 Apr 1995 13:01:46 GMT Lines: 2 I aneed methods to fluorescently label oligonucleotides on 5' and 3' ends, by covalent bonds. From owner-proteins@net.bio.net Tue Apr 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!pipex!oleane!jussieu.fr!univ-lyon1.fr!ghost.dsi.unimi.it!news.unige.it!moana.ibf.unige.it!biodev From: biodev@moana.ibf.unige.it (Gruppo Biodevices) Newsgroups: bionet.molbio.proteins Subject: heat proof enzymes and bioreactors Date: 5 Apr 1995 08:10:36 GMT Organization: Univ. of Genoa, Italy Lines: 18 Message-ID: <3ltj9s$s7p@alpha.cisi.unige.it> NNTP-Posting-Host: moana.ibf.unige.it X-Newsreader: TIN [version 1.2 PL2] Hi everybody! I'm searching for information on heat proof enzymes and their possible application in enzyme reactors. In particular, I would like to know if there are examples of reactions that is useful to conduct at high temperature (by "useful" I mean, for example, removal-degradation of undesired by-products by heating...or heat-driven reactions...). Please contact me at the e-mail address: biodev@ibf.unige.it thank you Marco Lanzi Institute of Biophysics School of Medicine Genova-Italy From owner-proteins@net.bio.net Tue Apr 04 23:00:00 1995 Path: biosci!daresbury!trane.uninett.no!Norway.EU.net!EU.net!howland.reston.ans.net!news.sprintlink.net!uunet!psinntp!barilvm!vms.huji.ac.il!wisipc.weizmann.ac.il!weizmann.weizmann.ac.il!BMPOUNY Newsgroups: bionet.molbio.proteins Subject: Start codons in E. coli translation. Message-ID: <1737792F5.BMPOUNY@weizmann.weizmann.ac.il> From: BMPOUNY@weizmann.weizmann.ac.il Date: Wed, 5 Apr 1995 10:27:01 GMT Sender: news@wisipc.weizmann.ac.il (News User) Organization: Weizmann Institute of Science X-Newsreader: NNR/VM S_1.3.2 Lines: 16 Dear all, As far as I know, AUG serves as a start codon for translation. It also codes for Met or when located at the first position, for formil-Met. Now, I found in a textbook that GUG codon which codes for Val, is ambiguous in that it codes both valine and methionine. Is that means that in some proteins the translation starts in Val residue ? If the answer is yes, than how the translation system "knows" when to add Met and when - Val ? Please send your answer to e-mail: bmpouny@weizmann.weizmann.ac.il thanks, Yoni From owner-proteins@net.bio.net Tue Apr 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!news.sprintlink.net!pipex!uknet!yama.mcc.ac.uk!avril!cmg From: cmg@sn2.ee.umist.ac.uk (-) Newsgroups: bionet.molbio.proteins Subject: Re: SDS PAGE and UREA in sample Date: 5 Apr 1995 10:21:58 GMT Organization: Dept. of EE&E, UMIST, Manchester, UK Lines: 28 Distribution: world Message-ID: <3ltr06$jin@yama.mcc.ac.uk> References: <1995Mar8.162256.44047@cc.usu.edu> NNTP-Posting-Host: avril.sn2.ee.umist.ac.uk X-Newsreader: TIN [version 1.2 PL2] Brian A. Hollander (afn08460@FREENET.UFL.EDU) wrote: : In my experience it does not as long as you *do not heat* your samples. : If you heat them the proteins in your samples will becone carbamylated : due to the ionized urea. This probably happens to some extent even : without boiling, but It hasn't seemed to affect migration if the samples : are processed and loaded at room temp. at least in my hands. : BAH : On 8 Mar 1995, NAME RAVI PRAKASH .D wrote: : > Dear Netters, : > Does the presence of Urea in the sample for SDS PAGE have any effect on the : > migration of the protein samples in the gel? : > The urea concentration is 5M in my protein and I loaded 15ul of this along : > with 5ul of 4X sample buffer(Laemli). Any suggestions will be helpfull. : > : > : >in my book urea act in the same way as mercaptoethanol and breaks s-s bond ( or is it h bonds) the point is that you have already sds in the gel and in the buffer this "denatures the protein" if you want to find out about the effect of urea on bond then there is a serise of papers due out on the avidity of immuno molecules for the detection of (a bacteria l carried by cats). If i were you i would mix a few gels with various concs of urea and run some test proteins select some multiunit ones and somne with known s-s bonds etc. Try mixind it with the same sameples and running them. Bye chris From owner-proteins@net.bio.net Tue Apr 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!pipex!warwick!yama.mcc.ac.uk!avril!cmg From: cmg@sn2.ee.umist.ac.uk (-) Newsgroups: bionet.molbio.proteins Subject: Re: native PAGE gels Date: 5 Apr 1995 10:27:01 GMT Organization: Dept. of EE&E, UMIST, Manchester, UK Lines: 15 Message-ID: <3ltr9l$jin@yama.mcc.ac.uk> References: <3jfgru$ddm@susscsc1.rdg.ac.uk> NNTP-Posting-Host: avril.sn2.ee.umist.ac.uk X-Newsreader: TIN [version 1.2 PL2] S. Khuri (abskhuri@reading.ac.uk) wrote: : Does anyone out there have any information about native PAGE gels? : The books only seem to skim over the subject, are there any Idiot's Guides : out there that I could have a look at? : Thanks!! : Sawsan Khuri what do you want to know. we did some with substrates included in the gel(protein) and ran proteinase on it. The result of this was that the protein had to munch its way throught the protein to move down the gel. We just made thte gel without the sds according to the instructions replace the SDS with a voulme of something to keep the % composition right There are a lot of papers on the subject, the first one on PAGE is quoted by every one t From owner-proteins@net.bio.net Tue Apr 04 23:00:00 1995 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!scripps.edu!riscsm!carl_hoeger From: carl_hoeger@qm.salk.edu (Carl Hoeger) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Conjugation/labeling using carbodiimide-HELP Date: Tue, 4 Apr 95 10:01:31 GMT Organization: The Scripps Research Institute, La Jolla, CA Lines: 21 Message-ID: NNTP-Posting-Host: mac6797.salk.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Newsreader: VersaTerm Link v1.1 Xref: biosci bionet.molbio.methds-reagnts:26834 bionet.molbio.proteins:4192 I am currently involved in a research project and I need your help! Do you do cojugations, labelings, etc. to proteins using water soluble carbodiimides, especially EDC? If so, or if you know of procedures for doing so, would you please take a few moments and either send me your exact procedure or a reference to the one you use? I am especially interested in hearing from those of you who are linking amine functions to Asp or Glu in a protein. Please include in your reponse the following: -pH used; -time of reaction; -concentration of EDC or WSCDI used; -concentration of protein/labeling agent; and -temp used. I am posting this in the hope of getting as wide a variety of procedures as possible. I will send to all who repond a summary of the procedures I get, as well as a summary of the results of our experiments (NOTE: I am not selling anything, nor will I be!). Thanks! Carl (carl_hoeger@qm.salk.edu) Carl Hoeger (carl_hoeger@qm.salk.edu) "Nothing is foolproof, as fools are so ingenious" :-] From owner-proteins@net.bio.net Tue Apr 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!pipex!warwick!yama.mcc.ac.uk!avril!cmg From: cmg@sn2.ee.umist.ac.uk (-) Newsgroups: bionet.molbio.proteins Subject: Re: Ca specific electrode, Ca chelators BAPTA or APTRA Date: 5 Apr 1995 10:09:55 GMT Organization: Dept. of EE&E, UMIST, Manchester, UK Lines: 40 Message-ID: <3ltq9j$jin@yama.mcc.ac.uk> References: NNTP-Posting-Host: avril.sn2.ee.umist.ac.uk X-Newsreader: TIN [version 1.2 PL2] Valery F. Thompson (valeryt@ag.arizona.edu) wrote: : I am interested in using a Ca2+ specific electrode for measurement of free : Ca2+ in solutions. I have read that it is possible to measure down to 1µM : Ca2+. I am primarily interested in the range of 1-1000µM. How reliable are : the values at the 1µM level? Is it possible to go lower? : I am also interested in hearing about possible interferences with the : measurements. I know Tris interferes, but at what level? : I have tried using some of the chelators such as 4F- APTRA and 5,5'-dibromo : BAPTA, but without much success. The absorbance of : these chelators does not seem to change very much (only about 0.02AU) when : going from "Ca free" to "Ca satd" at a chelator concentration that would be : acceptable for my experiments (about 25µM). The purpose is to measure the : free Ca conc in a protein solution, and 25µM of my protein is about : 2.5mg/ml (I am interested in the Ca binding properties of the protein). : Unfortunately I only have about 10mg of it and it is very difficult to purify. : Does anyone know how stable these chelators are in solution? : I have soaked all of my glassware and plasticware (except pipet tips and : microcentrifuge tubes) in 10% HNO3 and rinsed them with high purity water. I : have previously measured the Ca contamination of my stock reagents by AA and : found them to be below the detection limit. : If anyone has some information on the Ca specific electrode (I have requested : literature from several companies, but haven't received it yet) or on the use : of the chelators 5,5'-dibromo BAPTA or 4F-APTRA, I would appreciate hearing : about it. : Thanks. : Valery Thompson : Muscle Biology Group : University of Arizona : Tucson, AZ 85721 : valeryt@ag.arizona.edu. Dear val. I think that you could have the need for an ion selective electrode system. Using vancomycin as the selective agent it works much like the glass ph electrode. Im trying to remember this of the top of my head so i would look into it thurther. There should be some commercial devices available with use ISFET (ion selective field effect transistors) as the detection method. Work in this filed has been going on for years. Of course enzyme biosensors are the way forward. Ihope that helps you a little. bye chriss ss From owner-proteins@net.bio.net Tue