From owner-proteins@net.bio.net Sat Feb 01 22:00:00 1997 Path: biosci!daresbury!nntp-trd.UNINETT.no!nntp.uio.no!newsfeeds.sol.net!news.maxwell.syr.edu!insync!Gamma.RU!srcc!demos!news1.best.com!newshub1.home.com!news.home.com!ames!purdue!mozo.cc.purdue.edu!news From: alan friedman Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Thermotoga maritima DNA/cells Date: Sun, 02 Feb 1997 14:50:02 -0600 Organization: Purdue University Lines: 6 Message-ID: <32F4FB0D.85@bilbo.bio.purdue.edu> Reply-To: afried@bilbo.bio.purdue.edu NNTP-Posting-Host: friedman.bio.purdue.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (Macintosh; I; PPC) Xref: biosci bionet.molbio.methds-reagnts:54273 bionet.molbio.proteins:9890 Does anyone know a source of Thermotoga maritima genomic DNA suitable for PCR cloning? Does anyone have T. maritima cells? And are they hard to grow and extract DNA from? Thanks. alan friedman From owner-proteins@net.bio.net Sun Feb 02 22:00:00 1997 Path: biosci!agate!newsgate.cuhk.edu.hk!news.glink.net.hk!uunet!in2.uu.net!136.142.185.26!newsfeed.pitt.edu!mv29.pathology.pitt.edu!user From: pxpst2@vms.cis.pitt.edu (THE GREEK) Newsgroups: bionet.molbio.proteins Subject: human coagulation proteins Date: 3 Feb 1997 16:10:15 GMT Organization: USA Lines: 3 Message-ID: NNTP-Posting-Host: mv29.pathology.pitt.edu I need antibodies and probes for EPR-1, factor V, Factor IX, Factor VII, Factor X, and tissue factor. Any help would be greatly appreciated Peter Pediaditakis pxpst2@vms.cis.pitt.edu From owner-proteins@net.bio.net Sun Feb 02 22:00:00 1997 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!cam-news-hub1.bbnplanet.com!howland.erols.net!ix.netcom.com!news From: Philip Onigman Newsgroups: bionet.molbio.proteins Subject: Amicon now part of Millipore Date: Tue, 04 Feb 1997 03:12:09 GMT Organization: Netcom Lines: 8 Message-ID: <5d68od$kiq@dfw-ixnews11.ix.netcom.com> NNTP-Posting-Host: bos-ma6-16.ix.netcom.com X-NETCOM-Date: Mon Feb 03 9:00:29 PM CST 1997 X-Newsreader: NETCOMplete/3.0 Amicon and Millipore are now one company. This new organization should be better able to supply biochemists purifying proteins with up to date tools etc. See http://www.millipore.com for info and to post questions. Philip Onigman Millipore Corp Philip_Onigman@millipore.com From owner-proteins@net.bio.net Sun Feb 02 22:00:00 1997 Path: biosci!agate!howland.erols.net!news.nacamar.de!uunet!in2.uu.net!128.250.1.21!munnari.OZ.AU!metro!metro!news From: Phillip Robinson Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: Protein Labeling? Casein Kinase? Date: Tue, 04 Feb 1997 00:00:07 +1100 Organization: Information Technology Services, The University of Sydney, NSW, Australia Lines: 13 Distribution: inet Message-ID: <32F5E157.53FD@mail.usyd.edu.au> References: <5cm8jd$n4j@news.fas.harvard.edu> <32F42C77.3BAC@students.wisc.edu> NNTP-Posting-Host: modem-a-215.mp.usyd.edu.au Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0 (Win95; I) Xref: biosci bionet.molbio.methds-reagnts:54284 bionet.molbio.proteins:9891 > I have used Casein Kinase II to lable a protein from cytosol, and then > was able to immunoprecipitate it, producing a nicely labled band. My > protein has four CKII consensus sites (minimum D/E-X-X-S/T), with > several other acidic residues in close proximity. Check if your protein > has any CKII sites, and if so, CKII is commercially available (I use > B-Mannhein), and should work well. I have no experience with CKI. E-mail > if you need CKII rnx conditions. Funny, my memory says that the acidic amino acids should be in the +3 position, not the -3. In other words CKII consensus site minimum S/T-X-X-D/E. Phil From owner-proteins@net.bio.net Sun Feb 02 22:00:00 1997 Path: biosci!agate!usenet.kornet.nm.kr!xfer.kren.nm.kr!hammer.uoregon.edu!hunter.premier.net!newsfeeds.sol.net!uwm.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!news From: "Thomas J. Albert" Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: Protein Labeling? Casein Kinase? Date: Mon, 03 Feb 1997 22:49:52 -0600 Organization: UW-Madison Lines: 14 Message-ID: <32F6BFF0.51FD@students.wisc.edu> References: <5cm8jd$n4j@news.fas.harvard.edu> <32F42C77.3BAC@students.wisc.edu> <32F5E157.53FD@mail.usyd.edu.au> Reply-To: tjalbert@students.wisc.edu NNTP-Posting-Host: f182-164.net.wisc.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0 (Win95; I) Xref: biosci bionet.molbio.methds-reagnts:54322 bionet.molbio.proteins:9896 Phillip Robinson wrote: > > Funny, my memory says that the acidic amino acids should be in the +3 > position, not the -3. In other words CKII consensus site minimum > S/T-X-X-D/E. > > Phil I appologize, My dislexia is showing itself, S/T-X-X-D/E is correct, sorry about any misinformation. Tom From owner-proteins@net.bio.net Sun Feb 02 22:00:00 1997 Path: biosci!NBRF.GEORGETOWN.EDU!POSTMASTER From: POSTMASTER@NBRF.GEORGETOWN.EDU Newsgroups: bionet.molbio.proteins Subject: Re: Short sequence search? Date: 3 Feb 1997 13:37:05 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 12 Sender: daemon@net.bio.net Distribution: world Message-ID: <01IEZI1WVIYE9BVDCQ@NBRF.Georgetown.Edu> NNTP-Posting-Host: net.bio.net On 01 Feb 1997, Chihara asked > Is there a program that will search genbank or prosite etc. for short > amino acid sequnces? i.e. 5-7 residues? To search the PIR Protein Sequence Databases you can try http://www-nbrf.georgetown.edu/nbrf/scan.html ------------------------------------------------------------------------ Dr. John S. Garavelli Associate Director Protein Information Resource National Biomedical Research Foundation Washington, DC 20007 POSTMASTER@NBRF.GEORGETOWN.EDU From owner-proteins@net.bio.net Sun Feb 02 22:00:00 1997 Path: biosci!CS.Arizona.EDU!news.Arizona.EDU!uunet!in1.uu.net!128.6.21.17!dziuxsolim.rutgers.edu!amenti.rutgers.edu!not-for-mail From: ghall@rci.rutgers.edu (Gene Hall) Newsgroups: bionet.molbio.proteins Subject: Retinol binding protein Date: 1 Feb 1997 22:43:54 -0500 Organization: Rutgers University Lines: 7 Message-ID: <5d12hq$m2b@amenti.rutgers.edu> NNTP-Posting-Host: amenti.rutgers.edu Keywords: retinol binding protein Can someone help with what is retinol binding protein function is the human body. Why does it show up in urine and why is it in plasma? Thanks in advance, Eugene From owner-proteins@net.bio.net Mon Feb 03 22:00:00 1997 Path: biosci!rutgers!gatech!howland.erols.net!ix.netcom.com!netcom.net.uk!nntpfeed.doc.ic.ac.uk!sunsite.doc.ic.ac.uk!warwick!leicester!usenet From: "Dr E. Buxbaum" Newsgroups: bionet.molbio.proteins Subject: Re: Big Problems with ELISA Date: 4 Feb 1997 17:59:36 GMT Organization: University of Leicester, UK (PCFS User) Lines: 12 Message-ID: <5d7te8$9c9@falcon.le.ac.uk> References: <01bc0a46$022997c0$6a039386@schmidtdw.rz.ruhr-uni-bochum.de> <5d77an$d7q@info-server.surrey.ac.uk> NNTP-Posting-Host: pc47.msb.le.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) >>Another problem is that I want to make the ELISA by binding >>a polypeptid to the microtiter-plate wells (Maxisorp, Nunc). >>But I have the impression that the binding to the plate is extremely poor. >>I did it in PBS and 0,2 M Carbonate-Buffer pH 9,3. >>What can I do to increase the peptid´s binding? There are plates available with -COOH, -NH2 or -SH groups at their surface. These can be used to chemically couple your protein to the plate with appropriate crosslinkers. This may give better results than simple adsorption, especially for small proteins or peptides. I once used the -COOH variety with DCCD as crosslinker and got very reproducible results. From owner-proteins@net.bio.net Mon Feb 03 22:00:00 1997 Path: biosci!bcm.tmc.edu!cs.utexas.edu!news.sprintlink.net!news-peer.sprintlink.net!news.maxwell.syr.edu!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!cam-news-hub1.bbnplanet.com!howland.erols.net!vixen.cso.uiuc.edu!uwm.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!daemon From: klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins Subject: Re: enzyme quantification by OD? Date: Wed, 05 Feb 97 02:35:41 GMT Organization: UW-Madison Lines: 29 Message-ID: <5d8rom$2pga@news.doit.wisc.edu> References: <5c2shp$3l04@news.doit.wisc.edu> <1997Feb4.112309.93237@cc.usu.edu> NNTP-Posting-Host: f182-167.net.wisc.edu Mime-Version: 1.0 Content-Type: text/plain; charset=KOI8-R Content-Transfer-Encoding: 8bit X-Newsreader: News Xpress 2.0 X-No-Archive: Yes In article , debi@monmouth.com (Glen Ramsay) wrote: #In article <1997Feb4.112309.93237@cc.usu.edu>, arsphys@cc.usu.edu (Phil #Harrison) wrote: # #> klenchin@facstaff.wisc.edu (Dima Klenchin) wrote: #> #> >In article , #ammacleo@sfu.ca (Alasdair MacLeod) wrote: #> ># I am trying to figure out the best way to accurately determine the #> >#concentration of a mutant enzyme and can't use an active-site titrant. #> >#I am leary of dye-binding and the Lowry method to accurately quantify this #> >#protein. Is anyone else of the opinion that absorbance OD280 (ex. method #> >#of Scopes) is any better? #> #> >Well, if you have both proteins in pure form AND mutation does not replace #> >aromatic aa (esp. tryptophan), this is, IMHO, the best way to go. #> # #How about this: use amino acid analysis to obtain a rock solid measure of #a single stock of your protein, and using this concentration calculate the #extinction coefficient for the protein at 280 nm. This will eliminate #troubles from varying coefficients between different protein, and the #amino acid analysis will have to be done only once. ^^^^^ Twice. With different protein preparations and different aa analysis facilities... From owner-proteins@net.bio.net Mon Feb 03 22:00:00 1997 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.erols.net!worldnet.att.net!news.maxwell.syr.edu!news.bc.net!uvaix3e1.comp.UVic.CA!hera.bioc.uvic.ca!cupton From: cupton@uvic.ca (Chris Upton) Newsgroups: bionet.molbio.proteins,bionet.software Subject: pI calculation Followup-To: bionet.molbio.proteins Date: Wed, 5 Feb 97 00:49:28 GMT Organization: University of Victoria Lines: 22 Message-ID: NNTP-Posting-Host: hera.bioc.uvic.ca X-Newsreader: VersaTerm Link v1.1.6 Xref: biosci bionet.molbio.proteins:9910 bionet.software:17787 Hi, I'm looking for a way to query a protein database (eg Swiss-Prot) by pI. ie get all proteins with pI > 8. I'm not sure if there's programs to allow calc of pI for all proteins in the database. I guess output file could be large :-) Then I might want to sort them.... Hmm, alternatively it would be nice to at least be able to get the pI for 1-200 proteins. Is there anything that will allow batch processing? I seem to think I did this in the very distant past with 200 proteins, either with GCG or IG. Any advice appreciated.... Cheers, Chris Upton Chris Upton Biochemistry & Microbiology University of Victoria phone 250-721-6507 BC Canada fax 250-721-8855 From owner-proteins@net.bio.net Mon Feb 03 22:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!europa.clark.net!worldnet.att.net!feed1.news.erols.com!howland.erols.net!rill.news.pipex.net!pipex!warwick!bignews.shef.ac.uk!usenet From: G.E.Martin@shef.ac.uk (G E Martin) Newsgroups: bionet.molbio.proteins Subject: Non aqueous solvent for enzyme + substrate Date: 4 Feb 1997 15:36:18 GMT Organization: Molecular Biology & Biotechnology, University of Sheffield , UK Lines: 10 Message-ID: <5d7l1i$482@bignews.shef.ac.uk> NNTP-Posting-Host: pc065133.shef.ac.uk Mime-Version: 1.0 Content-Type: Text/Plain; charset=US-ASCII X-Newsreader: WinVN 0.99.7 I have a peripheral membrane protein with a hydrophobic substrate and have been studying simple enzyme kinetics in dilute detergent solutions. If I do away with the detergent the substrate precipitates. As the protein may form dimers and tetramers I would like to assay without detergents to see whether their is any cooperativity for example. Does anyone have ideas for alternative solvents ?? Many thanks Giles. From owner-proteins@net.bio.net Mon Feb 03 22:00:00 1997 Path: biosci!LUCANO.UCO.ES!bb2claus From: bb2claus@LUCANO.UCO.ES (Peter) Newsgroups: bionet.molbio.proteins Subject: electrophoresis at pH 8,0 Date: 4 Feb 1997 07:08:08 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 5 Sender: daemon@net.bio.net Distribution: world Message-ID: <32F76DA6.6E7A@lucano.uco.es> NNTP-Posting-Host: net.bio.net Does anybody know about a protein-electrophoresis-system that is able to resolve and focus satisfying at pH 8.0. I tried Tris-Acetate-System (0.05 M) and the results were