Apr 04 23:00:00 1995 Path: biosci!adam.cc.sunysb.edu!news.sprintlink.net!howland.reston.ans.net!news2.near.net!das-news2.harvard.edu!oitnews.harvard.edu!news.dfci.harvard.edu!usenet From: ming@mbcrr.harvard.edu (Ding Ming) Newsgroups: bionet.molbio.proteins Subject: Use of CTAB in acidic electrophoretic system of proteins? Date: 5 Apr 1995 19:01:17 GMT Organization: Dana Farber Cancer Institute Lines: 6 Sender: -Not-Authenticated-[3542] Message-ID: <3lupdt$fhc@cisunix1.dfci.harvard.edu> NNTP-Posting-Host: 155.52.84.88 X-Posted-From: InterNews 1.0.6@155.52.84.88 Xdisclaimer: No attempt was made to authenticate the sender's name. Did anyone ever use CTAB in an acidic system of protein electrophoresis just like SDS in an alkalinic system? Ding Ming ming@mbcrr.harvard.edu From owner-proteins@net.bio.net Tue Apr 04 23:00:00 1995 Path: biosci!adam.cc.sunysb.edu!news.sprintlink.net!howland.reston.ans.net!news.cac.psu.edu!news.pop.psu.edu!psuvax1!news.eecs.nwu.edu!news.acns.nwu.edu!uicvm.uic.edu!u12201 Organization: University of Illinois at Chicago, ADN Computer Center Date: Wed, 5 Apr 1995 10:19:06 CDT From: Message-ID: <95095.101906U12201@uicvm.uic.edu> Newsgroups: bionet.molbio.proteins Subject: Re: Need Serine Kinase inhibitor Distribution: world References: Lines: 3 Have you checked the Pierce Signal Transduction catalog ? PierceChem@mcimail.com Keld. From owner-proteins@net.bio.net Tue Apr 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!news.cac.psu.edu!news.pop.psu.edu!psuvax1!news.eecs.nwu.edu!news.acns.nwu.edu!uicvm.uic.edu!u12201 Organization: University of Illinois at Chicago, ADN Computer Center Date: Wed, 5 Apr 1995 17:08:03 CDT From: Message-ID: <95095.170803U12201@uicvm.uic.edu> Newsgroups: bionet.molbio.proteins Subject: Re: fluorescent labeling of oligonucleotide References: Lines: 11 The Pierce OligoLink kit chemically changes the 5' phosphate to either a free amine (NH2) or a free sulfhydryl (SH) depending on the protocol you pick. This can then be used to link the oligo to NHS-Fluorescein for example (also Pierce). You can E-mail Pierce at PierceChem@mcimail.com. Remember to give your address and phone/fax# since their information is not avaialable as e-mail, only mail or fax. In article , "Krzysztot W. Lazowski" says: > >I aneed methods to fluorescently label oligonucleotides on 5' and 3' >ends, by covalent bonds. From owner-proteins@net.bio.net Tue Apr 04 23:00:00 1995 Path: biosci!botany.uq.oz.au!J.Marcus From: J.Marcus@botany.uq.oz.au ("Marcus, Dr J.") Newsgroups: bionet.molbio.proteins Subject: Ammonium Sulfate Precipitation Date: 5 Apr 1995 18:38:25 -0700 Organization: Dept of Botany, Univ of Qld Lines: 24 Sender: daemon@net.bio.net Distribution: world Message-ID: <32B139677FA@botany.uq.oz.au> NNTP-Posting-Host: net.bio.net Dear netters, Does anyone have information regarding what precipitates at various ammonium sulfate concentrations? I would like to use the precipitation to get rid of as much non-proteinaceous material as possible and be left with the maximum amount of protein. I will post a summary in ~3 weeks. Thanks in advance for your help. Regards, John Marcus _________________________________________________________ John Marcus Marcus@tpp.uq.oz.au (Dr J.Marcus) Cooperative Research Centre for Tropical Plant Pathology 5th Level John Hines Building University of Queensland St. Lucia, QLD 4072 AUSTRALIA Fax: 61-7-365-4771 Phone: 61-7-365-4764 From owner-proteins@net.bio.net Tue Apr 04 23:00:00 1995 Path: biosci!daresbury!trane.uninett.no!nac.no!Norway.EU.net!EU.net!howland.reston.ans.net!spool.mu.edu!umn.edu!newsstand.tc.umn.edu!gold.tc.umn.edu!bucc0003 From: bucc0003@gold.tc.umn.edu (Paul A Bucciaglia) Newsgroups: bionet.molbio.proteins Subject: Re: Elution of proteins from polyacrylamide gel Date: 5 Apr 1995 14:23:12 -0500 Organization: University of Minnesota Lines: 37 Message-ID: References: NNTP-Posting-Host: gold.tc.umn.edu You can also stain SDS-PAGels with CuCl2 (0.3M). This is a negative stain which allows one to visualize the protein bands as cleared zones agianst an opaque background. Excise the gel slice, destain in 250 mM EDTA, 100mM TrisCL pH7.6, and electroelute. The protein is completly soluble after destaining, as it avoids any kind of MeOH or acetic acid as in Coomassie. Its more sensitive than Coomassie, although the gels are not as easy to interpret (IMOP). Be sure to visualize against a black background. paul bucciaglia udbl119@bay.cc.kcl.ac.uk (Mandy Johnstone) writes: >In article tberthe@gandalf.crihan.fr (Thierry Berthe) writes: >>Subject: Elution of proteins from polyacrylamide gel >>From: tberthe@gandalf.crihan.fr (Thierry Berthe) >>Date: Mon, 3 Apr 1995 09:17:03 >>What is the best way to elute proteins from polyacrylamide gel ???? >Stain the gel with Coomassie Blue (0.1%BB R-250, 10% MeOH, 0.5% Acetic Acid) >for about 15-30 mins depending on gel thickness and then destain with 10% >MeOH. Cut the desired band out with a razor blade and then electroelute the >protein from the acrylamide. We normally use an Amicon Centrilutor >Microelectroelutor which works great. The benefits of using such a low concn >of acetic acid in the stain is that high levels of acetic acid cause the >proteins to precipitate in the gel and make it incredibly difficult to >electroelute. Hope this helps. Good Luck. >M. >_______________ >Mandy Johnstone >(M.Johnstone@bay.cc.kcl.ac.uk) >King's College, London. From owner-proteins@net.bio.net Tue Apr 04 23:00:00 1995 Path: biosci!adam.cc.sunysb.edu!news.sprintlink.net!pipex!sunsite.doc.ic.ac.uk!alder.cc.kcl.ac.uk!bay.cc.kcl.ac.uk!udbl119 Newsgroups: bionet.molbio.proteins Subject: Re: Elution of proteins from polyacrylamide gel Message-ID: From: udbl119@bay.cc.kcl.ac.uk (Mandy Johnstone) Date: Wed, 5 Apr 1995 10:12:49 LOCAL References: Organization: King's College London Nntp-Posting-Host: pgw2.rai.kcl.ac.uk X-Newsreader: Trumpet for Windows [Version 1.0 Rev B final beta #4] Lines: 23 In article tberthe@gandalf.crihan.fr (Thierry Berthe) writes: >Subject: Elution of proteins from polyacrylamide gel >From: tberthe@gandalf.crihan.fr (Thierry Berthe) >Date: Mon, 3 Apr 1995 09:17:03 >What is the best way to elute proteins from polyacrylamide gel ???? Stain the gel with Coomassie Blue (0.1%BB R-250, 10% MeOH, 0.5% Acetic Acid) for about 15-30 mins depending on gel thickness and then destain with 10% MeOH. Cut the desired band out with a razor blade and then electroelute the protein from the acrylamide. We normally use an Amicon Centrilutor Microelectroelutor which works great. The benefits of using such a low concn of acetic acid in the stain is that high levels of acetic acid cause the proteins to precipitate in the gel and make it incredibly difficult to electroelute. Hope this helps. Good Luck. M. _______________ Mandy Johnstone (M.Johnstone@bay.cc.kcl.ac.uk) King's College, London. From owner-proteins@net.bio.net Tue Apr 04 23:00:00 1995 Path: biosci!rutgers!uwm.edu!spool.mu.edu!howland.reston.ans.net!news.sprintlink.net!uunet!boulder!stormo From: stormo@beagle.Colorado.EDU (Gary Stormo) Newsgroups: bionet.molbio.proteins Subject: Re: Start codons in E. coli translation. Date: 5 Apr 1995 16:04:43 GMT Organization: University of Colorado at Boulder Lines: 41 Message-ID: <3luf2r$36l@lace.Colorado.EDU> References: <1737792F5.BMPOUNY@weizmann.weizmann.ac.il> NNTP-Posting-Host: beagle.colorado.edu BMPOUNY@weizmann.weizmann.ac.il writes: >Dear all, > As far as I know, AUG serves as a start codon for translation. > It also codes for Met or when located at the first position, for > formil-Met. Now, I found in a textbook that GUG codon which codes > for Val, is ambiguous in that it codes both valine and methionine. > Is that means that in some proteins the translation starts in Val > residue ? If the answer is yes, than how the translation system > "knows" when to add Met and when - Val ? > Please send your answer to e-mail: > bmpouny@weizmann.weizmann.ac.il > thanks, > Yoni There are two Met-tRNAs in E. coli, one for translating internal AUG codons and one for initiation. The initiator tRNA always begins translation with a formyl-Met. This tRNA usually decodes AUG (the most common start codon) but it will also decode GUG and UUG, with less efficiency. In fact there is one documented case of an AUA initiation codon and mutations have been identified with CUG and ACG initiation codons. These are much less efficient than the standard initiation codons, but still do function at a low level. In summary, all proteins start with formyl-Met (which is often removed from the final protein product). This is usually encoded by an AUG start codon but other codons can also be decoded by the initiator tRNA. In cases where GUG or some other codon serves as the start of translation, it is still translated as formyl-Met rather than what that codon would be translated as internally. Hope that helps. -- Gary Stormo | MCD Biology | Keep in mind that to the advertising industry, Univ. of Colorado | every day is April Fool's Day. Boulder, CO 80309 | From owner-proteins@net.bio.net Wed Apr 05 23:00:00 1995 Path: biosci!daresbury!hgmp.mrc.ac.uk!news From: Pete Jenner Newsgroups: bionet.molbio.proteins Subject: Re: Help with Protein Introns Date: 6 Apr 1995 14:27:18 GMT Organization: NIMR Lines: 32 Message-ID: <3m0to6$ike@mercury.hgmp.mrc.ac.uk> References: <1995Apr4.161608.37742@waikato.ac.nz> Reply-To: p-jenner@nimr.mrc.ac.uk NNTP-Posting-Host: nimp326.nimr.mrc.ac.uk X-Newsreader: WinVN 0.92.2 In article <1995Apr4.161608.37742@waikato.ac.nz>, mbdinger@waikato.ac.nz says: > >Hello, > >I'm currently researching "Protein Introns", and am in need of information >on this subject. > >If anybody is involved in this field could provide any information, or knows >of references that I can look up, it would be greatly appreciated. >Abstract searches on the libraries CD-ROMs revealed nothing. > >Thanks. > >Any replies to: mbdinger@waikato.ac.nz. > > I've responded in detail by e-mail to Marcel, but have recieved mail from others wanting to know what inteins are. Inteins are in-frame insertions in genes that are excised at the protein level by a process called protein splicing. The two ends of the external protein (or extein) are ligated to give a fully functional mature protein. Examples have been found in mycobacteria, yeast, and archaebacteria. The inteins differ from each other in sequence, apart from characteristic splice site motifs, and they also have LAGLI-DADG signatures. About 10 examples have been found so far, and as others are found they will help us answer a number of questions about their function, origin, and occurance. Peter Jenner (Dept Mycobacterial Research NIMR, Mill Hill, London,NW7 1AA) From owner-proteins@net.bio.net Wed Apr 05 23:00:00 1995 Path: biosci!UNIXG.UBC.CA!eastmana From: eastmana@UNIXG.UBC.CA (Ann Eastman) Newsgroups: bionet.molbio.proteins Subject: Re: Elution of proteins from polyacrylamide gel Date: 6 Apr 1995 02:28:35 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 10 Sender: daemon@net.bio.net Distribution: world Message-ID: References: NNTP-Posting-Host: net.bio.net I've had great results electoeluting proteins from CBB-stained gels using an Amicon Centrilutor with Centricon microconcentrators. Unless you need to use a really low MW cutoff unit, the dye passes through the concentrator, and in any case there is no concentration of buffer components so you don't have to dialyze or lyphylize the sample afterwards. The recovered protein can be readily concentrated by centrifugation (although in my hands this took longer than indicated in the product info).> > AE From owner-proteins@net.bio.net Wed Apr 05 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!Germany.EU.net!zib-berlin.de!informatik.tu-muenchen.de!lrz-muenchen.de!OC3GRA!bca20 From: bca20@OC3GRA (Luettgen Holger) Newsgroups: bionet.molbio.proteins Subject: Re: pichia pastoris protease deficient Date: 6 Apr 1995 10:17:32 GMT Organization: Leibniz-Rechenzentrum, Muenchen (Germany) Lines: 11 Distribution: world Message-ID: <3m0f3s$s4m@sunserver.lrz-muenchen.de> References: NNTP-Posting-Host: oc3gra.org.chemie.tu-muenchen.de X-Newsreader: TIN [version 1.2 PL1] Judith A. Partaledis (partaledis@vpharm.com) wrote: : I am looking for a protease deficient strain of Pichia pastoris. Actually there is patent about the construction and use of protease deficient strain owned by Gleeson and Howard, Pat.No.:05324660, 28.6.1994 I am not familiar with american patent laws, but I think the strains described have to be availble for evaluation and there is no need for a licence, if you want to use them for research. Holger Luettgen From owner-proteins@net.bio.net Wed Apr 05 23:00:00 1995 Path: biosci!ACS.UCALGARY.CA!jbooth From: jbooth@ACS.UCALGARY.CA (Joseph Booth) Newsgroups: bionet.molbio.proteins Subject: protein mass spec Date: 6 Apr 1995 14:17:57 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 22 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Hi, I have purified a protein using anion exchange chromatography followed by RP-HPLC on a C18 column with an acetonitrile/water gradient (both containing 0.1% TFA). Re-running aliquots of my active fractions on the C18 column gives a peak with the same elution time confirming the presence of my protein. My problem arises when analyzing the protein by electrospray mass spectrometry. The protein has a predicted mass of about 6.5 kD and is quite acidic with a pI of about 4.0. I have sent samples (200-400 pmoles) of the protein to two different institutions for analysis and in both cases they were unable to obtain a signal in either positive or negative ion modes. It was suggested that residual TFA remaining after lyophilization might be causing interference but desalting the samples (PD-10 columns) against water prior to lyophilization did not seem to help. Any suggestions as to what might be causing this problem and possible solutions would be greatly appreciated. Thanks. Joe From owner-proteins@net.bio.net Wed Apr 05 23:00:00 1995 Path: biosci!adam.cc.sunysb.edu!news.sprintlink.net!cs.utexas.edu!swrinde!emory!usenet From: djdlab@bimcore.emory.edu (Dean Danner Lab) Newsgroups: bionet.molbio.proteins,bionet.molbio.yeast Subject: Protein interactions- none, now what??? Date: 6 Apr 1995 22:36:33 GMT Organization: Biomolecular Computing Resource, Emory University Lines: 7 Distribution: world Message-ID: <3m1qdh$5eu@emory.mathcs.emory.edu> Reply-To: mlanterm@gmm.gen.emory.edu NNTP-Posting-Host: bimcore.emory.edu Xref: biosci bionet.molbio.proteins:4205 bionet.molbio.yeast:2725 So- I would like to rack everyone's brain. I have proteins interacting with themselves (ie X with X, and Y with Y) in the yeast two hybrid system, and functioning in vivo in a yeast model system. SO what if I make a mutation (deletion or single aa change) and the protein no longer interact of function? What would be the best way to assess why I have lost interaction/function. The proteins I am studying function is a multienzyme complex so they form 24mers, or tetramers depending on which ones we are talking about. Margaret Lanterman mlanterm@gmm.gen.emory.edu From owner-proteins@net.bio.net Wed Apr 05 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!news.moneng.mei.com!uwm.edu!msunews!netnews.upenn.edu!obgynjm25.obgyn.upenn.edu!user From: ggerton@obgyn.upenn.edu (George L. Gerton) Newsgroups: bionet.molbio.proteins Subject: Re: Use of CTAB in acidic electrophoretic system of proteins? Date: Thu, 06 Apr 1995 07:57:25 -0500 Organization: University of Pennsylvania Lines: 19 Message-ID: References: <3lupdt$fhc@cisunix1.dfci.harvard.edu> NNTP-Posting-Host: obgynjm25.obgyn.upenn.edu In article <3lupdt$fhc@cisunix1.dfci.harvard.edu>, ming@mbcrr.harvard.edu (Ding Ming) wrote: > Did anyone ever use CTAB in an acidic system of protein electrophoresis > just like SDS in an alkalinic system? > > Ding Ming > > ming@mbcrr.harvard.edu Yes. See, for example: Fairbanks, G. and Avruch, J. 1972. Four gel systems for electrophoretic fractionation of membrane proteins using ionic detergents. J. Supramol. Struct. 1:66-75. -- George L. Gerton ggerton@obgyn.upenn.edu From owner-proteins@net.bio.net Wed Apr 05 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!ns1.faseb.org!darwin.sura.net!nih-csl!loglady.ninds.nih.gov!johnk From: johnk@spasm.niddk.nih.gov (John Kuszewski) Subject: 3-10 helix backbone angles Message-ID: <1995Apr6.222835.11457@alw.nih.gov> Sender: postman@alw.nih.gov (AMDS Postmaster) Nntp-Posting-Host: spasm.niddk.nih.gov Reply-To: johnk@spasm.niddk.nih.gov (John Kuszewski) Organization: National Insts. of Health Date: Thu, 6 Apr 1995 22:28:35 GMT Lines: 16 Can anyone tell me the standard backbone phi and psi angles for a 3-10 helix? -- _____________ | ___/_ | |/ / -- /\ // /-- || || / /|| || || / / || || ||/ / || John Kuszewski || |/ /| || johnk@spasm.niddk.nih.gov || / /|| || \/ / / || \/ that's MISTER protein G to you! |/__/| | /_________| From owner-proteins@net.bio.net Wed Apr 05 23:00:00 1995 Path: biosci!adam.cc.sunysb.edu!news.sprintlink.net!howland.reston.ans.net!swrinde!emory!usenet From: djdlab@bimcore.emory.edu (Dean Danner Lab) Newsgroups: bionet.molbio.proteins Subject: Re: Tropix vs. ECL Date: 6 Apr 1995 22:16:29 GMT Organization: Biomolecular Computing Resource, Emory University Lines: 7 Distribution: world Message-ID: <3m1p7t$5eu@emory.mathcs.emory.edu> References: <9503301209.aa28735@jwi-adm.jwci.org> Reply-To: djdlab@bimcore.emory.edu NNTP-Posting-Host: bimcore.emory.edu I started with Tropix and went to ECL which was wonderful and more sensitive (and still is). I recommend sticking with ECL- it is the best Margaret Lanterman no I don't work for Amersham From owner-proteins@net.bio.net Wed Apr 05 23:00:00 1995 Path: biosci!MANI.CBS.UMN.EDU!npv From: npv@MANI.CBS.UMN.EDU (Nora Plesofsky-Vig) Newsgroups: bionet.molbio.proteins Subject: electroblotting of proteins Date: 6 Apr 1995 15:12:34 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 18 Sender: daemon@net.bio.net Distribution: world Message-ID: <9504062313.AA04770@mani.cbs.umn.edu> Reply-To: nora@biosci.cbs.umn.edu NNTP-Posting-Host: net.bio.net I have a problem concerning the electrophoretic blotting of proteins from a polyacrylamide gel to a small pore PVDF membrane, using a Hoeffer Transfor apparatus, TE 50. I do the transfer in a 10 mM CAPS transfer buffer, pH 11, 10% methanol, since I wish to sequence the transferred peptides. I cool the buffer to 10-12oC, use a stir bar, make every attempt to remove bubbles, but still I get protein bands swirling within the membrane as though local ion currents have been set up. This only occurs in certain (unpredictable) areas, but obviously it would be desirable to avoid this. Does anyone have any experience with this problem? Any likely solutions? Thanks for any suggestions. Nora Plesofsky-Vig nora@biosci.cbs.umn.edu From owner-proteins@net.bio.net Wed Apr 05 23:00:00 1995 Path: biosci!daresbury!trane.uninett.no!due.unit.no!nac.no!Norway.EU.net!EU.net!howland.reston.ans.net!pipex!lyra.csx.cam.ac.uk!moose.bioc.cam.ac.uk!user From: nef@mole.bio.cam.ac.uk (Nick Fisher) Newsgroups: bionet.molbio.proteins Subject: Re: Use of CTAB in acidic electrophoretic system of proteins? Date: Thu, 06 Apr 1995 22:01:52 +0000 Organization: Cambridge University (Biochemistry Department) Lines: 27 Message-ID: References: <3lupdt$fhc@cisunix1.dfci.harvard.edu> NNTP-Posting-Host: moose.bioc.cam.ac.uk In article <3lupdt$fhc@cisunix1.dfci.harvard.edu>, ming@mbcrr.harvard.edu (Ding Ming) wrote: > Did anyone ever use CTAB in an acidic system of protein electrophoresis > just like SDS in an alkalinic system? > > Ding Ming > > ming@mbcrr.harvard.edu I have tried this system...CTAB is useable, but not particulary water soluble (a 1% w/v solution will crystallise out in a day or so). Suitable buffers for a discontinuous gel system are acetic acid/KOH at pH 6.8 for the stacking gel and pH 4.3 for the separating gel.