not satisfying (bands do not focus well enough). Is there a system that makes a stacking like the Laemmli-System? From owner-proteins@net.bio.net Mon Feb 03 22:00:00 1997 Message-ID: <32F74BA4.3F54@sgiclu.chemie.uni-konstanz.de> Date: Tue, 04 Feb 1997 15:45:56 +0100 From: Marcus Macht X-Mailer: Mozilla 3.01 (X11; I; IRIX 5.3 IP22) MIME-Version: 1.0 Newsgroups: bionet.molbio.proteins Subject: Re: MALDI-TOF is suitable to identify whether a protein is a dimer or a trimer? References: <32E843A6.28FF@plaza.snu.ac.kr> Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit NNTP-Posting-Host: sg17.chemie.uni-konstanz.de Organization: University of Constance, Germany Lines: 40 Path: biosci!daresbury!nntp-trd.UNINETT.no!online.no!sn.no!www.nntp.primenet.com!nntp.primenet.com!su-news-hub1.bbnplanet.com!news.bbnplanet.com!cam-news-hub1.bbnplanet.com!uunet!in1.uu.net!160.45.4.4!fu-berlin.de!news.belwue.de!news.uni-konstanz.de!sg17.chemie.uni-konstanz.de Lee, Ji Hyun wrote: > > MALDI-TOF analysis is suitable to identifying whether a protein is a > homodimer or a homotrimer? > > I have a small DNA-binding protein(6,100 dalton) quite well purified. > Through size exclusion HPLC gel chromatography, I found its effective > molecular weight of the protein in the absence of its binding DNA > sequence is about 19,000 dalton, close to homotrimer. > But I am not sure that it is a trimer based on only the result becuase I > didn't ever hear about homotrimer DNA binding protein; so I need some > additional experiments confirming this fact. > > Thank you for your time and expertise, > > Lee, Ji Hyun I would not suggest MALDI-MS for your problem. Normal acidic matrix solution conditions are far away from native conditions. Furthermore you perform a MALDI analysis normally from crystallized matrix. I think Electrospray-MS will be a much better method to determine if your protein is a monomer, dimer or trimer. But you will have to look very closely to the results because absence of dimer or trimer will not mean that there is no such oligomer because the relation between monomer and hihger adducts strongly depends on the design of the instrument, the solution conditions etc. Another possibility for determination of the aggregation state of your protein would be analytical ultracentrifugation. What about crosslinking and SDS-PAGE? I think there are some more (and probably much better) possibilities than MALDI-MS for determination of non-covalent oligomers. I hope I could help, yours sincerely Marcus Macht * Marcus Macht, AG Przybylski, Faculty Chemistry, University of Konstanz * PO Box 5560 M 732, 78464 Konstanz, Germany * Tel:++49-07531-883436/ Fax:++49-07531-883097 * Email: macht@sgiclu.chemie.uni-konstanz.de * WWW: http://www.ag-przybylski.chemie.uni-konstanz.de/~macht From owner-proteins@net.bio.net Mon Feb 03 22:00:00 1997 Path: biosci!agate!spool.mu.edu!uwm.edu!cs.utexas.edu!howland.erols.net!wits.uni.net.za!csir.uni.net.za!und.uni.net.za!und.ac.za!bchm1 From: morty@biochem.unp.ac.za (Rory Morty) Newsgroups: bionet.molbio.proteins Subject: Peptide hormones Date: Tue, 04 Feb 97 12:35:48 GMT Organization: University of Natal Lines: 18 Message-ID: <855074313.887212@unpsun3.cc.unp.ac.za> NNTP-Posting-Host: unpsun3.cc.unp.ac.za X-Newsreader: News Xpress 2.0 Beta #0 Keyword: peptide hormone, regulatory peptide, hormone Cache-Post-Path: unpsun3.cc.unp.ac.za!unknown@bchm1.bchm.unp.ac.za Hi all, I would be most grateful if somebody could direct me to any organisation, company or individual who may be willing to perform peptide hormone assays. The compounds I would like assayed for are: (1) Neurotensin (2) Vasoactive Intestinal Polypeptide (3) Atrial Natriuretic Factor (4) Glucagon (5) B-melanocyte stimulating hormone (6) B-endorphin PLease e-mail me. Many thanks in advance. Rory Morty e-mail: morty@bchm.unp.ac.za From owner-proteins@net.bio.net Mon Feb 03 22:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!europa.clark.net!arclight.uoregon.edu!news.bbnplanet.com!su-news-hub1.bbnplanet.com!newsout1.alt.net!news1.alt.net!news.aros.net!news.cs.utah.edu!cc.usu.edu!cc.usu.edu!nntp Newsgroups: bionet.molbio.proteins Subject: Re: enzyme quantification by OD? Message-ID: <1997Feb4.112309.93237@cc.usu.edu> From: arsphys@cc.usu.edu (Phil Harrison) Date: Tue, 04 Feb 1997 18:48:33 GMT References: <5c2shp$3l04@news.doit.wisc.edu> Nntp-Posting-Host: rm16.frrl.usu.edu X-Newsreader: Forte Free Agent 1.0.82 Lines: 30 klenchin@facstaff.wisc.edu (Dima Klenchin) wrote: >In article , ammacleo@sfu.ca (Alasdair MacLeod) wrote: ># I am trying to figure out the best way to accurately determine the >#concentration of a mutant enzyme and can't use an active-site titrant. >#I am leary of dye-binding and the Lowry method to accurately quantify this >#protein. Is anyone else of the opinion that absorbance OD280 (ex. method >#of Scopes) is any better? >Well, if you have both proteins in pure form AND mutation does not replace >aromatic aa (esp. tryptophan), this is, IMHO, the best way to go. >- Dima I have been using OD 280 with a rule-of-thumb that 1 absorbance unit was equal to 1 mg/ml protein. I used this in early stages of enzyme purification from a plant extract. I recently checked this out by comparison with the coomassie blue dye-binding (Bradford) assay. I found that the OD method overestimated the protein concentration by about 5-fold. This was after ammonium sulfate and desalting, so no free amino acids or anything else under ca. 5-10,000 would have been present. I suppose you could say on the other hand that the Bradford assay underestimated the protein concentration. But you have to believe in something, don't you? Phil Harrison arsphys@cc.usu.edu From owner-proteins@net.bio.net Mon Feb 03 22:00:00 1997 Path: biosci!agate!spool.mu.edu!howland.erols.net!quagga.ru.ac.za!ru.uni.net.za!und.uni.net.za!und.ac.za!bchm1 From: morty@biochem.unp.ac.za (Rory Morty) Newsgroups: bionet.molbio.proteins,bionet.general,bionet.molbio.methds-reagnts Subject: peptide hormone assays Date: Tue, 04 Feb 97 12:32:43 GMT Organization: University of Natal Lines: 19 Message-ID: <855074128.699433@unpsun3.cc.unp.ac.za> NNTP-Posting-Host: unpsun3.cc.unp.ac.za X-Newsreader: News Xpress 2.0 Beta #0 Keyword: peptide hormone, regulatory peptide, hormone Cache-Post-Path: unpsun3.cc.unp.ac.za!unknown@bchm1.bchm.unp.ac.za Xref: biosci bionet.molbio.proteins:9898 bionet.general:25480 bionet.molbio.methds-reagnts:54340 Hi! I would be most grateful if somebody could direct me to any person, organisation or company who may be willing to perform peptide hormone assays for me. The peptide hormes that I would like assay are: (1) B-melanocyte stimulating hormone (2) Neurotensin (3) Adrenocorticotropic hormone (4) B-nedorphin (5) Glucagon (6) Vasoactive Intestinal Polypeptide Any information would be much appreciated. Please e-mail me. Many thanks Rory Morty e-mail: morty@bchm.unp.ac.za From owner-proteins@net.bio.net Mon Feb 03 22:00:00 1997 Path: biosci!rutgers!gatech!www.nntp.primenet.com!nntp.primenet.com!mr.net!netnews.com!howland.erols.net!ix.netcom.com!netcom.net.uk!nntpfeed.doc.ic.ac.uk!sunsite.doc.ic.ac.uk!warwick!leicester!usenet From: "Dr E. Buxbaum" Newsgroups: bionet.molbio.proteins Subject: Re: electrophoresis at pH 8,0 Date: 4 Feb 1997 18:02:02 GMT Organization: University of Leicester, UK (PCFS User) Lines: 11 Message-ID: <5d7tiq$9c9@falcon.le.ac.uk> References: <32F76DA6.6E7A@lucano.uco.es> NNTP-Posting-Host: pc47.msb.le.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) bb2claus@LUCANO.UCO.ES (Peter) wrote: >Does anybody know about a protein-electrophoresis-system that is able to >resolve and focus satisfying at pH 8.0. I tried Tris-Acetate-System >(0.05 M) and the results were not satisfying (bands do not focus well >enough). Is there a system that makes a stacking like the >Laemmli-System? You could use DISK-electrophoresis with barbiturate buffer. It is a non-denaturating technique, don't know wether it can be used with detergent to. From owner-proteins@net.bio.net Mon Feb 03 22:00:00 1997 Path: biosci!CMU.CHIANGMAI.AC.TH!mdbci001 From: mdbci001@CMU.CHIANGMAI.AC.TH (Prachya Kongtawelert) Newsgroups: bionet.molbio.proteins Subject: Re: Big Problems with ELISA Date: 4 Feb 1997 17:20:20 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 68 Sender: daemon@net.bio.net Distribution: world Message-ID: References: <5d77an$d7q@info-server.surrey.ac.uk> NNTP-Posting-Host: net.bio.net On Tue, 4 Feb 1997, Pete wrote: > "Thorsten Schmidt" wrote: >=20 > >Dear Reader! >=20 > >I have big problems with ELISA. > >The main problem is that the color does not develop satisfactorally. >=20 > >=20 > >Where is the problem? What can I do? > >Why didn=B4t I get more color development? >=20 > Only you can determine precisely what is happening, but are you sure > that your stock H2O2 is OK and not degraded to just H2O? Have you > tried a different batch of enzyme label? And finally, are you sure > that you have no residual azide in you buffers (includind wash > buffers) Another thing that should be aware is citrate buffer, especially the pH=20 of this buffer which is should be around 5-5.5. We have used and kept=20 H2O2 for nearly 5 years in the fridge and it is still working well! >=20 > >Which OPD-solution do you use? >=20 > For many years we have now not used OPD at all. All our colorimetric > assays now use TMB as substrate. This is because it is not > carcinogenic like OPD, and the colour yield is about double that of > OPD. >=20 I am so sure that TMB (benzidine?) is not carcinogenic and have read one=20 paper comparison of the peroxidase substrate, and they reported that the=20 opd is the best (have to have a look that paper again!) anyway the point=20 is can individual show a good standard and dose response curve? > >Another problem is that I want to make the ELISA by binding > >a polypeptid to the microtiter-plate wells (Maxisorp, Nunc). > >But I have the impression that the binding to the plate is extremely poo= r. > >I did it in PBS and 0,2 M Carbonate-Buffer pH 9,3. > >What can I do to increase the peptid=B4s binding? >=20 > Here you could have two different problems. It is not unknown for > small peptides to be deformed in the adsorption process to as to "bend > out of shape" the antigenic epitope - thus reducing binding and > therefore signal. Or yo may as you suggest be getting poor binding - > try a whole series of buffers for binding bicarbonate/PBS/TBS/anything > handy at a range of pH's from 5 to about 10 (or even more exteme at > both ends if it looks useful). This should give you an idea of what's > best - and you could also try increasing the molarity of the binding > buffer if you start getting desperate! >=20 > >It would be too kind of you if you=B4ll send me an answer! >=20 > >Thank you in advance >=20 > >Thorsten Schmidt >=20 > Pete >=20 >=20 >=20 Prachya From owner-proteins@net.bio.net Mon Feb 03 22:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!www.nntp.primenet.com!nntp.primenet.com!news.bbnplanet.com!su-news-hub1.bbnplanet.com!su-news-feed4.bbnplanet.com!jeeves.usfca.edu!news From: Carol Chihara Newsgroups: bionet.molbio.proteins Subject: Thanks Date: Tue, 04 Feb 1997 15:31:30 -0700 Organization: USF Department of Biology Lines: 1 Message-ID: <32F7B8C2.4617@usfca.edu> NNTP-Posting-Host: chiharac.usfca.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0 (Macintosh; I; 68K) Many thanks to all the responders to my query re short peptide search! From owner-proteins@net.bio.net Mon Feb 03 22:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!www.nntp.primenet.com!nntp.primenet.com!news.maxwell.syr.edu!insync!uunet!in3.uu.net!205.164.220.8!news.monmouth.com!fh-ppp39.monmouth.com!user From: debi@monmouth.com (Glen Ramsay) Newsgroups: bionet.molbio.proteins Subject: Re: enzyme quantification by OD? Date: Tue, 04 Feb 1997 18:28:46 -0500 Organization: Aviv Instruments Lines: 27 Message-ID: References: <5c2shp$3l04@news.doit.wisc.edu> <1997Feb4.112309.93237@cc.usu.edu> NNTP-Posting-Host: fh-ppp19.monmouth.com X-Newsreader: Yet Another NewsWatcher 2.1.8 In article <1997Feb4.112309.93237@cc.usu.edu>, arsphys@cc.usu.edu (Phil Harrison) wrote: > klenchin@facstaff.wisc.edu (Dima Klenchin) wrote: > > >In article , ammacleo@sfu.ca (Alasdair MacLeod) wrote: > ># I am trying to figure out the best way to accurately determine the > >#concentration of a mutant enzyme and can't use an active-site titrant. > >#I am leary of dye-binding and the Lowry method to accurately quantify this > >#protein. Is anyone else of the opinion that absorbance OD280 (ex. method > >#of Scopes) is any better? > > >Well, if you have both proteins in pure form AND mutation does not replace > >aromatic aa (esp. tryptophan), this is, IMHO, the best way to go. > How about this: use amino acid analysis to obtain a rock solid measure of a single stock of your protein, and using this concentration calculate the extinction coefficient for the protein at 280 nm. This will eliminate troubles from varying coefficients between different protein, and the amino acid analysis will have to be done only once. Of course this assumes that nothing is done to the protein to change to coefficient (mutation, ligand binding, solvent conditions). Glen From owner-proteins@net.bio.net Mon Feb 03 22:00:00 1997 Path: biosci!daresbury!nntp-trd.UNINETT.no!nntp.uio.no!newsfeeds.sol.net!uwm.