[AcH] is 5 mM in the stacker and 29 mM in the separating gel. Remember to exchange the bromophenol blue in the loading buffer for methylene blue or someother cationic dye, and to run the gel towards the cathode ;) TEMED is not an especially good polymerisation catalyst at low pH, so try riboflavin instead. Read chapter 1 of Hames and Rickwoods book (Gel Electrophoresis of Proteins, 2e 1990, IRL press) if you don't already have it for a good description of "non-Laemlli" polyacrylamide gel systems. Nick Fisher Biochemistry Dept. Cambridge University UK From owner-proteins@net.bio.net Wed Apr 05 23:00:00 1995 Path: biosci!daresbury!trane.uninett.no!nac.no!Norway.EU.net!EU.net!uknet!bhamcs!comlab.ox.ac.uk!oxuniv!oxuniv!nntp Newsgroups: bionet.molbio.proteins Subject: Protein Crosslinking by UV Message-ID: <1995Apr6.133249.31068@oxvaxd> From: sg@physiol.ox.ac.uk (Simon Golding) Date: 6 Apr 95 13:32:49 +0100 Organization: Physiology, Oxford University, UK Nntp-Posting-Host: pc-transport.physiol X-Newsreader: WinVN 0.93.14 MIME-Version: 1.0 Lines: 19 I am hoping to identify and isolate a membrane protein using a radio-iodinated/ tritiated inhibitor protein that is known to bind. The protocol I was going to follow involves covalently binding the 2 proteins (once initial binding has taken place) using UV. Is this as easy as it sounds, can this UV technique be applied to all proteins or does it have specific structural requirements and which radio isotope would be better (assuming that iodination doesn't alter the binding of the 2 proteins in the first place)? Thanks for the help Simon (No signature quotation, sorry!) From owner-proteins@net.bio.net Thu Apr 06 23:00:00 1995 Newsgroups: bionet.molbio.proteins,bionet.molbio.yeast Path: biosci!newshost.lanl.gov!news.ttu.edu!aurora.LaTech.edu!darwin.sura.net!nih-csl!loglady.ninds.nih.gov!johnk From: johnk@spasm.niddk.nih.gov (John Kuszewski) Subject: Re: Protein interactions- none, now what??? Message-ID: <1995Apr7.163308.26538@alw.nih.gov> Sender: postman@alw.nih.gov (AMDS Postmaster) Nntp-Posting-Host: spasm.niddk.nih.gov Reply-To: johnk@spasm.niddk.nih.gov (John Kuszewski) Organization: National Insts. of Health References: <3m1qdh$5eu@emory.mathcs.emory.edu> Date: Fri, 7 Apr 1995 16:33:08 GMT Lines: 36 Xref: biosci bionet.molbio.proteins:4211 bionet.molbio.yeast:2731 In article <3m1qdh$5eu@emory.mathcs.emory.edu>, djdlab@bimcore.emory.edu (Dean Danner Lab) writes: |> So- I would like to rack everyone's brain. |> I have proteins interacting with themselves (ie X with X, and Y with Y) in the yeast two hybrid system, and functioning in vivo in a yeast model system. |> SO what if I make a mutation (deletion or single aa change) and the protein no longer interact of function? What would be the best way to assess why I have lost interaction/function. The proteins I am studying function is a multienzyme complex so they f|> orm 24mers, or tetramers depending on which ones we are talking about. |> (Please try to limit yourself to 80 characters/line!) If you want to know if your mutation has destabilized each monomer so much that it's unfolded the protein, have a look at the CD or NMR spectra. Calorimetry would also show changes in the stability. If you want to see if you've just destabilized the interaction surfaces, do some ultracentrifugation or light scattering measurements of the monomer-polymer equilibria. If you see no changes in the free energy of monomer-monomer interactions and you haven't unfolded the protein, then perhaps your mutations are right in the active site. Hope this helps. -- _____________ | ___/_ | |/ / -- /\ // /-- || || / /|| || || / / || || ||/ / || John Kuszewski || |/ /| || johnk@spasm.niddk.nih.gov || / /|| || \/ / / || \/ that's MISTER protein G to you! |/__/| | /_________| From owner-proteins@net.bio.net Thu Apr 06 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!newshost.lanl.gov!news.ttu.edu!aurora.LaTech.edu!darwin.sura.net!nih-csl!NewsWatcher!user From: newitt@ncifcrf.gov (John A. Newitt) Subject: Re: electroblotting of proteins Message-ID: Sender: postman@alw.nih.gov (AMDS Postmaster) Nntp-Posting-Host: 128.231.113.35 Organization: National Institutes of Health References: <9504062313.AA04770@mani.cbs.umn.edu> Date: Fri, 7 Apr 1995 13:08:50 GMT Lines: 25 In article <9504062313.AA04770@mani.cbs.umn.edu>, nora@biosci.cbs.umn.edu wrote: > I have a problem concerning the electrophoretic blotting of proteins > from a polyacrylamide gel to a small pore PVDF membrane, using a > Hoeffer Transfor apparatus, TE 50. I do the transfer in a 10 mM CAPS > transfer buffer, pH 11, 10% methanol, since I wish to sequence the > transferred peptides. I cool the buffer to 10-12oC, use a stir bar, > make every attempt to remove bubbles, but still I get protein bands > swirling within the membrane as though local ion currents have been > set up. This only occurs in certain (unpredictable) areas, but > obviously it would be desirable to avoid this. > I've never used this buffer system, so I can't think of any specific problems, but here's a suggestion. I've had bubble problems sometimes when the transfer was running at high current conditions. When the blot sandwich is placed in the apparatus near the cathode (-) it is subjected to more bubbles (2e- + 2 H2O -> 2OH- + H2) than when placed near the anode (+) (H2O -> 2H= + 1/2 O2 + 2e-). So when I have the choice, I always set up the sandwiches in the slot closest to the anode. In theory, the stirring should prevent the bubbles from causing problems in your case, but it won't hurt to try it this way. John A. Newitt National Institutes of Health Bethesda, Maryland USA From owner-proteins@net.bio.net Thu Apr 06 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!daresbury!sunsite.doc.ic.ac.uk!doc.news.pipex.net!pipex!oleane!jussieu.fr!univ-lyon1.fr!swidir.switch.ch!scsing.switch.ch!rzusuntk.unizh.ch!NewsWatcher!user From: maga@vetbio.unizh.ch (Giovanni Maga) Subject: Re: Protein interactions- none, now what??? Message-ID: Followup-To: bionet.molbio.proteins,bionet.molbio.yeast Sender: newsadm@rzu-news.unizh.ch (CNEWS ADMINISTRATION) Organization: University of Zurich Irchel- Biochemistry References: <3m1qdh$5eu@emory.mathcs.emory.edu> Date: Fri, 7 Apr 1995 14:19:45 GMT Lines: 21 In article <3m1qdh$5eu@emory.mathcs.emory.edu>, djdlab@bimcore.emory.edu (Dean Danner Lab) wrote: > So- I would like to rack everyone's brain. > I have proteins interacting with themselves (ie X with X, and Y with Y) in the yeast two hybrid system, and functioning in vivo in a yeast model system. > SO what if I make a mutation (deletion or single aa change) and the protein no longer interact of function? What would be the best way to assess why I have lost interaction/function. The proteins I am studying function is a multienzyme complex so they form 24mers, or tetramers depending on which ones we are talking about. > > Margaret Lanterman > mlanterm@gmm.gen.emory.edu Well, if you can show that aa mutation = loss of function and that loss of function follows from loss of monomers interaction it's already a fact. Being the only difference between wt and mut one aa, it is not so azardous to speculate that it was the reason (BTW, a single aa mutation which abolishes protein protein interation should be *very* critical). I would suggest to test this in other systems than two-hybrid, just as a confirmation. Since your multienzyme is forming 4mer or even 24mers, it could be easy to distinguish the case of subassemblies or only monomers in solution by gel-filtration or native PAGE. Then, you can speculate about the role of your aa. maga@vetbio.unizh.ch From owner-proteins@net.bio.net Thu Apr 06 23:00:00 1995 Path: biosci!rutgers!gatech!howland.reston.ans.net!news.sprintlink.net!uunet!world!news.bu.edu!rmandel From: rmandel@bu.edu (Richard Mandel) Newsgroups: bionet.molbio.proteins Subject: Re: Tropix vs. ECL Date: 7 Apr 1995 19:22:55 GMT Organization: Boston University Lines: 37 Distribution: world Message-ID: <3m43ef$h1r@news.bu.edu> References: <9503301209.aa28735@jwi-adm.jwci.org> NNTP-Posting-Host: acs.bu.edu X-Newsreader: TIN [version 1.2 PL0] Dale Yuzuki's Work Account (dale@JWCI.ORG) wrote: : Dear netters, : I've been using Amersham's ECL system for my Westerns for a few months : now; recently I've come across Tropix's `Western-Light Plus' tertiary : detection system. How does it compare to the ECL? : (For those not familiar with Tropix's system, it uses a biotinylated : secondary antibody, and a tertiary strep-avidin linked to AP, and : developed with their CSPD substrate.) : FWIW I'm looking for the highest sensitivity around. My mouse sera : gives me very low signals of polyclonal Ab in my system. Thanks in : advance. The sensitivity of the detection can be increased in the ECL system by combining it with Pierce Chemical Co.'s ABC kit. This kit uses biotin conjugated secondary antibody, avidin and biotin conjugated peroxidase or AP. The amplification comes from the multiple biotin binding sites of avidin and the multiple biotins attaced to the enzyme. A complex between avidin and the biotinylated enzyme is first formed which is then reacted with the primary-secondary-biotinylated complex. Out