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!martinlab From: klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins Subject: Re: enzyme quantification by OD? Date: Tue, 04 Feb 97 21:13:57 GMT Organization: UW-Madison Lines: 51 Message-ID: <5d8cdo$36di@news.doit.wisc.edu> References: <5c2shp$3l04@news.doit.wisc.edu> <1997Feb4.112309.93237@cc.usu.edu> NNTP-Posting-Host: martinlab.biochem.wisc.edu X-Newsreader: News Xpress Version 1.0 Beta #4 X-No-Archive: Yes In article <1997Feb4.112309.93237@cc.usu.edu>, arsphys@cc.usu.edu (Phil Harrison) wrote: ->klenchin@facstaff.wisc.edu (Dima Klenchin) wrote: -> ->>In article , ammacleo@sfu.ca (Alasdair MacLeod) wrote: ->># I am trying to figure out the best way to accurately determine the ->>#concentration of a mutant enzyme and can't use an active-site titrant. ->>#I am leary of dye-binding and the Lowry method to accurately quantify this ->>#protein. Is anyone else of the opinion that absorbance OD280 (ex. method ->>#of Scopes) is any better? -> ->>Well, if you have both proteins in pure form AND mutation does not replace ^^ ^^^^ ->>aromatic aa (esp. tryptophan), this is, IMHO, the best way to go. -> ->>- Dima -> -> ->I have been using OD 280 with a rule-of-thumb that 1 absorbance unit ->was equal to 1 mg/ml protein. I used this in early stages of enzyme ->purification from a plant extract. I recently checked this out by ->comparison with the coomassie blue dye-binding (Bradford) assay. I ->found that the OD method overestimated the protein concentration by ->about 5-fold. This was after ammonium sulfate and desalting, so no ->free amino acids or anything else under ca. 5-10,000 would have been ->present. How about nucleic acids? You don't need much of them... ->I suppose you could say on the other hand that the Bradford assay ->underestimated the protein concentration. But you have to believe in ->something, don't you? 1. Did I ever say this is best way to go for crude preps? 2. 1 O.U. at 280 = 1 mg/ml isn't good estimate but OK for estimates +/- several-fold. - Dima __ / /\ Dima Klenchin / / \ / / /\ \ klenchin@facstaff.wisc.edu / / /\ \ \ tel. (608)263-1163 / /_/__\ \ \ FAX (608)262-3453 /________\ \ \ \___________\/ From owner-proteins@net.bio.net Mon Feb 03 22:00:00 1997 Path: biosci!agate!nntpfeed.doc.ic.ac.uk!sunsite.doc.ic.ac.uk!info-server.surrey.ac.uk!usenet From: bss3pk@surrey.ac.uk (Pete) Newsgroups: bionet.molbio.proteins Subject: Re: Big Problems with ELISA Date: Tue, 04 Feb 1997 13:04:20 GMT Organization: University of Surrey, UK Lines: 49 Message-ID: <5d77an$d7q@info-server.surrey.ac.uk> References: <01bc0a46$022997c0$6a039386@schmidtdw.rz.ruhr-uni-bochum.de> NNTP-Posting-Host: sbspcpk2.sbs.surrey.ac.uk X-Newsreader: Forte Free Agent 1.0.82 "Thorsten Schmidt" wrote: >Dear Reader! >I have big problems with ELISA. >The main problem is that the color does not develop satisfactorally. >Where is the problem? What can I do? >Why didn´t I get more color development? Only you can determine precisely what is happening, but are you sure that your stock H2O2 is OK and not degraded to just H2O? Have you tried a different batch of enzyme label? And finally, are you sure that you have no residual azide in you buffers (includind wash buffers) >Which OPD-solution do you use? For many years we have now not used OPD at all. All our colorimetric assays now use TMB as substrate. This is because it is not carcinogenic like OPD, and the colour yield is about double that of OPD. >Another problem is that I want to make the ELISA by binding >a polypeptid to the microtiter-plate wells (Maxisorp, Nunc). >But I have the impression that the binding to the plate is extremely poor. >I did it in PBS and 0,2 M Carbonate-Buffer pH 9,3. >What can I do to increase the peptid´s binding? Here you could have two different problems. It is not unknown for small peptides to be deformed in the adsorption process to as to "bend out of shape" the antigenic epitope - thus reducing binding and therefore signal. Or yo may as you suggest be getting poor binding - try a whole series of buffers for binding bicarbonate/PBS/TBS/anything handy at a range of pH's from 5 to about 10 (or even more exteme at both ends if it looks useful). This should give you an idea of what's best - and you could also try increasing the molarity of the binding buffer if you start getting desperate! >It would be too kind of you if you´ll send me an answer! >Thank you in advance >Thorsten Schmidt Pete From owner-proteins@net.bio.net Tue Feb 04 22:00:00 1997 Path: biosci!bloom-beacon.mit.edu!howland.erols.net!cam-news-hub1.bbnplanet.com!news.bbnplanet.com!cpk-news-hub1.bbnplanet.com!cpk-news-feed4.bbnplanet.com!maze.dpo.uab.edu!usenet From: Matthew Parker Newsgroups: bionet.molbio.proteins Subject: Re: [Q] Elution from Affi-Gel Blue? Date: Wed, 05 Feb 1997 13:23:30 -0600 Organization: University of Alabama at Birmingham Lines: 13 Message-ID: <32F8DE32.2FD@topaz.microbio.uab.edu> References: <5dabh8$rmm@uni.library.ucla.edu> NNTP-Posting-Host: fenway.microbio.uab.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0 (Win95; I) Sergio Rotstein wrote: > > I have a protein that binds ferociously to an Affi-Gel Blue column by > Bio-Rad. Thanks to that I can get a pretty pure sample of protein. The > problem is that the only way I can elute the protein is by adding ADP > and no matter how much I dialize later on, I can't get rid of all the > ADP If your protein can be refolded, try eluting it in urea (6-8 M) or guanidine (maybe 5 or 6 M). This should denature the protein, which should pull it off the column. Refold by dialyzing out the denaturant. The latter step might take some experimentation to get conditions which give full return of activity. You might have to dialyze out the denaturant slowly: e.g., into 5M, then 4M, etc. Good luck! From owner-proteins@net.bio.net Tue Feb 04 22:00:00 1997 Path: biosci!agate!howland.erols.net!news.sprintlink.net!news-peer.sprintlink.net!news.maxwell.syr.edu!news.bc.net!info.ucla.edu!nnrp.info.ucla.edu!usenet From: sergio@harker.mbi.ucla.edu (Sergio Rotstein) Newsgroups: bionet.molbio.proteins Subject: [Q] Elution from Affi-Gel Blue? Date: 5 Feb 1997 16:12:24 GMT Organization: University of California, Los Angeles Lines: 15 Message-ID: <5dabh8$rmm@uni.library.ucla.edu> NNTP-Posting-Host: harker.mbi.ucla.edu X-Newsreader: knews 0.9.6 I have a protein that binds ferociously to an Affi-Gel Blue column by Bio-Rad. Thanks to that I can get a pretty pure sample of protein. The problem is that the only way I can elute the protein is by adding ADP and no matter how much I dialize later on, I can't get rid of all the ADP (which then interferes with protein activity). My question to the group is: Do you know of any other way to get the protein out of the column? I've tried 10mM NaCl up to 5M salt and it still doesn't come out until I add the dreaded ADP!!! Thanks for any help. Sergio. sergio@mbi.ucla.edu From owner-proteins@net.bio.net Tue Feb 04 22:00:00 1997 Path: biosci!agate!howland.erols.net!vixen.cso.uiuc.edu!newsfeed.internetmci.com!hunter.premier.net!uunet!in1.uu.net!142.77.1.4!news.uunet.ca!rcogate.rco.qc.ca!altitude!usenet From: "Achim Recktenwald, PhD" Newsgroups: bionet.molbio.proteins Subject: Re: A broad and strong A280 peak in a HIC chromatogram Date: Wed, 05 Feb 1997 08:48:30 -0500 Organization: IBEX Technologies, Inc., Biochemistry, 5485 Pare, Montreal, PQ, H4P 1P7, Canada Lines: 37 Message-ID: <32F88FAB.44AA@ibex.ca> References: NNTP-Posting-Host: ibex.hip.cam.org Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Macintosh; I; PPC) Leemor Joshua-Tor wrote: > > >Hi Dear Netters: > >This really confused me. I used a HIC (Butyl Sepharose 4 Fast Flow from > >Pharmacia) as the last purification step (preceded by DEAE and CM Sephadex), > >and I observed a broad and strong A280 peak (from fraction #7 to #31, 2ml > >each) in passed and washed fractions. The absorbance of 280 nm reached 2.499, > >the limit of the spectrophotometer. I ran a SDS-PAGE and did not see any > >protein bands by silver stain. In addition, I measured the protein > >concentration by Bradford reagent and it did not show very very strong > >reaction, either. Although it did not bother the protein I wanted which was > >eluted by a negative linear gradient, it did confuse me for the discussion. I > >don't think I've got this high amount of proteins after two ion exchanges, and > >I did not see this strong peak in those two columns, either. Plus, ammonium > >sulfate (2.4M) should not interfere the 280 nm readings. What else?? Any > >reasonable discussion is welcome. Thanks a lot in advance. > > > >Ming > > Do you have Triton in there? Triton has a peak at 277 nm. > > ******************************************************************* > Leemor Joshua-Tor, Ph.D. > Assistant Investigator > Keck Structural Biology > Cold Spring Harbor Laboratory Tel. (516) 367 8821 > 1 Bungtown Road Fax (516) 367 8873 > Cold Spring Harbor, NY 11724 e-mail: leemor@cshl.org > ******************************************************************* Another source could be free amino acids. Why don't you list the buffers used during your purification. Achim From owner-proteins@net.bio.net Tue Feb 04 22:00:00 1997 Path: biosci!RORQUAL.CC.METU.EDU.TR!asen From: asen@RORQUAL.CC.METU.EDU.TR (alaattin sen) Newsgroups: bionet.molbio.proteins Subject: NATO ADVANCED STUDY INSTITUTE Date: 5 Feb 1997 06:42:32 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 120 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net MEETING INFORMATION -Please helpus to publicize this NATO ASI by forwarding or posting this announcement on bulletin boards, list-serv, www sites, society publication calendars, and your own distribution lists. -Thank you for your interest, cooperation and help THE PRELIMINARY ANNOUNCEMENT NATO Advanced Study Institute on MOLECULAR AND APPLIED ASPECTS OF OXIDATIVE DRUG METABOLIZING ENZYMES August 31-September 11, 1997 Tekirova, ANTALYA-TURKEY OBJECTIVE AND SCOPE: The purpose of the Institute is to provide an international forum for dissemination of advanced scientific knowledge on molecular and applied aspects of oxidative drug metabolizing enzymes, significance of these enzymes in human drug metabolism, genotoxicology, chemical carcinogenesis, insecticide resistance and drug development. LOCATION: The Meeting will take place in a five star Hotel and Holiday village, CORINTHIA in Tekirova, ANTALYA-TURKEY on the Mediterranean Coast. This area is called as Turkish Riviera which possesses one of the finest settings of Mediterranean. It is also littered with ancient ruins. The city, Antalya has an International Airport which is readily accessible by flights from Europe, Israel, as well as Istanbul, Ankara and Izmir. SCIENTIFIC ORGANIZING COMMITTEE: Emel ARINC (Turkey), Alan R. Boobis (UK) Ernest Hodgson (USA) John B. Schenkman (USA) LECTURERS Emel ARINC (Turkey) Steven L. KELLY (UK) Alan R. Boobis (UK) Anthony Y.H. Lu (USA) David EATON (USA) Franz OESCH (Germany) Frank J. GONZALEZ (USA) Richard M. PHILPOT (USA) Israel HANUKOGLU (Israel) John B. SCHENKMAN (USA) Ernest HODGSON (USA) Volker ULLRICH (Germany) Note: This is a preliminary announcement. ASI will have 3 or 4 more lecturer ORGANIZATION and PROGRAMME The lectures will usually be between 9:00 and 12:30 in the mornings and 16:00 and 19:30 in the afternoons with brief breaks for coffee or tea. This schedule allows sufficient time for the scientific sessions and other activities. The morning sessions will be devoted