experience is that it greater than 10x more sensitive than ECL by itself. The cost of the ABC kit, however, is high ($340) so if your antibodies are free and you have enough antigen it is better to use the ECL system straight. -- Ciao, Rich --------------------------------------------------------------------------- Richard Mandel | E-mail address: rmandel@acs.bu.edu Boston Univ. School of Medicine | Phone No. 617-638-4512 Boston, MA 02118 | Fax No. 617-638-4085 ----------------------------------------------------------------------------- From owner-proteins@net.bio.net Thu Apr 06 23:00:00 1995 Path: biosci!rutgers!uwm.edu!vixen.cso.uiuc.edu!howland.reston.ans.net!newsserver.jvnc.net!netnews.sb.com!NewsWatcher!user From: William_P_Prichett%notes@SB.COM (William P. Prichett) Newsgroups: bionet.molbio.proteins Subject: Re: electroblotting of proteins Followup-To: bionet.molbio.proteins Date: 7 Apr 1995 17:38:15 GMT Organization: SmithKline Beecham Pharmacueticals Lines: 23 Distribution: world Message-ID: References: <9504062313.AA04770@mani.cbs.umn.edu> NNTP-Posting-Host: 139.136.69.95 > I have a problem concerning the electrophoretic blotting of proteins > from a polyacrylamide gel to a small pore PVDF membrane, using a > Hoeffer Transfor apparatus, TE 50. I do the transfer in a 10 mM CAPS > transfer buffer, pH 11, 10% methanol deleted but still I get protein bands > swirling within the membrane as though local ion currents have been > set up. deleted > Thanks for any suggestions. > > Nora Plesofsky-Vig > nora@biosci.cbs.umn.edu Nora, I use these conditions for my blots and a biorad transblot tank apparatus. It works well for me. The problem that you are describing sounds like you are still getting bubbles in your Scotch brite pads. I use 150-200mA overnight, this gives me about 25 V (gels size is about 15 X 15cm). You may also try the semidry blotters. I use the one from Millipore fairly often also, it works good to. Good Luck, Bill Prichett From owner-proteins@net.bio.net Thu Apr 06 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!vixen.cso.uiuc.edu!uwm.edu!reuter.cse.ogi.edu!engr.orst.edu!odo.PEAK.ORG!gaia.ucs.orst.edu!news.uidaho.edu!usenet From: magn8831@uidaho.edu (Tim Magnuson) Newsgroups: bionet.molbio.proteins Subject: Pigments binding to enzymes Date: 7 Apr 1995 06:23:55 GMT Organization: University of Idaho Lines: 11 Message-ID: <3m2lpr$6ie@newshound.uidaho.edu> NNTP-Posting-Host: xslip23.csrv.uidaho.edu Mime-Version: 1.0 Content-Type: text/plain; charset=US-ASCII Content-Transfer-Encoding: 7bit X-Newsreader: WinVN 0.90.5 I am working with an extracellular enzyme secreted by a Streptomyces spp. My organism produces a fair amount of pigments, and these interfere with purification and analysis. I've tried PVPP, Biopol, polyethylene- imine etc. with not much luck to separate the pigments from the enzyme. Standard purification procedures (MonoQ, Rotofor etc.) purify activity somewhat, but the protein still has gunk on it. I don't want to have to resort to gunk-out carb cleaner; any tips, advice, methods would be greatly appreciated. Please respond to above address. Thanx T. S. Magnuson From owner-proteins@net.bio.net Thu Apr 06 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!swrinde!pipex!warwick!yama.mcc.ac.uk!zippy.dct.ac.uk!dundee.ac.uk!dundee.ac.uk!not-for-mail Newsgroups: bionet.molbio.proteins Subject: Re: How to deglycosylate fetuin? Message-ID: <3lrjf9$gqn@dux.dundee.ac.uk> From: hjtreuma@dux.dundee.ac.uk (H.J. Treumann Biochemistry ext 4301) Date: 4 Apr 1995 15:01:13 +0100 References: Organization: The University, Dundee, DD1 4HN, Scotland, UK. NNTP-Posting-Host: dux.dundee.ac.uk X-Newsreader: TIN [version 1.2 PL1] Lines: 24 Stefan, why do you want to do it with an enzyme? There are several chemical methods for deglycosylation of O-linkeds. One way to do it would be an acid treatment with trifluoromethane sulfonic acid (TFMS). Alternatively you could do an alkaline beta elimination (followed by a reduction if you are interested in the glycans). Mail me if you need more details - I should be able to look up references for you. Good luck, Achim A. Treumann Carbohydrate Research Centre Department of Biochemistry, University of Dundee, Dundee DD1 4HN email a.treumann@dundee.ac.uk, fax +44-382-322583, tel 344301 Stefan.Rosen@mbioekol.lu.se wrote: : Hello! : I want to enzymatically pile off the O-linked sugars (totally) in fetuin : (from fetal calf serum). From owner-proteins@net.bio.net Thu Apr 06 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!swrinde!howland.reston.ans.net!news.moneng.mei.com!uwm.edu!news.alpha.net!solaris.cc.vt.edu!swiss.ans.net!paperboy.amoco.com!cronkite!usenet From: wmmounts@amoco.com (Bill Mounts) Newsgroups: bionet.molbio.proteins Subject: Re: 3-10 helix backbone angles Date: 7 Apr 1995 13:31:33 GMT Organization: Amoco Corporation Lines: 17 Message-ID: <3m3erl$kom@cronkite.amoco.com> References: <1995Apr6.222835.11457@alw.nih.gov> Reply-To: wmmounts@amoco.com In article 11457@alw.nih.gov, johnk@spasm.niddk.nih.gov (John Kuszewski) writes: >Can anyone tell me the standard backbone phi and >psi angles for a 3-10 helix? From Voet & Voet's Biochemistry (1990) For a right handed 3(10) helix phi = -49 degrees psi = -26 degrees Bill Mounts Vysis, Inc. Downers Grove, IL USA From owner-proteins@net.bio.net Thu Apr 06 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!newshost.lanl.gov!news.ttu.edu!aurora.LaTech.edu!darwin.sura.net!fconvx.ncifcrf.gov!mack From: mack@ncifcrf.gov (Joe Mack) Subject: Re: Protein Crosslinking by UV Message-ID: Organization: Frederick Cancer Research and Development Center References: <1995Apr6.133249.31068@oxvaxd> Date: Fri, 7 Apr 1995 14:52:29 GMT Lines: 32 In article <1995Apr6.133249.31068@oxvaxd> sg@physiol.ox.ac.uk (Simon Golding) writes: >I am hoping to identify and isolate a membrane protein using a >radio-iodinated/ tritiated inhibitor protein that is known to >bind. The protocol I was going to follow involves covalently >binding the 2 proteins (once initial binding has taken place) >using UV. > >Is this as easy as it sounds, The yields can be poor and the residues linked can be non-specific (although you may not care). The UV tends to fry the protein before it can cross link. A better version, although much higher tech is to use UV laser cross linking, one pulse does it. From a poster I saw, I believe there is a set up at OXford to do this, however I've forgotten the name. However Peter von Hippel (in Oregon) should know who he is. Another guy is Buc (Buch?) in Paris. Joe Mack mack@ncifcrf.gov .sig under construction From owner-proteins@net.bio.net Thu Apr 06 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!bcm!cs.utexas.edu!news.sprintlink.net!uunet!in1.uu.net!world!NewsWatcher!user From: nestgrp@world.std.com (Amos Heckendorf) Subject: Re: Ammonium Sulfate Precipitation Message-ID: Sender: news@world.std.com (Mr Usenet Himself) Nntp-Posting-Host: world.std.com Organization: The Nest Group, Inc. References: <32B139677FA@botany.uq.oz.au> Date: Fri, 7 Apr 1995 19:42:58 GMT Lines: 32 In article <32B139677FA@botany.uq.oz.au>, J.Marcus@botany.uq.oz.au ("Marcus, Dr J.") wrote: > Dear netters, > Does anyone have information regarding what > precipitates at various ammonium sulfate concentrations? I > would like to use the precipitation to get rid of as much > non-proteinaceous material as possible and be left with > the maximum amount of protein. I will post a summary in > ~3 weeks. John Marcus: The best indirect reference to what would ppt in ammonium sulfate would be the reciprocal of the elution pattern from HIC (hydrophobic interaction chromatography). The last to come out is the most hydrophobic and would come down first in a batch pptn mode. This would give you an objective reference. Since most people run these columns from 1.8M sulfate to 100 mM phosphate buffer, no sulfate the variables would be temperature, pH and sample composition (how much salt is in the sample), all of which would affect the retention times and skew the results. Just an idea so you can research the published lit and get a handle on a broad question. regards, -- Amos Heckendorf (nestgrp@world.std.com) Tel: 800-347-6378 or 508-481-6223; FAX: 508-485-5736. Value Added Resellers of HPLC columns and Pre-Cast Gels for Peptide and Protein Purification and Nucleic Acid Purification Kits. From owner-proteins@net.bio.net Thu Apr 06 23:00:00 1995 Path: biosci!daresbury!trane.uninett.no!Norway.EU.net!EU.net!howland.reston.ans.net!math.ohio-state.edu!jussieu.fr!oleane!sct.fr!NewsWatcher!user From: danyv@world-net.sct.fr (Vauterin D) Newsgroups: bionet.biophysics,bionet.cellbiol,bionet.molbio.proteins,sci.bio.microbiology Subject: Esterase activity Date: Fri, 07 Apr 1995 23:00:41 +0100 Organization: World-Net - Dialup Internet - Paris - France Lines: 7 Message-ID: NNTP-Posting-Host: client13.sct.fr Xref: biosci bionet.biophysics:827 bionet.cellbiol:2024 bionet.molbio.proteins:4218 sci.bio.microbiology:676 Hi, I search informations about ESTERASE ACTIVITY after gama irradiations. -- Paris From owner-proteins@net.bio.net Thu Apr 06 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!vixen.cso.uiuc.edu!uwm.edu!uwvax!uchinews!kimbark!sferguso From: sferguso@kimbark.uchicago.edu (Stacy Ferguson) Subject: Re: bcl-2 antibodies- need help!!!!!! X-Nntp-Posting-Host: midway.uchicago.edu Message-ID: Sender: news@midway.uchicago.edu (News Administrator) Reply-To: sferguso@midway.uchicago.edu Organization: The University of Chicago References: <3m3eof$l8t@detroit.freenet.org> Date: Fri, 7 Apr 1995 20:26:49 GMT Lines: 17 In article <3m3eof$l8t@detroit.freenet.org>, Jay Kulkarni wrote: > >Hi!! >I am looking for bcl-2 antibody which will react with chicken bcl-2, >does anyone have it? if so would you please let the the source? >you help will be appreciated and ackowledged. >thanx for you time. >jay Try Don Ewert at the Wistar Institute in Philadelphia. Stacy From owner-proteins@net.bio.net Thu Apr 06 23:00:00 1995 Newsgroups: bionet.molbio.proteins Path: biosci!bcm!news.msfc.nasa.gov!sol.ctr.columbia.edu!howland.reston.ans.net!torn!watserv2.uwaterloo.ca!watserv3.uwaterloo.ca!peptidarus.UWaterloo.ca!ltaylor From: ltaylor@peptidarus.UWaterloo.ca (Lorne Taylor) Subject: Re: protein mass spec Message-ID: Sender: news@watserv3.uwaterloo.ca Nntp-Posting-Host: peptidarus.uwaterloo.ca Organization: University of Waterloo References: Date: Fri, 7 Apr 1995 14:04:17 GMT Lines: 47 Joe...do you have the experimental details of the two failed attempts? It would be interesting to see what kind of solvent/buffer system they both used? Whenever I have a troublesome sample I try a) dissolve sample in water or water/acetonitrile (as little ACN as possible!) b) apply sample to a C18 disposable cartridge c) wash C18 with copious amounts of water using compressed air to remove water d) elute with neat formic acid e) cross fingers and shoot sample You might also want to try a MALDI MS run as MALDI is a lot more tolerant to small amounts of contaminants than ESMS is. Good Luck Lorne Taylor Protein Analysis Lab University of Waterloo In article , jbooth@ACS.UCALGARY.CA (Joseph Booth) writes: > Hi, > > I have purified a protein using anion exchange chromatography > followed by RP-HPLC on a C18 column with an acetonitrile/water gradient > (both containing 0.1% TFA). Re-running aliquots of my active fractions > on the C18 column gives a peak with the same elution time confirming the > presence of my protein. > My problem arises when analyzing the protein by > electrospray mass spectrometry. The protein has a predicted mass of > about 6.5 kD and is quite acidic with a pI of about 4.0. I have sent > samples (200-400 pmoles) of the protein to two different institutions for > analysis and in both cases they were unable to obtain a signal in either > positive or negative ion modes. It was suggested that residual TFA > remaining after lyophilization might be causing interference but > desalting the samples (PD-10 columns) against water prior to > lyophilization did not seem to help. > Any suggestions as to what might be causing this problem and > possible solutions would be greatly appreciated. Thanks. > > Joe > > From owner-proteins@net.bio.net Thu Apr 06 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!news.cac.psu.edu!news.pop.psu.edu!hudson.lm.com!godot.cc.duq.edu!ddsw1!infoserv.illinois.net!news.nd.edu!news1.oakland.edu!detroit.freenet.org!detroit.freenet.org!ag457 From: ag457@detroit.freenet.org (Jay Kulkarni) Newsgroups: bionet.molbio.proteins Subject: bcl-2 antibodies- need help!!!!!! Date: 7 Apr 1995 13:29:51 GMT Organization: The Greater Detroit Free-Net Lines: 7 Message-ID: <3m3eof$l8t@detroit.freenet.org> NNTP-Posting-Host: detroit.freenet.org Hi!! I am looking for bcl-2 antibody which will react with chicken bcl-2, does anyone have it? if so would you please let the the source? you help will be appreciated and ackowledged. thanx for you time. jay From owner-proteins@net.bio.net Sat Apr 08 23:00:00 1995 Path: biosci!newshost.lanl.gov!news.ttu.edu!seas.smu.edu!convex!insosf1.infonet.net!news-feed-1.peachnet.edu!hobbes.cc.uga.edu!cssun.mathcs.emory.edu!emory!swrinde!howland.reston.ans.net!news.cac.psu.edu!news.pop.psu.edu!psuvax1!news.eecs.nwu.edu!news.acns.nwu.edu!uicvm.uic.edu!u12201 Organization: University of Illinois at Chicago, ADN Computer Center Date: Sat, 8 Apr 1995 10:33:34 CDT From: Message-ID: <95098.103334U12201@uicvm.uic.edu> Newsgroups: bionet.molbio.proteins Subject: Re: Protein Crosslinking by UV References: <1995Apr6.133249.31068@oxvaxd> Lines: 3 If you need photocrosslinking, Pierce Chemical has a large selection of these. Also other xlinkers that would do what you need. E-mail: PierceChem@mcimail.com From owner-proteins@net.bio.net Sat Apr 08 23:00:00 1995 Path: biosci!bloom-beacon.mit.edu!gatech!howland.reston.ans.net!cs.utexas.edu!geraldo.cc.utexas.edu!daisy.cc.utexas.edu!not-for-mail From: rajeshk@daisy.cc.utexas.edu (rajesk) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Help ! Protein synthesis/transduction Date: 9 Apr 1995 16:48:24 -0500 Organization: The University of Texas at Austin; Austin, Texas Lines: 20 Message-ID: <3m9kn8$dnt@daisy.cc.utexas.edu> NNTP-Posting-Host: daisy.cc.utexas.edu Keywords: signal transduction, second messenger,protein synthesis, actinomycin D Xref: biosci bionet.molbio.methds-reagnts:26970 bionet.molbio.proteins:4225 Hi, I am posting this for a friend. Please post the responses here or mail to vaishali@jove.acs.unt.edu Many research papers on signal transduction, especially, second messengers, have one experiment when trying to elucidate activation of second messenger in response to any growth factor: Inhibition of protein synthesis with the help of general transcrition inhibitor like actinomycin D. The results of these experiments say that active protein synthesis is necessary for mediating second messenger response. The question is, what can one directly tell from this experiment? If it is second messenger response, how can we say that it requires protein synthesis as actinomycin will inhibit synthesis of all proteins ? Thanks Rajesh From owner-proteins@net.bio.net Sat Apr 08 23:00:00 1995 Path: biosci!agate!dog.ee.lbl.gov!news.cs.utah.edu!cc.usu.edu!rm323.vsb.usu.edu!gr8bn Newsgroups: bionet.molbio.proteins Subject: protein amount determination Message-ID: From: gr8bn@cc.usu.edu (I-Jen Huang) Date: Sun, 9 Apr 1995 17:21:25 GMT Organization: Program in Molecular Biology Nntp-Posting-Host: rm323.vsb.usu.edu Lines: 8 Dear netter Is there a good method to determin the amount of protein in a insoluble fraction ? Thanks for your help. I.Huang gr8bn@cc.usu.edu From owner-proteins@net.bio.net Sat Apr 08 23:00:00 1995 Path: biosci!daresbury!trane.uninett.no!Norway.EU.net!EU.net!howland.reston.ans.net!vixen.cso.uiuc.edu!uwm.edu!rutgers!oitnews.harvard.edu!cmcl2!is.NYU.EDU!beavis From: beavis@is.nyu.edu (beavis) Newsgroups: bionet.molbio.proteins Subject: Re: protein mass spec Date: 8 Apr 1995 19:26:24 GMT Organization: New York University Lines: 61 Message-ID: <3m6o10$iba@cmcl2.NYU.EDU> References: NNTP-Posting-Host: is.nyu.edu X-Newsreader: TIN [version 1.2 PL2] Lorne Taylor (ltaylor@peptidarus.UWaterloo.ca) wrote: : Joe...do you have the experimental details of the two failed attempts? : It would be interesting to see what kind of solvent/buffer system they both used? : Whenever I have a troublesome sample I try : a) dissolve sample in water or water/acetonitrile (as little ACN as possible!) : b) apply sample to a C18 disposable cartridge : c) wash C18 with copious amounts of water using compressed air to remove water : d) elute with neat formic acid : e) cross fingers and shoot sample : You might also want to try a MALDI MS run as MALDI is a lot more : tolerant to small amounts of contaminants than ESMS is. : Good Luck : Lorne Taylor : Protein Analysis Lab : University of Waterloo : In article , jbooth@ACS.UCALGARY.CA (Joseph Booth) writes: : > Hi, : > : > I have purified a protein using anion exchange chromatography : > followed by RP-HPLC on a C18 column with an acetonitrile/water gradient : > (both containing 0.1% TFA). Re-running aliquots of my active fractions : > on the C18 column gives a peak with the same elution time confirming the : > presence of my protein. : > My problem arises when analyzing the protein by : > electrospray mass spectrometry. The protein has a predicted mass of : > about 6.5 kD and is quite acidic with a pI of about 4.0. I have sent : > samples (200-400 pmoles) of the protein to two different institutions for : > analysis and in both cases they were unable to obtain a signal in either : > positive or negative ion modes. It was suggested that residual TFA : > remaining after lyophilization might be causing interference but : > desalting the samples (PD-10 columns) against water prior to : > lyophilization did not seem to help. : > Any suggestions as to what might be causing this problem and : > possible solutions would be greatly appreciated. Thanks. : > : > Joe : > : > I would have to agree with Lorne. Acidic proteins usually aggregate in solution because of traces of divalent cations. The electrospray process aggrevates this situation because of the drying of the droplets in the ion source interface region. MALDI would probably work better for this type of material. Ron Beavis Protein Chemistry Laboratory The Skirball Institute of Biomolecular Medicine New York University Medical Center email: beavis@is.nyu.edu home page: http://128.122.10.3/ From owner-proteins@net.bio.net Sat Apr 08 23:00:00 1995 Path: biosci!daresbury!trane.uninett.no!nntp.uio.no!nac.no!Norway.EU.net!EU.net!sun4nl!news.iaf.nl!biochem!gertjan From: gertjan@biochem.iaf.nl (Gert-Jan Euverink) Newsgroups: bionet.molbio.proteins Subject: Re: Pigments binding to enzymes Distribution: world Message-ID: <797282110.0snx@biochem.iaf.nl> References: <3m2lpr$6ie@newshound.uidaho.edu> Date: Fri, 07 Apr 95 19:15:10 GMT Reply-To: gertjan@biochem.iaf.nl Lines: 23 In article <3m2lpr$6ie@newshound.uidaho.edu> magn8831@uidaho.edu writes: > >I am working with an extracellular enzyme secreted by a Streptomyces spp. >My organism produces a fair amount of pigments, and these interfere >with purification and analysis. I've tried PVPP, Biopol, polyethylene- >imine etc. with not much luck to separate the pigments from the >enzyme. Standard purification procedures (MonoQ, Rotofor etc.) purify >activity