to lectures only. The late afternoon sessions will also include discussion, poster sessions and a limited number of paper presentations. The following lectures will be given: -Drug Metabolizing Enzymes: An overview (J. B. Schenkman) -Molecular Biology of Cytochrome P450: P450 Genes, Their Expression and Regulation (F. J. Gonzalez) -Chemical Principles of Heme Thiolate Catalysis (V. Ullrich) -Other Components of P450 Dependent System (E. Arinc) -Mitochondrial P450 System: Function and Components and Structures (I. Hanukoglu) -Protein-Protein Interactions in the Multicomponent Monooxygenase Reactions (J. B. Schenkman) -Biochemical Aspects of FMOs (E. Hodgson) -Molecular Aspects of FMOs (R. M. Philpot) -Molecular Basis of Chemical Carcinogenesis (F. J. Gonzalez) -Human Cytochromes P450 (A. R. Boobis) -Human Cytochromes P450 Polymorphism: Role in Cancer Susceptibility (F. J. Gonzalez) -In Vitro Studies of Human P450 Enzymes (A. R. Boobis) -Aflatoxin Biotransformations, Toxicity and Carcinogenicity (D. Eaton) -The Biochemistry and Physiology of Prostacyclin and Thromboxane Synthase (V. Ullrich) -Insect P450 Isozymes, Functions and P450-Mediated Insecticide Resistance (E. Hodgson) -Drug-Drug, Drug-Xenobiotic Interactions (F. Oesch) -Biotechnological Applications of Cytochrome P450 Isozymes (S. L. Kelly) -Drug Metabolism Studies in Drug Dicovery and Development (A.H.Y. Lu) PROCEEDINGS: Lecture nots written in a review type will be published shortly after the Meeting by PLENUM Press and one copy will be distributed to each participants and lecturer free of charge. PARTICIPANTS: Advanced Study Institute will accomodate about 85 graduate students, post-doctoral fellows, senior scientist from universities, governmental organizations and the industry. Full attendance to the Institute is required. FINANCES: Participants are expected to cover their own travel and living expenses. Partial support is available. The total cost of ASI including abstract book, full-board, coffee servings, all you can drink (Turkish alcoholic beverages), whole day snacks, sauna and Turkish bath and some other activities is 770 US dollars per person sharing a double room. Accomodations for the participants' family members are available at the same cost as for the participants. Children at 0-6 years will be free of charge. Children 7-12 years old 35 US dollars per night staying with the parents. EXHIBITS and MANUFACTURER'S FORUM: A limited amount of space is available for exhibiting. If you are interested in exhibiting or advertising in the Conference Documentation or sponsoring the Manufacturer's Forum, contact to Dr. E. Arinc. FOR FURTHER INFORMATION AND APPLICATION FORMS Contact: Dr. Emel ARINC Middle East Technical University, ODTU TR-06531, Ankara-TURKEY E-mail:earinc@metu.edu.tr From owner-proteins@net.bio.net Tue Feb 04 22:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!europa.clark.net!news.maxwell.syr.edu!insync!Gamma.RU!srcc!Radio-MSU.net!news.dfn.de!newsjunkie.ans.net!newsfeeds.ans.net!lantana.singnet.com.sg!peony.singnet.com.sg!nuscc.nus.sg!bortas.ee.nus.sg!yuyi From: yuyi@bortas.ee.nus.sg (Yu Yi) Newsgroups: bionet.molbio.proteins Subject: Protein structure prediction Date: 5 Feb 1997 07:52:11 GMT Organization: National University of Singapore Lines: 5 Message-ID: <5d9e7b$g8s@nuscc.nus.sg> Reply-To: yuyi@ee.nus.sg NNTP-Posting-Host: yuyi%@bortas.ee.nus.sg X-Newsreader: TIN [version 1.2 PL2] Can anybody tell me what's the result of 2nd protein structure prediction contest? I thought it should be out last December. Yu Yi From owner-proteins@net.bio.net Tue Feb 04 22:00:00 1997 Path: biosci!agate!howland.erols.net!cam-news-hub1.bbnplanet.com!news.bbnplanet.com!cpk-news-hub1.bbnplanet.com!cpk-news-feed1.bbnplanet.com!uky.edu!usenet From: Wayne Rice Newsgroups: bionet.molbio.proteins Subject: Hatching enzyme antibody Date: Wed, 05 Feb 1997 16:31:29 -0500 Organization: University of Kentucky Lines: 7 Message-ID: <32F8FC31.48B2@pop.uky.edu> NNTP-Posting-Host: 128.163.40.133 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (Win95; I) Does anyone know of a commercially available antibody to any of the teleost or amphibian hatching enzymes? I can't seem to find one, so any suggestions would be appreciated. Wayne Rice Graduate student University of Kentucky From owner-proteins@net.bio.net Tue Feb 04 22:00:00 1997 Path: biosci!agate!howland.erols.net!www.nntp.primenet.com!nntp.primenet.com!arclight.uoregon.edu!news.bc.net!uvaix3e1.comp.UVic.CA!usenet From: tbuckley@sol.uvic.ca (Tom Buckley) Newsgroups: bionet.molbio.proteins Subject: Silver Staining Reveals Contaminating Bands in Sample Buffer Date: 5 Feb 1997 21:43:34 GMT Organization: University of Victoria Lines: 7 Message-ID: <5dauu6$2rdc@uvaix3e1.comp.UVic.CA> Reply-To: tbuckley@sol.uvic.ca NNTP-Posting-Host: toxin4.bioc.uvic.ca Mime-Version: 1.0 X-Newsreader: WinVN 0.93.11 Dear Readers, I am looking for a way to clean up contaminating bands in sample buffer that show up on silver stained polyacrylamide gels. I would appreciate hearing from anyone who has had similar problems. Please send reply to klnelson@uvic.ca. Thanks From owner-proteins@net.bio.net Tue Feb 04 22:00:00 1997 Path: biosci!agate!spool.mu.edu!howland.erols.net!portc02.blue.aol.com!audrey01.news.aol.com!not-for-mail From: mctmsalvu@aol.com (MCTMSalvu) Newsgroups: bionet.molbio.proteins Subject: Re: [Q] Elution from Affi-Gel Blue? Date: 6 Feb 1997 01:00:22 GMT Organization: AOL http://www.aol.com Lines: 8 Message-ID: <19970206010000.UAA07163@ladder01.news.aol.com> References: <5dabh8$rmm@uni.library.ucla.edu> NNTP-Posting-Host: ladder01.news.aol.com X-Admin: news@aol.com Dear Sergio, After you elute your protein from the dye column with ADP, try precipitating the protein with ammonium and then desalt or dialyze the resuspended protein. Some nucleotide-binding proteins require this treatment in order to free their sites of bound nucleotides. Good luck...................MIKE From owner-proteins@net.bio.net Tue Feb 04 22:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!www.nntp.primenet.com!nntp.primenet.com!uwm.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!martinlab From: klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins Subject: Re: [Q] Elution from Affi-Gel Blue? Date: Wed, 05 Feb 97 15:54:46 GMT Organization: UW-Madison Lines: 38 Message-ID: <5dae3c$e5e@news.doit.wisc.edu> References: <5dabh8$rmm@uni.library.ucla.edu> NNTP-Posting-Host: martinlab.biochem.wisc.edu X-Newsreader: News Xpress Version 1.0 Beta #4 X-No-Archive: Yes In article <5dabh8$rmm@uni.library.ucla.edu>, sergio@harker.mbi.ucla.edu (Sergio Rotstein) wrote: ->I have a protein that binds ferociously to an Affi-Gel Blue column by ->Bio-Rad. Thanks to that I can get a pretty pure sample of protein. The ->problem is that the only way I can elute the protein is by adding ADP ->and no matter how much I dialize later on, I can't get rid of all the ->ADP Why? Does the protein bind ADP/ATP? (Cause if not then the dialysis is done incorrectly and the problem does not exist). ->(which then interferes with protein activity). My question to the ->group is: Do you know of any other way to get the protein out of the ->column? I've tried 10mM NaCl up to 5M salt and it still doesn't come ->out until I add the dreaded ADP!!! I'd elute continue eluting with ADP. If it binds ADP too strongly, then any of the following might work: 1. eluting with analogs that hopefully bind weaker (AMP, ATP, ADPbetaS) 2. precipitating eluted protein under conditions that disrupt nucleotide binding but preserve protein's activity (ammonium sulphate, PEG, acidic buffers, etc - all depends on protein). 3. either alone or in conjunction with 2): dialysis under conditions that weaken ADP binding - high salt and/or lower pH should do it. Cheers, - Dima __ / /\ Dima Klenchin / / \ / / /\ \ klenchin@facstaff.wisc.edu / / /\ \ \ tel. (608)263-1163 / /_/__\ \ \ FAX (608)262-3453 /________\ \ \ \___________\/ From owner-proteins@net.bio.net Tue Feb 04 22:00:00 1997 Path: biosci!agate!spool.mu.edu!uwm.edu!newsfeeds.sol.net!news.maxwell.syr.edu!news.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.ums.edu!news.umbc.edu!haven.umd.edu!hecate.umd.edu!ram From: ram@mbisgi.umd.edu (Ram Samudrala) Newsgroups: bionet.molbio.proteins Subject: Re: Protein structure prediction Date: 5 Feb 1997 21:27:08 GMT Organization: The Centre for Advanced Research in Biotechnology Lines: 15 Message-ID: <5datvc$8t7@hecate.umd.edu> References: <5d9e7b$g8s@nuscc.nus.sg> Reply-To: me@ram.org NNTP-Posting-Host: indigo7.carb.nist.gov X-Newsreader: TIN [version 1.2 PL0] Yu Yi (yuyi@bortas.ee.nus.sg) wrote: >Can anybody tell me what's the result of 2nd protein structure prediction >contest? I thought it should be out last December. Everyone lost! <-: Seriously, assuming you're talking about CASP, you should try the prediction centre link from . I am not sure if the results are publicly accessible, however. --Ram me@ram.org || http://www.ram.org || http://www.twisted-helices.com/th Minds that have nothing to confer find little to perceive. ---Wordsworth From owner-proteins@net.bio.net Wed Feb 05 22:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!www.nntp.primenet.com!nntp.primenet.com!news.bbnplanet.com!cpk-news-hub1.bbnplanet.com!cpk-news-feed4.bbnplanet.com!maze.dpo.uab.edu!bmg146.cmc.uab.edu!user From: siyer@bmg.bhs.uab.edu (Sai) Newsgroups: bionet.molbio.proteins Subject: Re: Fusion protein/DTT Date: Thu, 06 Feb 1997 12:51:00 -0500 Organization: Biochem& Molec.genetics, UAB Lines: 24 Message-ID: References: <5dd3hc$o6t@news.ox.ac.uk> NNTP-Posting-Host: bmg146.cmc.uab.edu In article <5dd3hc$o6t@news.ox.ac.uk>, rgrant@molbiol.ox.ac.uk.remove_this_bit wrote: > Recently I read about a fusion protein system that uses DTT for cleavage - > does anyone have any experience of this? > > If emailing, please > Reply-To: rgrant@molbiol.ox.ac.uk > > -- > Richard P. Grant MA DPhil University of Oxford > Nuffield Department Obstetrics and Gynaecology FFPGP > http://users.ox.ac.uk/~lady0266 http://www.molbiol.ox.ac.uk/~rgrant > ------------------------# "Assimilate THIS!" #------------------------ yes...its a system called IMPACT from new england biolabs. i think it's at their web page (www.neb.com). cheers... -- Sai Iyer siyer@bmg.bhs.uab.edu graduate student; dept of biochemistry and molecular genetics university of alabama at birmingham From owner-proteins@net.bio.net Wed Feb 05 22:00:00 1997 Path: biosci!agate!nntpfeed.doc.ic.ac.uk!sunsite.doc.ic.ac.uk!warwick!lyra.csx.cam.ac.uk!news.ox.ac.uk!worf.molbiol.ox.ac.uk!rgrant From: rgrant@worf.molbiol.ox.ac.uk.remove_this_bit (Richard P Grant) Newsgroups: bionet.molbio.proteins Subject: Fusion protein/DTT Date: 6 Feb 1997 17:14:20 GMT Organization: Oxford University Lines: 11 Message-ID: <5dd3hc$o6t@news.ox.ac.uk> Reply-To: rgrant@molbiol.ox.ac.uk.remove_this_bit NNTP-Posting-Host: worf.molbiol.ox.ac.uk X-Newsreader: TIN [version 1.2 PL2] Recently I read about a fusion protein system that uses DTT for cleavage - does anyone have any experience of this? If emailing, please Reply-To: rgrant@molbiol.ox.ac.uk -- Richard P. Grant MA DPhil University of Oxford Nuffield Department Obstetrics and Gynaecology FFPGP http://users.ox.ac.uk/~lady0266 http://www.molbiol.ox.ac.uk/~rgrant ------------------------# "Assimilate THIS!" #------------------------ From owner-proteins@net.bio.net Wed Feb 05 22:00:00 1997 Path: biosci!daresbury!nntp-trd.UNINETT.no!nntp.uio.no!news.maxwell.syr.edu!europa.clark.net!arclight.uoregon.edu!leto.ou.edu!news.ou.edu!news.ecn.uoknor.edu!REX.RE.uokhsc.edu!usenet From: Bob Steinberg Newsgroups: bionet.molbio.proteins Subject: Re: Silver Staining Reveals Contaminating Bands in Sample Buffer Date: Thu, 06 Feb 1997 10:45:02 -0800 Organization: University of Oklahoma H.S.C. Lines: 12 Message-ID: <32FA26AE.728C@ETOWAH.UOKHSC.EDU> References: <5dauu6$2rdc@uvaix3e1.comp.UVic.CA> NNTP-Posting-Host: 157.142.56.147 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.02 (Win16; I) Tom Buckley wrote: > > Dear Readers, > I am looking for a way to clean up contaminating bands in sample > buffer that show up on silver stained polyacrylamide gels. I would > appreciate hearing from anyone who has had similar problems. Please send > reply to klnelson@uvic.ca. > Thanks We had this problem in the past-- if I remember correctly, the source of the bands was the 2-mercaptoethanol in the sample buffer (probably proteins extracted from skin?). From owner-proteins@net.bio.net Wed Feb 05 22:00:00 1997 Path: biosci!agate!howland.erols.net!news.bbnplanet.com!cam-news-hub1.bbnplanet.com!bloom-beacon.mit.edu!news.starnet.net!news.starnet.net!news.inlink.com!mckinnie.inlink.com!user From: remckinnie@aol.com (Russell E McKinnie) Newsgroups: bionet.molbio.proteins Subject: Re: enzyme quantification by OD? Date: Thu, 06 Feb 1997 11:48:10 -0600 Organization: Unemployed Biochemist Lines: 83 Message-ID: References: <5c2shp$3l04@news.doit.wisc.edu> <1997Feb4.112309.93237@cc.usu.edu> <5dd09t$plj@falcon.le.ac.uk> NNTP-Posting-Host: mckinnie.inlink.com X-Newsreader: Yet Another NewsWatcher 2.1.7 In article <5dd09t$plj@falcon.le.ac.uk>, "Dr E. Buxbaum" wrote: > debi@monmouth.com (Glen Ramsay) wrote: > > >How about this: use amino acid analysis to obtain a rock solid measure of > >a single stock of your protein, and using this concentration calculate the > >extinction coefficient for the protein at 280 nm. This will eliminate > >troubles from varying coefficients between different protein, and the > >amino acid analysis will have to be done only once. Of course this > >assumes that nothing is done > >to the protein to change to coefficient (mutation, ligand binding, solvent > >conditions). > > > I know a former colleque who tested this approach. He made a solution of > BSA, gave samples of it to different laboratories for amino acid > analysis and compared the results. The results were... disappointing (to > use a polite term). > > However, there are by now at least two different methods to calculate the > molar absorbance of a protein from the sequence: > Perkins: Eur. J. Biochem. 