somewhat, but the protein still has gunk on it. I don't want >to have to resort to gunk-out carb cleaner; any tips, advice, methods >would be greatly appreciated. Please respond to above address. > >Thanx >T. S. Magnuson > The best way to deal with the problem is to find a medium in which your Streptomyces does not produce pigments. The pigments are usually produced in the stationary phase. If your protein is also synthesized in the exponential growth phase you have separated your protein from the pigments. Maybe a cation exchanger or a hydrophobic interaction column binds the pigments mores stronger than your protein. Did you also try to dialyze against a high salt buffer ? From owner-proteins@net.bio.net Sat Apr 08 23:00:00 1995 Path: biosci!daresbury!trane.uninett.no!nac.no!Norway.EU.net!EU.net!news.sprintlink.net!cs.utexas.edu!swrinde!emory!news-feed-1.peachnet.edu!news.duke.edu!godot.cc.duq.edu!newsfeed.pitt.edu!uunet!in1.uu.net!bloom-beacon.mit.edu!mojo.eng.umd.edu!cs.umd.edu!news.umbc.edu!haven.umd.edu!purdue!mozo.cc.purdue.edu!bc211.biochem.purdue.edu!user From: mooret@aclcb.purdue.edu (hunk monger) Newsgroups: bionet.molbio.proteins Subject: Re: electroblotting of proteins Followup-To: bionet.molbio.proteins Date: Fri, 07 Apr 1995 09:11:03 +0800 Organization: Organization of Bored Grad Students Lines: 8 Distribution: world Message-ID: References: <9504062313.AA04770@mani.cbs.umn.edu> NNTP-Posting-Host: bc211.biochem.purdue.edu I had this problem several years ago with S & S's 0.1 µ nitrocellulose. The company sales rep refused to admit that it could happen but the problem was quite reproducible. The solution: don't use the very small pore size membrane. Use at least the 22 µ membranes and you should have no problem. Steve Broyles Purdue University From owner-proteins@net.bio.net Sat Apr 08 23:00:00 1995 Path: biosci!agate!news.mindlink.net!news.bc.net!unixg.ubc.ca!cellse.nce.ubc.ca!user From: flo@unixg.ubc.ca (Roger Graham) Newsgroups: bionet.molbio.proteins Subject: Enzyme Kinetics-Hehre Mech.--Help Followup-To: bionet.molbio.proteins Date: Sun, 09 Apr 1995 17:23:22 -0700 Organization: UBC Lines: 13 Message-ID: NNTP-Posting-Host: cellse.nce.ubc.ca I was wondering if there is anyone who is familiar with hydrolysis by glycosidases of glycosyl fluorides having the wrong anomeric configuration(Hehre mechanism). With this type of mechanism, hydrolysis is substrate activated and two molecules of substrate must bind in the active site first. Plots of RAte vs substrate concentration and 1/rate vs 1/substrate give upwardly concave curves. What I would like to know is what is the equation to describe these types of kinetics and if possible, how is it derived. A reference would be very helpful also. Please mail me directly. Thanks Howard howie@unixg.ubc.ca From owner-proteins@net.bio.net Sat Apr 08 23:00:00 1995 Path: biosci!agate!howland.reston.ans.net!cs.utexas.edu!geraldo.cc.utexas.edu!daisy.cc.utexas.edu!not-for-mail From: rajeshk@daisy.cc.utexas.edu (rajesk) Newsgroups: bionet.cellbiol,bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Help! Date: 9 Apr 1995 17:49:06 -0500 Organization: The University of Texas at Austin; Austin, Texas Lines: 21 Message-ID: <3m9o92$e4n@daisy.cc.utexas.edu> NNTP-Posting-Host: daisy.cc.utexas.edu Keywords: signal transduction, second messenger,protein synthesis, actinomycin Xref: biosci bionet.cellbiol:2035 bionet.molbio.methds-reagnts:26972 bionet.molbio.proteins:4226 Hi, I am posting this for a friend. Please post the responses here or mail to vaishali@jove.acs.unt.edu Many research papers on signal transduction, especially, second messengers, have one experiment when trying to elucidate activation of second messenger in response to any growth factor: Inhibition of protein synthesis with the help of general transcrition inhibitor like actinomycin D. The results of these experiments say that active protein synthesis is necessary for mediating second messenger response. The question is, what can one directly tell from this experiment? If it is second messenger response, how can we say that it requires protein synthesis as actinomycin will inhibit synthesis of all proteins ? Thanks Rajesh From owner-proteins@net.bio.net Sun Apr 09 23:00:00 1995 Path: biosci!rutgers!gatech!howland.reston.ans.net!agate!news.mindlink.net!vanbc.wimsey.com!unixg.ubc.ca!yossi From: yossi@unixg.ubc.ca (Yossef AvGay) Newsgroups: bionet.molbio.proteins Subject: what is the highest % of triton x-100 alowed in SDSPAGE Date: 10 Apr 1995 22:29:33 GMT Organization: University of British Columbia, Vancouver, B.C., Canada Lines: 8 Message-ID: <3mcbgd$af3@nnrp.ucs.ubc.ca> NNTP-Posting-Host: interchg.ubc.ca X-Newsreader: TIN [version 1.2 PL2] In the sample that i am applying to SDSPAGE i have about 0.2% triton before conc. I wondered wether higher % of triton will disturb migration of my protein ...... -- Dr. Yossef Av-Gay Microbiology Dept. U.B.C Vancouver B.C. Canada From owner-proteins@net.bio.net Sun Apr 09 23:00:00 1995 Path: biosci!rutgers!concert!gatech!howland.reston.ans.net!news.cac.psu.edu!news.tc.cornell.edu!travelers.mail.cornell.edu!newsstand.cit.cornell.edu!usenet From: "Tim Triche, Jr." Newsgroups: bionet.molbio.proteins Subject: Heat shock proteins and exercise Date: 10 Apr 1995 21:45:47 GMT Organization: Cornell University Lines: 17 Sender: tjt3@cornell.edu (Verified) Message-ID: <3mc8ub$j84@newsstand.cit.cornell.edu> NNTP-Posting-Host: j309003052.resnet.cornell.edu Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1b3 (Macintosh; I; 68K) X-URL: news:bionet.molbio.proteins/3673-4172#jpwscyto-0304941646290001@line06.kemp-du.pavilion.co.uk I was wondering, after having read the proceedings of the 1992 conference on activity and health, what new developments have been reported in the lit and elsewhere regarding production/expression of heat shock proteins in cardiac muscle? I'm new to the topic, but very interested, as I am a pre-med student with ACE/ACSM certification as a trainer and a neurobiology concentration in my major. So I am very curious as to the crossings over between brain functions under stress and chaperone proteins, and their relation to lipid peroxidation (as an indicator of potential peroxidation, not as a directly related phenomenon) during intense aerobic exercise. Thanks! the artiste formerly known as jabbo [] "Anyone who cannot explain his finger, ph, mailto:tjt3@cornell.edu [] work to a fourteen-year-old editor of the sci.med.nutrition FAQ [] is a charlatan." http://www.cen.uiuc.edu/cgi-bin/ryl [] Jacob Berzelius From owner-proteins@net.bio.net Sun Apr 09 23:00:00 1995 Path: biosci!HELIX.NIH.GOV!geetha From: geetha@HELIX.NIH.GOV (Geetha Vasudevan) Newsgroups: bionet.molbio.proteins Subject: Re: 3-10 helix backbone angles... Date: 10 Apr 1995 11:25:08 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 23 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net johnk@spasm.niddk.nih.gov (John Kuszewski) writes: >Can anyone tell me the standard backbone phi and >psi angles for a 3-10 helix? The mean observed phi,psi for 3-10 helices are -71,-18 deg. according to Barlow etal., 1988. It is -49,-26 deg. according to Perutz, 1951; It is -74, 4 deg. according to Pauling etal.,1951; Hope this helps. --GV | GEETHA VASUDEVAN Ph.#: (301)-402-0506 (w) | | Post Doctoral Visiting Fellow Fax#: (301)-496-2172 (w) | | Anal.Bio.Sec., Lab.of Str.Biol. E-mail: geetha@helix.nih.gov | | Bldg.12A/Room 2041,DCRT, NIH, Bethesda, MD 20892 | From owner-proteins@net.bio.net Sun Apr 09 23:00:00 1995 Path: biosci!bloom-beacon.mit.edu!newsserver.pixel.kodak.com!clpd-newsserver!news.sprintlink.net!howland.reston.ans.net!vixen.cso.uiuc.edu!uwm.edu!lll-winken.llnl.gov!venus.sun.com!nntp-hub2.barrnet.net!news3.near.net!yale!usenet From: abonvin@volta.csb.yale.edu ("Alexandre Bonvin") Newsgroups: bionet.molbio.proteins Subject: X-PLOR www pages Date: 10 Apr 1995 17:47:24 GMT Organization: Yale University, Department of Computer Science, New Haven, CT Lines: 17 Message-ID: <3mbqvc$iib@babyblue.cs.yale.edu> NNTP-Posting-Host: volta.csb.yale.edu Dear netters, Xplor has now a www home page featuring the X-PLOR manual and tutorial files on-line. The address is: http://pauli.csb.yale.edu Alexandre ========================================================================== | Alexandre Bonvin PhD | Phone: (203) 432-5066 | | Mol. Biophys. & Biochemistry | Fax: (203) 432-6946 | | Yale University, PO Box 208114 | Email: abonvin@laplace.csb.yale.edu | | New Haven CT 06511, USA | | ========================================================================== From owner-proteins@net.bio.net Sun Apr 09 23:00:00 1995 Path: biosci!rutgers!gatech!howland.reston.ans.net!vixen.cso.uiuc.edu!uwm.edu!lll-winken.llnl.gov!venus.sun.com!nntp-hub2.barrnet.net!news3.near.net!yale!usenet From: abonvin@volta.csb.yale.edu ("Alexandre Bonvin") Newsgroups: bionet.molbio.proteins Subject: X-PLOR www pages (correction) Date: 10 Apr 1995 17:54:23 GMT Organization: Yale University, Department of Computer Science, New Haven, CT Lines: 17 Message-ID: <3mbrcf$ik1@babyblue.cs.yale.edu> NNTP-Posting-Host: volta.csb.yale.edu Dear netters, Xplor has now a www home page featuring the X-PLOR manual and tutorial files on-line. The official address is: http://xplor.csb.yale.edu (pauli in the previous posting should work as well) Alexandre ========================================================================== | Alexandre Bonvin PhD | Phone: (203) 432-5066 | | Mol. Biophys. & Biochemistry | Fax: (203) 432-6946 | | Yale University, PO Box 208114 | Email: abonvin@laplace.csb.yale.edu | | New Haven CT 06511, USA | | ========================================================================== From owner-proteins@net.bio.net