157 (1986) 169-180 > Gill & van Hippel: Anal. Biochem. 182 (1989) 319-326 > > I have checked the results of both procedures with Hsc70 and found a > difference of about 6% between them. Considering that the difference > between for example Lowry and Bradford assay, depending on sample and > standard protein, can be 16-fold (sic!), that is not too bad. > Obviously you need a pure protein for this. > > If this approach is not feasible for some reason, you may want to > consider the BCA method. The colour yield depends much less on the kind > of protein than with other methods. Hi Guys, There is a whole literature on determining protein absorptivities (ASTM word for extinction coefficients). See the article by Nick Pace et al in Protein Science (1995, vol 4 pp 2411-2423) on the latest way to calculate absorptivities. His methods should work very well on comparing protein variants. Usually, there are only minor changes in absorptivity in protein variants. You only get into trouble if the change is an Trp or Tyr or the protein unfolds. Personally, I prefer to use amino acid analysis to get protein concentrations. It can be very accurate if done properly. For the absorptivity, measure both absorbance and AAA at multiple protein concentrations. The absorptivity is the slope and will have less uncertainty than a single point determination. You can get into problems with the recoveries of the individual amino acids in the AAA. Try to get the person analyzing the AAA to normalize the results to the ³well recovered² amino acids (see Sittampalam, et al 1988, J Assoc Off Anal Chem vol 71, pp 833-838). I have used this overall method to measure several protein pharmaceutical absorptivities with great success. The hardest part is working out the conditions to get complete hydrolysis of the protein. Other issues to consider: PROTEIN PURITY - If your protein is over 90% ( or so ) pure all methods will work fine. For less pure samples (crude extracts, purification process samples) use some guess (1 OD = 1 mg/mL) and don¹t worry about the absorptivity. Worry about getting the protein pure. The other concentration methods will work better on some crude samples because of interferences from buffer additives. PROTEIN CONCENTRATION - To use the A280 method your protein must be ~ 1 mg/mL (+/- an order of magnitude or two). You can play tricks with the pathlength and get a little more range, but not much. Some of the dye binding assays have much improved sensitivity, especially fluorescence methods. Hope this helps. Russell ============================================================ Russell E McKinnie, PhD remckinnie@aol.com Unemployed Biochemist ------------------------------------------------------------ Expertise in Biophysical Chemistry / Analytical Biochemistry Resume available, consulting possible ============================================================ From owner-proteins@net.bio.net Wed Feb 05 22:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!compuserve.com!news.production.compuserve.com!news From: DSF <76042.532@CompuServe.COM> Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: DNA Sequencing Date: 6 Feb 1997 16:09:05 GMT Organization: Cadus Lines: 3 Message-ID: <5dcvn1$p37$1@mhade.production.compuserve.com> Xref: biosci bionet.molbio.methds-reagnts:54469 bionet.molbio.proteins:9926 Has anyone out there used the upgrades for the ABI 373 or 377 DNA sequencers? What are your impressions, opinions, whatever..? Thanks- David From owner-proteins@net.bio.net Wed Feb 05 22:00:00 1997 Newsgroups: bionet.molbio.embldatabank,bionet.molbio.proteins,bionet.molecules.peptides,bionet.general,ebi.servers,ebi.announce,embnet.general Path: biosci!daresbury!hgmp.mrc.ac.uk!ebi.ac.uk!saturn.ebi.ac.uk!apweiler From: apweiler@saturn.ebi.ac.uk (Rolf Apweiler) Subject: E. coli and M. pneumoniae protein sequences Sender: news@ebi.ac.uk (usenet news) Message-ID: Date: Thu, 6 Feb 1997 12:39:46 GMT Lines: 28 Reply-To: apweiler@saturn.ebi.ac.uk (Rolf Apweiler) Organization: European Bioinformatics Institute (EMBL) - UK X-Newsreader: mxrn 6.18-31 Xref: biosci bionet.molbio.embldatabank:762 bionet.molbio.proteins:9925 bionet.molecules.peptides:654 bionet.general:25509 We got some request for the protein sequences of the complete genomes of E. coli and M. pneumoniae. So we created preliminary TREMBL entries of all CDS in the complete E. coli and M. pneumoniae genomes (EMBL/DDBJ/Genbank AC numbers for E.coli AE000111-510 and AE000001-63 for M. pneumoniae). We made them publicly available on the EBI FTP server: ftp.ebi.ac.uk/pub/databases/trembl/genomes/ Please note that these are no official TREMBL entries! Most of these preliminary entries are redundant against corresponding SWISS-PROT + TREMBL entries. In two weeks the next TREMBL release will be publicly available, and the E. coli and M. pneumoniae entries will be integrated in the release (except the redundant entries which will be eliminated). ======================================================================= Rolf Apweiler | SWISS-PROT Coordinator EMBL Outstation | Email:apweiler@ebi.ac.uk European Bioinformatics Institute (EBI) | URL: http://www.ebi.ac.uk Wellcome Trust Genome Campus, Hinxton | Tel: +44 (1223) 494435 Cambridge CB10 1SD, UK | Fax: +44 (1223) 494968 ======================================================================== From owner-proteins@net.bio.net Wed Feb 05 22:00:00 1997 Path: biosci!agate!spool.mu.edu!uwm.edu!newsfeeds.sol.net!hunter.premier.net!uunet!in1.uu.net!160.45.4.4!fu-berlin.de!news-ber1.dfn.de!news-lei1.dfn.de!news-nue1.dfn.de!uni-erlangen.de!winx03!wpxx02!not-for-mail From: krasel@wpxx02.toxi.uni-wuerzburg.de (Cornelius Krasel) Newsgroups: bionet.molbio.proteins Subject: Re: [Q] Elution from Affi-Gel Blue? Date: Thu, 6 Feb 1997 13:43:29 +0100 Organization: University of Wuerzburg, Germany Lines: 22 Message-ID: References: <5dabh8$rmm@uni.library.ucla.edu> NNTP-Posting-Host: wpxx02.toxi.uni-wuerzburg.de X-Newsreader: TIN [UNIX 1.3 950824BETA PL0] [Sorry, didn't see original message. This is edited from a reply by Matthew Parker.] Sergio Rotstein wrote: > I have a protein that binds ferociously to an Affi-Gel Blue column by > Bio-Rad. Thanks to that I can get a pretty pure sample of protein. The > problem is that the only way I can elute the protein is by adding ADP > and no matter how much I dialize later on, I can't get rid of all the > ADP Even gel filtration might not get rid of all of the ADP. What you could try is to use other dye resins which bind your protein of interest less strongly. I am pretty successful with Reactive Yellow 86, sold by Sigma. However, note that elution with salt will be much less specific than with ADP, so it is likely that your protein will be less pure. --Cornelius. -- /* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */ /* D-97078 Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de SP3 */ /* "Science is the game we play with God to find out what His rules are." */ From owner-proteins@net.bio.net Wed Feb 05 22:00:00 1997 Path: biosci!agate!howland.erols.net!news.sprintlink.net!news-peer.sprintlink.net!uunet!in1.uu.net!128.250.1.21!munnari.OZ.AU!metro!metro!news From: Phillip Robinson Newsgroups: bionet.molbio.proteins Subject: Re: Short sequence search? Date: Thu, 06 Feb 1997 21:20:21 +1100 Organization: Information Technology Services, The University of Sydney, NSW, Australia Lines: 22 Distribution: inet Message-ID: <32F9B065.473B@mail.usyd.edu.au> References: <01IEZI1WVIYE9BVDCQ@NBRF.Georgetown.Edu> NNTP-Posting-Host: modem-a-151.mp.usyd.edu.au Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0 (Win95; I) POSTMASTER@NBRF.GEORGETOWN.EDU wrote: > > On 01 Feb 1997, Chihara asked > > Is there a program that will search genbank or prosite etc. for short > > amino acid sequnces? i.e. 5-7 residues? > To search the PIR Protein Sequence Databases you can try > http://www-nbrf.georgetown.edu/nbrf/scan.html Thanks for the tip on this web site, John. Its very good. I went through this problem extensively (I thought), and I only came up with the program Scrutineer, which is in the WISCONSIN PACKAGE, (at least I think its in the same package, ours is Version 8.1.0, March 1996). I don't know how you get access to it, but we have an account with a service that has the whole package. We access it (Unix) via a telnet session (PC or Mac). The real beauty of this program is that you can use very short sequences (2 amino acids if you must) and can use regular expressions or wildcards (eg "[RK].S" returns either RXS or KXS, which will obviously be a lot a sequences). Hope this helps, Phil CMRI, Sydney From owner-proteins@net.bio.net Wed Feb 05 22:00:00 1997 Path: biosci!agate!spool.mu.edu!uwm.edu!news-peer.gsl.net!news.gsl.net!howland.erols.net!rill.news.pipex.net!pipex!uunet!in2.uu.net!128.250.1.21!munnari.OZ.AU!metro!metro!news From: Phillip Robinson Newsgroups: bionet.molbio.proteins Subject: Re: pI calculation Date: Thu, 06 Feb 1997 21:05:18 +1100 Organization: Information Technology Services, The University of Sydney, NSW, Australia Lines: 19 Distribution: inet Message-ID: <32F9ACDE.26B7@mail.usyd.edu.au> References: NNTP-Posting-Host: modem-a-151.mp.usyd.edu.au Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0 (Win95; I) Chris Upton wrote: > I'm looking for a way to query a protein database (eg Swiss-Prot) by pI. > ie get all proteins with pI > 8. > I'm not sure if there's programs to allow calc of pI for all proteins in the > database. I guess output file could be large :-) > Then I might want to sort them.... > Hmm, alternatively it would be nice to at least be able to get the pI for > 1-200 proteins. Is there anything that will allow batch processing? > I seem to think I did this in the very distant past with 200 proteins, > either with GCG or IG. I found this last week. It is the answer to your problem. Works great! My conditions returned almost 1200 proteins. (My refined re-run was much better - only 250). "TagIdent is a tool which allows the retrieval of the SWISS-PROT entries closest to a given pI and MW." Go to http://expasy.hcuge.ch/www/guess-prot.html Phil From owner-proteins@net.bio.net Wed Feb 05 22:00:00 1997 Path: biosci!agate!nntpfeed.doc.ic.ac.uk!sunsite.doc.ic.ac.uk!warwick!leicester!usenet From: "Dr E. Buxbaum" Newsgroups: bionet.molbio.proteins Subject: Re: enzyme quantification by OD? Date: 6 Feb 1997 16:19:09 GMT Organization: University of Leicester (PCFS User) Lines: 32 Message-ID: <5dd09t$plj@falcon.le.ac.uk> References: <5c2shp$3l04@news.doit.wisc.edu> <1997Feb4.112309.93237@cc.usu.edu> NNTP-Posting-Host: pc47.msb.le.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) debi@monmouth.com (Glen Ramsay) wrote: >How about this: use amino acid analysis to obtain a rock solid measure of >a single stock of your protein, and using this concentration calculate the >extinction coefficient for the protein at 280 nm. This will eliminate >troubles from varying coefficients between different protein, and the >amino acid analysis will have to be done only once. Of course this >assumes that nothing is done >to the protein to change to coefficient (mutation, ligand binding, solvent >conditions). I know a former colleque who tested this approach. He made a solution of BSA, gave samples of it to different laboratories for amino acid analysis and compared the results. The results were... disappointing (to use a polite term). However, there are by now at least two different methods to calculate the molar absorbance of a protein from the sequence: Perkins: Eur. J. Biochem. 157 (1986) 169-180 Gill & van Hippel: Anal. Biochem. 182 (1989) 319-326 I have checked the results of both procedures with Hsc70 and found a difference of about 6% between them. Considering that the difference between for example Lowry and Bradford assay, depending on sample and standard protein, can be 16-fold (sic!), that is not too bad. Obviously you need a pure protein for this. If this approach is not feasible for some reason, you may want to consider the BCA method. The colour yield depends much less on the kind of protein than with other methods. From owner-proteins@net.bio.net Wed Feb 05 22:00:00 1997 Path: biosci!agate!spool.mu.edu!uwm.edu!newsfeeds.sol.net!news.maxwell.syr.edu!insync!uunet!in1.uu.net!128.250.1.21!munnari.OZ.AU!news.mel.connect.com.au!harbinger.cc.monash.edu.au!not-for-mail From: Dr Zhonglin Chai Newsgroups: bionet.molbio.proteins Subject: Antibodies to actin and microtubulin. Date: Fri, 07 Feb 1997 13:34:34 +1000 Organization: Monash University Medical School Lines: 14 Message-ID: <5de4bp$5um$2@harbinger.cc.monash.edu.au> NNTP-Posting-Host: tohoffice.path.monash.edu.au Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.02 (Macintosh; I; 68K) CC: bionet.molbio.methds-reagnts Hi there, Do you know any antibodies good for fluorescent antibody staining to localise actin and belta-microtubulin in cultured human cells? I want to co-localise my testing protein with either actin filament in contractile ring or the mitotic spindle microtubulins. All the information and suggestions are appreciated. Please reply by email to "zchai@cobra.path.monash.edu.au" Thanks in advance. Zhonglin Chai Monash Med. Sch. Melbourne, Australia From owner-proteins@net.bio.net Wed Feb 05 22:00:00 1997 Path: biosci!bloom-beacon.mit.edu!howland.erols.net!cam-news-hub1.bbnplanet.com!news.bbnplanet.com!cpk-news-hub1.bbnplanet.com!cpk-news-feed1.bbnplanet.com!uky.edu!usenet From: Wayne Rice Newsgroups: bionet.molbio.proteins Subject: Hatching enzyme antibody Date: Thu, 06 Feb 1997 17:36:56 -0500 Organization: University of Kentucky Lines: 7 Message-ID: <32FA5D08.7A4A@pop.uky.edu> NNTP-Posting-Host: 128.163.40.133 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (Win95; I) I am looking for any antibody, monoclonal or polyclonal, specific for any of the teleost or amphibian hatching enzymes. I have been unsuccessful in locating one in commercial catalogs, and any information would be greatly appreciated. Wayne Rice University of Kentucky From owner-proteins@net.bio.net Thu Feb 06 22:00:00 1997 Path: biosci!SCRIPPS.EDU!ryang From: ryang@SCRIPPS.EDU ("Ri-Yao Yang/SBR4/354.9980") Newsgroups: bionet.molbio.proteins Subject: GST-fusion protein expression in mammalian cells Date: 7 Feb 1997 21:29:33 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 5 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Does any body have experience and vectors for expressing GST-fusion proteins in mammalian cells. I would like to hear any comment on the usefulness of GST-fusion proteins in the studies of in vivo protein interactions in mammalain cells. From owner-proteins@net.bio.net Thu Feb 06 22:00:00 1997 Path: biosci!agate!howland.erols.net!torn!resunix.sickkids.on.ca!news From: Randy Willis Newsgroups: bionet.molbio.proteins Subject: Re: native gel electrophoresis of basic proteins Date: Fri, 07 Feb 1997 18:54:16 -0500 Organization: The Hospital For Sick Children Lines: 34 Message-ID: <32FBC0A8.167E@gandalf.psf.sickkids.on.ca> References: <5dfgds$ccn$1@montespan.pasteur.fr> NNTP-Posting-Host: gollum.psf.sickkids.on.ca Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0 (X11; I; IRIX 5.3 IP12) CC: demaccallum@ninewells.dundee.ac.uk david maccallum wrote: I am working on a large basic protein with a predicted pI of 10. I want to analyse it by native gel electrophoresis. Has anyone any suggestions about buffers which allow it to run the right way? To which michael mourez responded: There is a method described in Schagger and von Jagow, Anal. Biochem. (199)pp 223-231 and Schagger et al., Anal. Biochem., (217) pp 220-230 which use Coomassie blue to bind native membrane protein or soluble protein. For smaller protein the use of taurodeoxycholate is preferred to Coomassie Blue. To both of which, I respond: Another method which I have used successfully, is simply to run the native gel as you normally would but to run the gel in a low pH buffer system. For Pharmacia's Phast System, they recommend using a buffer recipe containing...4.4g beta-alanine and 4ml acetic acid in 100ml (H20) which gives you a buffer of pH~4. Don't forget to reverse the leads and you can use pyronin Y as a dye front marker. Good luck. Randall C Willis, Researcher Biochemistry Research Hosp for Sick Children 3522-555 University Ave Toronto, ON M5G 1X8 CANADA 416-813-5933 (ph) -5022 (fax) willis@gandalf.psf.sickkids.on.ca From owner-proteins@net.bio.net Thu Feb 06 22:00:00 1997 Path: biosci!agate!howland.erols.net!torn!resunix.sickkids.on.ca!news From: Randy Willis Newsgroups: bionet.molbio.proteins Subject: Re: Fusion protein/DTT Date: Fri, 07 Feb 1997 18:46:35 -0500 Organization: The Hospital For Sick Children Lines: 27 Message-ID: <32FBBEDB.41C6@gandalf.psf.sickkids.on.ca> References: <5dd3hc$o6t@news.ox.ac.uk> NNTP-Posting-Host: gollum.psf.sickkids.on.ca Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0 (X11; I; IRIX 5.3 IP12) To: rgrant@molbiol.ox.ac.uk Richard, The system which you seek is the IMPACT system supplied by New England Biolabs. We purchased it recently and found that it was insufficient for our needs, a protein structure lab using NMR, but I have heard that if overall yield is not too much of an issue, then the system is quite good. There are certain limitations as to what residues can precede the cysteine which is involved in a transesterification reaction with the DTT and the peptide backbone but these are relatively minor problems overall. As well, in their own manual, NEB describes the fact that they attempted about two dozen fusions with different eukaryotic proteins and only about half of them gave any decent results. I'm hoping that they are still working on improvements to the system as I would still be quite excited to hear about any large production fusion system which allows cleavage without a protease. Good luck. Randall C Willis, Researcher Biochemistry Research Hosp for Sick Children 3522-555 University Ave Toronto, ON M5G 1X8 CANADA 416-813-5933 (ph) -5022 (fax) willis@gandalf.psf.sickkids.on.ca From owner-proteins@net.bio.net Thu Feb 06 22:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!www.nntp.primenet.com!nntp.primenet.com!howland.erols.net!worldnet.att.net!cbgw2.lucent.com!news.bu.edu!usenet From: James Freeman Newsgroups: bionet.molbio.proteins Subject: Online Genomic Analysis Tool From The BMERC Date: Fri, 07 Feb 1997 14:15:21 -0500 Organization: BioMolecular Engineering Resource Center Lines: 52 Message-ID: <32FB7F49.31DFF4F5@darwin.bu.edu> NNTP-Posting-Host: hinshelwood.bu.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01Gold (X11; I; SunOS 4.1.4 sun4m) CC: jfreeman@darwin.bu.edu BMERC/MBCRR Bulletin: February 1997 WWW interface for searching several Genomes at once: (NEW--Includes E. Coli!) The E. Coli genome has just been sequenced as of the last week of January. At BMERC, we have added the E. Coli genome to our genome blast page. The E. Coli genome is of particular interests to Molecular Biologists because the vast majority of functions assigned to E. Coli have been done by experimentation. Most other genomes have their functions assigned by homology. The complete and nearly complete genomes of Saccharomyces Cerevisiae , Methanococcus Jannaschii, E. Coli, and Bacillus Subtilis are now available. Our WWW Blast interface allows you to search, using your sequence, against two subsets of the available putative open reading frames of these genomes using blastp. A Search Against Annotated ORF's and a Search Against Unannotated ORF's of these genomes are the search options available from this page. Your output will consist of detailed references for the significant blast matches and the raw blast output. The detailed references consist of a reference key, the annotation where available, the protein sequence, and the dna for the ORF's. We are also providing a tool for function keyword searching.We have built a table that relates E. Coli to the other three main genomes using blast with a Karlan Altschul score of < 10E-17. This keyword searching tool will print a list of every sequence identifier that is close to the E. Coli gene of the cluster where the keyword is found. The WWW address for our Blast Genome Analysis blast page is: http://bmerc-www.bu.edu/genome/genomeblastp.html The WWW address for the E Coli functional search is: http://bmerc-www.bu.edu/genome/ecoli-keyword.html If you have any questions/comments on this WWW page, please send them to Jim Freeman at jfreeman@darwin.bu.edu -- Jim Freeman P: mammon@tiac.net W: jfreeman@darwin.bu.edu Programmer/Analyst at Bio-Molecular Engineering Center at BU Etiam unum capillum umbram suam habet. http://www.tiac.net/users/mammon/index.html From owner-proteins@net.bio.net Thu Feb 06 22:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!europa.clark.net!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!cam-news-hub1.bbnplanet.com!howland.erols.net!rill.news.pipex.net!pipex!oleane!pasteur.fr!not-for-mail From: Michael MOUREZ Newsgroups: bionet.molbio.proteins Subject: Re: native gel electrophoresis of basic proteins Date: 7 Feb 1997 15:06:36 GMT Organization: Institut Pasteur Lines: 18 Message-ID: <5dfgds$ccn$1@montespan.pasteur.fr> References: NNTP-Posting-Host: 68bw1-2-100.dyn.pasteur.fr Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) To: demaccallum@ninewells.dundee.ac.uk X-URL: news:demaccallum-230197154757@pergolesi.medschool.dundee.ac.uk demaccallum@ninewells.dundee.ac.uk (david maccallum) wrote: >I am working on a large basic protein with a predicted pI of 10. I want to >analyse it by native gel electrophoresis. Has anyone any suggestions about >buffers which allow it to run the right way? There is a method described in Schagger and von Jagow, Anal. Biochem. (199)pp 223-231 and Schagger et al., Anal. Biochem., (217) pp 220-230 which use Coomassie blue to bind native membrane protein or soluble protein. This induce a charge shift and allow the native migration of these proteins even when they are basic. This method works best with 100 to 1000 kDa protein. For smaller protein the use of taurodeoxycholate is preferred to Coomassie Blue. Good luck. Michael MOUREZ From owner-proteins@net.bio.net Fri Feb 07 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!newsfeeds.sol.net!feed1.news.erols.