Sun Apr 09 23:00:00 1995 Path: biosci!rutgers!concert!gatech!howland.reston.ans.net!vixen.cso.uiuc.edu!ux1.cso.uiuc.edu!elarson From: elarson@ux1.cso.uiuc.edu (larson eric) Newsgroups: bionet.molbio.proteins Subject: Re: protein amount determination Date: 10 Apr 1995 17:18:47 GMT Organization: University of Illinois at Urbana Lines: 27 Message-ID: <3mbp9n$7s@vixen.cso.uiuc.edu> References: <9504101633.AA26169@bnlux1.bnl.gov.bnl.gov> NNTP-Posting-Host: ux1.cso.uiuc.edu berges@BNLUX1.BNL.GOV (John Berges) writes: >The BCA assay is badly messed up by any reducing compound such as >DTT and cysteine. The Bradford is not compatible with strong alkalis >or detergents. I have extraction buffers with both...and they have enough >NADH in them that abs(280) measurements are also tenuous. I'd be >happy to hear suggestions to overcome the problem. I currently TCA >precipitate sample, then resulspend in a satisfactory buffer. There is a >slight loss of sample, and the procedure is tedious. ========== Not to beat a dead horse, but if you add 0.0175% Sodium deoxycholate just before doing the TCA ppt., protein recovery is maximized (though you're now constrained to using the Lowry procedure I believe). Since Na deoxycholate breaks down in water (I don't know to what), the most convenient stock seems to be 1.75% NaDOC in DMSO with a 1:100 dilution into whatever buffer is to be carried forth into TCA ppt. I concur on the hassel of doing a ppt. prior to doing the Lowry (or any assay). -- Eric Larson | University of Illinois at Urbana-Champaign USDA/Agronomy | 190 PABL; 1201 W. Gregory; Urbana, IL 61801 elarson@ux1.cso.uiuc.edu | Voice 217.244.3079 Fax 217.244.4419 Fidonet: 1:233/4.1 | My opinions are my own, but correct :-) From owner-proteins@net.bio.net Sun Apr 09 23:00:00 1995 Path: biosci!rutgers!gatech!howland.reston.ans.net!vixen.cso.uiuc.edu!ux1.cso.uiuc.edu!elarson From: elarson@ux1.cso.uiuc.edu (larson eric) Newsgroups: bionet.molbio.proteins Subject: Re: protein amount determination Date: 10 Apr 1995 16:31:45 GMT Organization: University of Illinois at Urbana Lines: 34 Message-ID: <3mbmhh$l3m@vixen.cso.uiuc.edu> References: NNTP-Posting-Host: ux1.cso.uiuc.edu bucc0003@gold.tc.umn.edu (Paul A Bucciaglia) writes: >I use the BCA assay asay (Bicinchoninic acid/Cu), available as a kit from >Sigma (i don't have the original reference with me, write if you want >it). It tolerates up to 1% SDS, so you can solubilize your insoluble >fraction in SDS and then determine protein conc. Be sure to include your >solubilization buffer in the blank and your protein standards. Also be >sure the assay wil tolerate your particular solubilization components. >PS--I have found this assay to give a good standard curve, but that it >gives a protein concentration of about 1/10 of the reading from a >Bradford assay. Supposedly (and this is just hearsay) the BCA is more >accurate as it is much less sensitive to amino acid composition, so >perhaps this is the difference. Any net comments? >gr8bn@cc.usu.edu (I-Jen Huang) writes: >>Dear netter >>Is there a good method to determin the amount of protein in a insoluble >>fraction ? ============ The Lowry assay also works with 1% SDS present. You might try a procedure I am quite fond of: Larson EM, BT Howlett, AT Jagendorf (1986) Artificial reductant enhancement of the Lowry method for protein determination. Anal Biochem 155: 243-248. -- Eric Larson | University of Illinois at Urbana-Champaign USDA/Agronomy | 190 PABL; 1201 W. Gregory; Urbana, IL 61801 elarson@ux1.cso.uiuc.edu | Voice 217.244.3079 Fax 217.244.4419 Fidonet: 1:233/4.1 | My opinions are my own, but correct :-) From owner-proteins@net.bio.net Sun Apr 09 23:00:00 1995 Path: biosci!BNLUX1.BNL.GOV!berges From: berges@BNLUX1.BNL.GOV (John Berges) Newsgroups: bionet.molbio.proteins Subject: Re: protein amount determination Date: 10 Apr 1995 09:33:39 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 53 Sender: daemon@net.bio.net Distribution: world Message-ID: <9504101633.AA26169@bnlux1.bnl.gov.bnl.gov> NNTP-Posting-Host: net.bio.net On Mon, 10 Apr 1995 08:54:11 -0700 bucc0003@gold.tc.umn.edu (Paul A Bucciaglia) wrote: >Subject: Re: protein amount determination >I use the BCA assay asay (Bicinchoninic acid/Cu), available as a kit from >Sigma (i don't have the original reference with me, write if you want >it). It tolerates up to 1% SDS, so you can solubilize your insoluble >fraction in SDS and then determine protein conc. Be sure to include your >solubilization buffer in the blank and your protein standards. Also be >sure the assay wil tolerate your particular solubilization components. > >Good luck, >paul bucciaglia > >PS--I have found this assay to give a good standard curve, but that it >gives a protein concentration of about 1/10 of the reading from a >Bradford assay. Supposedly (and this is just hearsay) the BCA is more >accurate as it is much less sensitive to amino acid composition, so >perhaps this is the difference. Any net comments? I have pursued the issue of different protein assays giving different results a bit. The Bradford assay is more sensitive to arginine residues (better dye binding than a typical peptide bond), while the BCA and other copper- redox assays are very sensitive to tryptophan, tyrosine, etc. "Accuracy" is a tough one, because it depends on so many things. See: Berges et al. 1993. A comparison of Lowry, Bradford and Smith protein assays using different protein standards and protein isolated from the marine diatom Thalassiosira pseudonana. Marine Biology 115: 187-193 for a somewhat unsatisfying overview. The BCA assay is badly messed up by any reducing compound such as DTT and cysteine. The Bradford is not compatible with strong alkalis or detergents. I have extraction buffers with both...and they have enough NADH in them that abs(280) measurements are also tenuous. I'd be happy to hear suggestions to overcome the problem. I currently TCA precipitate sample, then resulspend in a satisfactory buffer. There is a slight loss of sample, and the procedure is tedious. ________________________________________________________________ _/_/_/ _/ _/ _/ Dr. John A. Berges _/ _/ _/ _/ _/ Oceanic and Atmospheric _/ _/ _/_/ _/ _/ Sciences Divison _/_/_/ _/ _/ _/ _/ Brookhaven National Lab _/ _/ _/ _/ _/ _/ Upton, NY 11973 _/ _/ _/ _/_/ _/ Phone: 516-282-4077 _/_/_/_/ _/ _/ _/_/_/_/ Email: berges@BNLUX1.BNL.GOV ________________________________________________________________ From owner-proteins@net.bio.net Sun Apr 09 23:00:00 1995 Path: biosci!rutgers!gatech!swrinde!howland.reston.ans.net!spool.mu.edu!umn.edu!newsstand.tc.umn.edu!gold.tc.umn.edu!bucc0003 From: bucc0003@gold.tc.umn.edu (Paul A Bucciaglia) Newsgroups: bionet.molbio.proteins Subject: Re: protein amount determination Date: 10 Apr 1995 10:25:05 -0500 Organization: University of Minnesota Lines: 28 Message-ID: References: NNTP-Posting-Host: gold.tc.umn.edu I use the BCA assay asay (Bicinchoninic acid/Cu), available as a kit from Sigma (i don't have the original reference with me, write if you want it). It tolerates up to 1% SDS, so you can solubilize your insoluble fraction in SDS and then determine protein conc. Be sure to include your solubilization buffer in the blank and your protein standards. Also be sure the assay wil tolerate your particular solubilization components. Good luck, paul bucciaglia PS--I have found this assay to give a good standard curve, but that it gives a protein concentration of about 1/10 of the reading from a Bradford assay. Supposedly (and this is just hearsay) the BCA is more accurate as it is much less sensitive to amino acid composition, so perhaps this is the difference. Any net comments? gr8bn@cc.usu.edu (I-Jen Huang) writes: >Dear netter >Is there a good method to determin the amount of protein in a insoluble >fraction ? >Thanks for your help. >I.Huang >gr8bn@cc.usu.edu From owner-proteins@net.bio.net Sun Apr 09 23:00:00 1995 Path: biosci!agate!howland.reston.ans.net!EU.net!uknet!strath-cs!st-and!Aberdeen!ort020 From: ort020@nof.abdn.ac.uk (a.vijayan) Newsgroups: bionet.molbio.proteins Subject: Re: electroblotting of proteins Date: 10 Apr 1995 13:28:22 GMT Organization: University of Aberdeen, Scotland Lines: 23 Distribution: world Message-ID: <3mbbpm$ij3@nof.abdn.ac.uk> References: <9504062313.AA04770@mani.cbs.umn.edu> NNTP-Posting-Host: med.abdn.ac.uk X-Newsreader: TIN [version 1.2 PL2] Nora Plesofsky-Vig (npv@MANI.CBS.UMN.EDU) wrote: : I have a problem concerning the electrophoretic blotting of proteins : from a polyacrylamide gel to a small pore PVDF membrane, using a : Hoeffer Transfor apparatus, TE 50. I do the transfer in a 10 mM CAPS : transfer buffer, pH 11, 10% methanol, since I wish to sequence the : transferred peptides. I cool the buffer to 10-12oC, use a stir bar, : make every attempt to remove bubbles, but still I get protein bands : swirling within the membrane as though local ion currents have been : set up. This only occurs in certain (unpredictable) areas, but : obviously it would be desirable to avoid this. : Does anyone have any experience with this problem? Any likely : solutions? : Thanks for any suggestions. : Nora Plesofsky-Vig : nora@biosci.cbs.umn.edu Try a different batch of memberanes. Before this you could try changing the gel compositions to a different percentage and see this makes any difference. From owner-proteins@net.bio.net Sun Apr 09 23:00:00 1995 Path: biosci!agate!howland.reston.ans.net!swrinde!pipex!sunsite.doc.ic.ac.uk!yama.mcc.ac.uk!io.salford.ac.uk!usenet From: R.Lauder@lancaster.ac.uk (Bob Lauder) Newsgroups: bionet.molbio.proteins Subject: Re: re.glycoproteins Date: 10 Apr 1995 10:35:58 GMT Organization: Lancaster University Lines: 21 Mess