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!cpk-news-feed4.bbnplanet.com!maze.dpo.uab.edu!bmg146.cmc.uab.edu!user From: siyer@bmg.bhs.uab.edu (Sai) Newsgroups: bionet.molbio.proteins Subject: Re: Fusion protein/DTT Date: Sat, 08 Feb 1997 13:19:20 -0500 Organization: Biochem& Molec.genetics, UAB Lines: 52 Message-ID: References: <5dd3hc$o6t@news.ox.ac.uk> <32FBBEDB.41C6@gandalf.psf.sickkids.on.ca> NNTP-Posting-Host: bmg146.cmc.uab.edu In article <32FBBEDB.41C6@gandalf.psf.sickkids.on.ca>, Randy Willis wrote: > Richard, > > The system which you seek is the IMPACT system supplied by New England > Biolabs. We purchased it recently and found that it was insufficient > for our needs, a protein structure lab using NMR, but I have heard that > if overall yield is not too much of an issue, then the system is quite > good. There are certain limitations as to what residues can precede the > cysteine which is involved in a transesterification reaction with the > DTT and the peptide backbone but these are relatively minor problems > overall. As well, in their own manual, NEB describes the fact that they > attempted about two dozen fusions with different eukaryotic proteins and > only about half of them gave any decent results. I'm hoping that they > are still working on improvements to the system as I would still be > quite excited to hear about any large production fusion system which > allows cleavage without a protease. > > Good luck. > > Randall C Willis, Researcher > Biochemistry Research > Hosp for Sick Children > 3522-555 University Ave > Toronto, ON > M5G 1X8 CANADA > > 416-813-5933 (ph) -5022 (fax) > willis@gandalf.psf.sickkids.on.ca to add to this, i just started using this system in our lab as well...crude extract analysis showed that my eukaryotic protein was expressed somewhat decently in BL21 (DE3) and XL-1 blue strains. i got about 50% soluble Vs insolube which is a damn good ratio when it comes to expressed proteins. i havent done a "real" purification yet to figure yeild, but this is soon to follow. as above, my case is also similar in the sense that i will be using the expressed protein thru this system for crystallography, so from the standpoint of not having to worry about post-purification modifications (like protease cleavage to obviate the recombinant his-tags or gst-tags), this system is worth the 300 bucks that we paid for it, if you can get your protein to express...its definitely worth it if you want to express prokaryotic proteins as the system seems to work real well with expressing prok. proteins (they state this in their manual)...good luck... -- Sai Iyer (sai@uab.edu) Pre-doctoral fellow i.e lowly graduate student (Dr.Gerald Hart's lab) Dept of Biochemistry and Molecular Genetics University of Alabama at Birmingham... From owner-proteins@net.bio.net Fri Feb 07 22:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!europa.clark.net!cpk-news-hub1.bbnplanet.com!cam-news-hub1.bbnplanet.com!news.bbnplanet.com!howland.erols.net!vixen.cso.uiuc.edu!news.stealth.net!uunet!in3.uu.net!128.250.1.21!munnari.OZ.AU!news.ecn.uoknor.edu!news.cis.okstate.edu!nntp.ksu.edu!not-for-mail From: scotbean@ksu.ksu.edu (Scott Bean) Newsgroups: bionet.molbio.proteins Subject: Reversible Staining of Non SDS gels at acid pH, help! Date: 9 Feb 1997 04:48:29 GMT Organization: kansas state university Lines: 18 Message-ID: <5djkut$7dj$1@cnn.ksu.ksu.edu> NNTP-Posting-Host: pc31gqu.usgmrl.ksu.edu Mime-Version: 1.0 Content-Type: Text/Plain; charset=US-ASCII X-Newsreader: WinVN 0.99.7 Hello; Does anyone know of a reversible stain that will work in an acid/urea gel (no SDS;pH 3.1) similar to the copper or zinc staining methods used with SDS PAGE? Every method I can find utilizes complexes between metal ions and SDS. I did find one method for gels containing 8 M urea and chilling to -70 C, but I don't have (or want) 8 M urea in my gels or a way to cool them to -70 C. The reference for the reversible copper stain says that it can be used without SDS but they only showed this at high pH, I've never been able to get it to work at a low pH. I've tried staining reference lanes and using them to try and cut out my bands, didn't work very well for me. I've also tried blotting and then staining with ponceau S and that didn't work either. Any ideas or suggestions? Thanks. Scott Bean Kansas State University From owner-proteins@net.bio.net Fri Feb 07 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!newsfeeds.sol.net!hammer.uoregon.edu!arclight.uoregon.edu!news.bc.net!rover.ucs.ualberta.ca!news.ucalgary.ca!molomac.bio.ucalgary.ca!user From: jbooth@acs.ucalgary.ca (Joe boothe) Newsgroups: bionet.molbio.proteins Subject: antibody conjugation Date: Sat, 08 Feb 1997 13:11:54 -0700 Organization: University of Calgary Lines: 21 Message-ID: NNTP-Posting-Host: @molomac.bio.ucalgary.ca X-Newsreader: Yet Another NewsWatcher 2.1.8 An experiment I am doing requires the production of a horseradish peroxidase primary antibody. To accomplish the conjugation I am following the sodium periodate method as outlined in "Current Protocols" section 11. My primary antibody (a rabbit polyclonal) was purified on a protein A column and concentrated to 1mg/ml. One ml of this antibody solution was mixed with 0.5 ml of sodium periodate-treated HRPO (2.5 mg) and the conjugation carried out in a disposable BioRad mini column containing 0.25g of G25 Sephadex. Elution, reduction and recovery of the antibody conjugate was as described in the protocol. All reagents were used fresh (and purchased recently from Sigma). Two attempts of this procedure have failed to give an active conjugate when tested on a western blot. Also, no evidence of conjugation can be seen when the antibodies are run on an SDS gel. However, the antibodies do retain their avidity for the antigen as determined by reaction of the western blot with HRPO-labeled secondary antibodies. I would appreciate hearing from anyone with experience with this procedure or with suggestions for solving the problem. Thanks. Joe From owner-proteins@net.bio.net Sat Feb 08 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!www.nntp.primenet.com!nntp.primenet.com!howland.erols.net!vixen.cso.uiuc.edu!news.stealth.net!uunet!in2.uu.net!136.142.185.26!newsfeed.pitt.edu!bb3.andrew.cmu.edu!andrew.cmu.edu!mustafa+ From: Mustafa Unlu Newsgroups: bionet.molbio.proteins Subject: Re: Silver Staining Reveals Contaminating Bands in Sample Buffer Date: Sun, 9 Feb 1997 10:26:54 -0500 Organization: Doctoral student, Chemistry, Carnegie Mellon, Pittsburgh, PA Lines: 15 Message-ID: <8mzSmyi00WB_04TGk0@andrew.cmu.edu> References: <5dauu6$2rdc@uvaix3e1.comp.UVic.CA> NNTP-Posting-Host: po9.andrew.cmu.edu In-Reply-To: <5dauu6$2rdc@uvaix3e1.comp.UVic.CA> Hi, I have had these bands appear also, right around 50kD or so. I think there is even a reference showing that they are human skin keratin. Even one of the original references for silver staining (sorry I don't have it here I'm writing from home) admits that "at maximum exposure" these bands appear. I can give you refs on Monday morning (EST) if you want. Regards, M. From owner-proteins@net.bio.net Sat Feb 08 22:00:00 1997 Path: biosci!biosci!not-for-mail From: Rong-i Hong Newsgroups: bionet.molbio.proteins,bionet.xtallography,bionet.software,bionet.molec-model,bionet.biophysics,sci.chem Subject: Protein 2nd structure assignment Date: 9 Feb 1997 14:00:30 -0800 Organization: Oxford University Lines: 18 Sender: daemon@net.bio.net Distribution: world Message-ID: <32FB0643.41C6@bioch.ox.ac.uk> References: <32E977EB.5290@plaza.snu.ac.kr> <32F0B455.43D4@chem.gla.ac.uk> NNTP-Posting-Host: net.bio.net Xref: biosci bionet.molbio.proteins:9946 bionet.xtallography:3183 bionet.software:17841 bionet.molec-model:1364 bionet.biophysics:2621 sci.chem:75051 Dear All, We are doing some work about assignments of protein secondary sturcture elements. Are there any programs, other than DSSP, DEFINE and STRIDE, to define secondary structure based on the atomic coordinates? If so, could you let me know where I might acquire such programs? Thanks in advance. ------------------------------------------------------------ Rong-I Hong Lab. of Molecular Biophysics EMail: rong@bioch.ox.ac.uk University of Oxford Tel: 01865-275 369 Rex Richards Building Fax: 01865-275 182 Oxford, OX1 3QU United Kingdom ------------------------------------------------------------ From owner-proteins@net.bio.net Sun Feb 09 22:00:00 1997 Path: biosci!metu.edu.tr!arinc From: arinc@metu.edu.tr (Faruk Arinc) Newsgroups: bionet.molbio.proteins Subject: First Announcement of the Int. Symposium, BIOTRANSPORT'98 Date: 10 Feb 1997 01:58:11 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 173 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net INTERNATIONAL CENTRE FOR HEAT AND MASS TRANSFER BIOTRANSPORT'98 INTERNATIONAL SYMPOSIUM ON HEAT AND MASS TRANSFER IN BIOLOGICAL AND MEDICAL ENGINEERING 8 - 12 June 1998 Kusadasi, Turkey FIRST ANNOUNCEMENT & CALL FOR PAPERS OBJECTIVE AND SCOPE The International Centre for Heat and Mass Transfer is pleased to announce BIO-TRANSPORT'98, an International Symposium on Heat and Mass Transfer in Biological Tissues. This symposium is a follow up of the highly successful First Symposium on Bioheat and Mass Transport held in 1991. The main objective of the Symposium is to bring together scientists and engineers from around the world involved in bioheat and mass transfer research and to provide a relaxed atmosphere for in-depth discussion and exchange of the state-of-the-art of related theory, experiments, and applications. SESSIONS * Heat and mass transport in tissue engineering processes. * Energy based surgical modalities: microwave, ultrasound, radio frequency, laser, cryosurgery. * Thermally coupled phenomena in living systems: laser tissue optics; hyperthermia cancer therapy; burn injury processes; tissue and organ cryopreservation * Mass transport limited function in the design and operation of artificial organs. * Measurement and modeling of the thermal influence of blood perfusion in living tissues; thermal methods to measure blood perfusion. * Microscale heat and mass transport phenomena in biological systems. * Design for commercial scale tissue processing protocols * Temperature-triggered drug delivery in living tissues INTERNATIONAL SCIENTIFIC COMMITTEE K.R. Diller, Chairman, Univ. of Texas, Austin, USA J.C. Chato, Univ. of Illinois, Urbana/Champaign, USA T.C. Hua, East China Tech. Univ. Shanghai, PRC H. Ishiguro, Univ. Tsukuba, Japan R.C. Lee, Univ. Chicago, USA D.E. Pegg, Univ. York, UK T.P. Ryan, Valley Lab/Pfizer, Inc., USA J.B. Saulnier, ENSMA, Futuroscope, France A. Shitzer, Technion, Haifa, Israel P. Tikuisis, DCIEM, Canada M. Toner, Harvard Medical School, Boston, USA B.X. Wang, Tsinguha Univ., Beijing PRC V.P. Zharov, Moscow State Tech. Univ. Russia LOCATION Kusadasi is a resort town on the Aegean coast of Turkey, about 90 km south of the metrapolitan city, Izmir. Many major airlines have direct flights to Izmir airport during summer time. There are several archeological sites closeby (Ephesus is only 18 km away.) ORGANIZATION & IMPORTANT DEADLINES There will be stand-up presentations of contributed papers as well as poster presentations at the Symposium. In addition, several keynote lectures will be delivered. It is planned that all Keynote lectures and contributed papers will be published in a dedicated Volume of Proceedings and may be considered for publication in an archival journal. A number of Poster sessions will be organized during the Symposium to provide a format for researchers to discuss their most recent work in a relaxed atmosphere. The abstracts of all presentations made at the symposium will be included in the Book of Abstracts available on site. Stand-up Presentations: * October 1, 1997: Preliminary Application Form due to ICHMT Secretariat * November 15, 1997: Four copies of full paper due to K.R. Diller * March 15, 1998: Paper reviews returned to authors for preparation of final versions * May 31, 1998: Final camera ready copy of paper due, plus an Extended Abstract for publication in the Book of Abstracts for the Symposium Poster Presentations: * March 31, 1998: Four copies of Extended Abstracts due to K.R. Diller * April 15, 1998: Abstract reviews returned to authors for preparation of final versions * May 31, 1998: Final camera ready copy of Extended Abstract due for publication in the Book of Abstracts for the Symposium IMPORTANT ADDRESSES AND NUMBERS Chairman: Professor Kenneth R. DILLER Biomedical Engineering Program The University of Texas at Austin ENS 612 Austin, TX 78712-1084, USA Tel: +1-512-471-7167 Fax: +1-512-471-0616 e-mail: kdiller@mail.utexas.edu ICHMT Secretariat: Professor Faruk ARINC Mechanical Engineering Department Middle East Technical University 06531 Ankara, Turkey Tel: +90-312-210-1429 Fax: +90-312-210-1331 or 1266 e-mail: arinc@metu.edu.tr http://ichmt.me.metu.edu.tr ---------------------------------------------------------------------------- BIOTRANSPORT'98 Int. Symposium on Heat and Mass Transfer in Biological and Medical Engineering PRELIMINARY APPLICATION FORM To: arinc@metu.edu.tr Title: __________________________ Name: __________________________________ Affiliation: _______________________________________________________________ Address: ___________________________________________________________________ ___________________________________________________________________ Telephone: _____________________________ Fax:_______________________________ E-mail: ____________________________________________________________________ [ ] I plan to attend the symposium [ ] I plan to submit a paper Tentative title of paper: __________________________________________________ [ ] I shall be accompanied by ____________________________________________ [ ] I am interested in the possibility of a post-conference tour in Turkey for____ persons to ____ Istanbul ____ or to Cappadocia. (Please indicate one or the other as the tours will be at the same time) ---------------------------------------------------------------------------- From owner-proteins@net.bio.net Sun Feb 09 22:00:00 1997 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.erols.net!newsxfer3.itd.umich.edu!su-news-hub1.bbnplanet.com!news.bbnplanet.com!news.pbi.net!news.dp.beckman.com!inet.dp.beckman.com!KLBRILLHART!klbrillhart Newsgroups: bionet.molbio.proteins Subject: RE:antibody conjugation Message-ID: From: klbrillhart@ccgate.dp.beckman.com (Kurt L Brillhart) Date: Sat, 8 Feb 1997 09:58:56 Distribution: world Organization: Beckman Instruments, Inc. NNTP-Posting-Host: 134.217.88.81 X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] Lines: 11 Need more details (buffer conditions, temp., etc.). One suggestion- it is a common practice to include a small volume of Tris base in the collection tubes when using acid elution from protein A in order to neutralize ASAP and retain as much Ab activity as possible. Tris contains a primary amine and will compete very nicely for the aldehydes you've generated on the HRP. If you haven't done so already try dialysing or desalting your Ab prep against your coupling buffer. Kurt Brillhart klbrillhart@ccgate.dp.beckman.com From owner-proteins@net.bio.net Sun Feb 09 22:00:00 1997 Path: biosci!daresbury!nntp-trd.UNINETT.no!nntp.uio.no!www.nntp.primenet.com!nntp.primenet.com!rill.news.pipex.net!pipex!uunet!in1.uu.net!155.229.2.176!metro.atlanta.com!cpk-news-feed3.bbnplanet.com!es.dupont.com!topgun.es.dupont.com!vanakete.es.dupont.com!user From: vanakete@a1.esvax.umc.dupont.com (Thomas E Van Aken) Newsgroups: bionet.molbio.proteins Subject: Re: antibody conjugation Date: 10 Feb 1997 19:38:50 GMT Organization: DuPont Central Research and Developement Lines: 5 Message-ID: References: NNTP-Posting-Host: vanakete.es.dupont.com Try using the glutaraldehyde linking method. "Antibodies A Laboratory Manual" by Ed Harlow and David Lane page 346-347 Tom From owner-proteins@net.bio.net Sun Feb 09 22:00:00 1997 Path: biosci!agate!mendel.Berkeley.EDU!tomt From: Thomas Tan Newsgroups: bionet.molbio.proteins Subject: Re: Silver Staining Reveals Contaminating Bands in Sample Buffer Date: Mon, 10 Feb 1997 19:21:43 -0800 Organization: data communication and networking services Lines: 16 Message-ID: References: <5dauu6$2rdc@uvaix3e1.comp.UVic.CA> <32FA26AE.728C@ETOWAH.UOKHSC.EDU> NNTP-Posting-Host: mendel.berkeley.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII In-Reply-To: <32FA26AE.728C@ETOWAH.UOKHSC.EDU> "Protein Methods" by Bollag et al. states, "SDS gels in which 2-mercaptoethanol is used may develop 2 horizontal lines at 60 kd and 67 kd. These can be eliminated by using less 2-mercaptoethanol (Marshall and Williams, 1984)." Tom Tan > Tom Buckley wrote: > > > > Dear Readers, > > I am looking for a way to clean up contaminating bands in sample > > buffer that show up on silver stained polyacrylamide gels. I would > > appreciate hearing from anyone who has had similar problems. Please send > > reply to klnelson@uvic.ca. > > Thanks > From owner-proteins@net.bio.net Sun Feb 09 22:00:00 1997 Path: biosci!daresbury!nntp-trd.UNINETT.no!sn.no!Oslo2.Norway.EU.net!Norway.EU.net!EU.net!howland.erols.net!rill.news.pipex.net!pipex!warwick!leicester!usenet From: "Dr E. Buxbaum" Newsgroups: bionet.molbio.proteins Subject: Re: Reversible Staining of Non SDS gels at acid pH, help! Date: 10 Feb 1997 16:19:58 GMT Organization: University of Leicester (PCFS User) Lines: 27 Message-ID: <5dnhre$17s@falcon.le.ac.uk> References: <5djkut$7dj$1@cnn.ksu.ksu.edu> NNTP-Posting-Host: pc47.msb.le.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) scotbean@ksu.ksu.edu (Scott Bean) wrote: >Hello; > >Does anyone know of a reversible stain that will work in an acid/urea gel >(no SDS;pH 3.1) similar to the copper or zinc staining methods used with SDS >PAGE? Every method I can find utilizes complexes between metal ions and SDS. I >did find one method for gels containing 8 M urea and chilling to -70 C, but I >don't have (or want) 8 M urea in my gels or a way to cool them to -70 C. The >reference for the reversible copper stain says that it can be used without SDS >but they only showed this at high pH, I've never been able to get it to work at >a low pH. I've tried staining reference lanes and using them to try and cut >out my bands, didn't work very well for me. I've also tried blotting and then >staining with ponceau S and that didn't work either. Any ideas or suggestions? Ponceau on a western should normaly work, and is reversible. You did remember that most proteins are positively charged at your pH, i.e. that the electrophoresis and blotting need to be done with reversed polarity? Do "non-reversible" stains like Coomassie work? Coomassie is reversible, just destain long enough in the presence of a dye scavenger like activated charcoal or foam. The other thing you could try is to use fluorescent staining of the protein, Molecular Probes have a suitable stain in their catalogue. What do you want to use the gels for? Immunisation? From owner-proteins@net.bio.net Sun Feb 09 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!cs.utexas.edu!swrinde!newsfeed.internetmci.com!news.msfc.nasa.gov!info.uah.edu!maze.dpo.uab.edu!juniper.cis.uab.edu!nntp.msstate.edu!maize.biochem.msstate.edu!user From: dsluthe@ra.msstate.edu (D.S. Luthe) Newsgroups: bionet.molbio.proteins Subject: Student needs post-doc Date: Mon, 10 Feb 1997 12:56:46 -0600 Organization: Mississippi State Univ. Lines: 2 Message-ID: NNTP-Posting-Host: maize.biochem.msstate.edu I have a Ph.D. student who needs a plant molecular biology/molecular biology post doc ASAP. Please contact me if you are interested. From owner-proteins@net.bio.net Sun Feb 09 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!www.nntp.primenet.com!nntp.primenet.com!mr.net!newsfeeds.sol.net!hammer.uoregon.edu!zephyr.texoma.net!uunet!in3.uu.net!136.142.185.26!newsfeed.pitt.edu!mv29.pathology.pitt.edu!user From: pxpst2@vms.cis.pitt.edu (THE GREEK) Newsgroups: bionet.molbio.proteins Subject: Re: Silver Staining Reveals Contaminating Bands in Sample Buffer Date: 10 Feb 1997 21:26:39 GMT Organization: USA Lines: 22 Message-ID: References: <5dauu6$2rdc@uvaix3e1.comp.UVic.CA> <8mzSmyi00WB_04TGk0@andrew.cmu.edu> NNTP-Posting-Host: mv29.pathology.pitt.edu > Hi, /> /> I have had these bands appear also, right around 50kD or so. I think /> there is even a reference showing that they are human skin keratin. /> /> Even one of the original references for silver staining (sorry I don't /> have it here I'm writing from home) admits that "at maximum exposure" /> these bands appear. /> //> I can give you refs on Monday morning (EST) if you want. > /> /> Regards, /> M. You guys are silly. I would be surprised if you did not see bands with silver staining. Getting pure proteins without denaturing (ie acetonitrile) is difficult to say the least. Consult a good protein purification book and you guys could learn a lot Peter Pediaditakis pxpst2@vms.cis.pitt.edu From owner-proteins@net.bio.net Sun Feb 09 22:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!europa.clark.net!cpk-news-hub1.bbnplanet.com!cpk-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!news.bbnplanet.com!arclight.uoregon.edu!news.bc.net!torn!hone!informer1.cis.McMaster.CA!fhs.csu.McMaster.CA!kothc From: Chris Koth Newsgroups: bionet.molbio.proteins Subject: RNA-DNA hybrids Date: Mon, 10 Feb 1997 15:56:48 -0500 Organization: McMaster University, Hamilton, Ontario, Canada (NewServer) Lines: 27 Message-ID: References: <5ctfhb$5hh@doom.itqb.unl.pt> NNTP-Posting-Host: fhs.csu.mcmaster.ca Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII In-Reply-To: <5ctfhb$5hh@doom.itqb.unl.pt> Hi I don't want to give too much unnecessary background, but I am working on transcription assays to study a particular protein and would like to examine the effects of stabilizing or destabilizing the RNA-DNA hybrid. I plan to use ITP in place of GTP to destabilize and bromo-uracil (in place of UTP) to stabilize. Can anyone recommend anything else to try? Also, I am having trouble locating a good source of ITP. Any ideas in that department? Thanks, Chris ******************************************************************************** Chris Koth Tel: 1-905-525-9140 x24115 Graduate Student - Biochemistry FAX: 1-905-546-9940 McMaster University eMAIL: koth@lords.com HSC Rm. 4H2 1200 Main St. W. Hamilton, Ontario Canada L8N 3Z5 ******************************************************************************** From owner-proteins@net.bio.net Sun Feb 09 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!newsfeeds.sol.net!newspump.sol.net!howland.erols.net!cam-news-hub1.bbnplanet.com!news.bbnplanet.com!uunet!in2.uu.net!199.232.56.18!news.ultranet.com!usenet From: "John P. McGrath" Newsgroups: bionet.molbio.proteins Subject: Re: Silver Staining Reveals Contaminating Bands in Sample Buffer Date: Tue, 11 Feb 1997 00:26:38 -0400 Organization: UltraNet Communications, Inc. Lines: 3 Message-ID: <32FFF4FB.659@ma.ultranet.com> References: <5dauu6$2rdc@uvaix3e1.comp.UVic.CA> NNTP-Posting-Host: d9.met.ma.ultra.net Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0 (Macintosh; I; PPC) To: tbuckley@sol.uvic.ca One solution to the problem caused by the 2-ME contaminant is the use of DTT as the reducing agent in the sample buffer. This results in a much lower background in the Ag-stained gel. From owner-proteins@net.bio.net Sun Feb 09 22:00:00 1997 From: Richard van Wegen Newsgroups: bionet.molbio.proteins Subject: N-terminal assay Date: Tue, 11 Feb 1997 10:19:55 -0800 Organization: The University of Adelaide Lines: 10 Message-ID: <3300B84B.53C6@chemeng.adelaide.edu.au> NNTP-Posting-Host: 129.127.6.172 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0 (Win16; I) Path: biosci!agate!spool.mu.edu!uwm.edu!cs.utexas.edu!www.nntp.primenet.com!nntp.primenet.com!howland.erols.net!swrinde!ihnp4.ucsd.edu!munnari.OZ.AU!news.mel.connect.com.au!news.ade.connect.com.au!duster.adelaide.on.net!ns.saard.net!hakea.adelaide.edu.au!usenet Hi everyone, I am trying to hydrolyse whey proteins with acid and am looking for a way to quantify the extent of hydrolysis. Someone suggested using an N-terminal assay. Could anyone give me a reference to such a procedure? I found one but it involved extremely nasty chemicals. Thanks for your help, Richard van Wegen Department of Chemical Engineering University of Adelaide, Australia From owner-proteins@net.bio.net Sun Feb 09 22:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!www.nntp.primenet.com!nntp.primenet.com!cpk-news-hub1.bbnplanet.com!su-news-hub1.bbnplanet.com!news.bbnplanet.com!newsxfer3.itd.umich.edu!cloudbreak.rs.itd.umich.edu!news.mtu.edu!msunews!NewsWatcher!user From: davilaap@pilot.msu.edu (Jenny Davila-Aponte) Newsgroups: bionet.molbio.proteins Subject: Re: Silver Staining Reveals Contaminating Bands in Sample Buffer Followup-To: bionet.molbio.proteins Date: Mon, 10 Feb 1997 16:55:38 -0400 Organization: Michigan State University Lines: 25 Message-ID: References: <5dauu6$2rdc@uvaix3e1.comp.UVic.CA> <8mzSmyi00WB_04TGk0@andrew.cmu.edu> NNTP-Posting-Host: 35.8.197.48 In article <8mzSmyi00WB_04TGk0@andrew.cmu.edu>, Mustafa Unlu wrote: > > Hi, > > I have had these bands appear also, right around 50kD or so. I think > there is even a reference showing that they are human skin keratin. > > Even one of the original references for silver staining (sorry I don't > have it here I'm writing from home) admits that "at maximum exposure" > these bands appear. > > I can give you refs on Monday morning (EST) if you want. > > > Regards, > M. I have had this problem with silver stain gels and finally got rid of them by wearing gloves whenever I am making or using any solutions that come in contact with my gels and by preparing fresh buffers for my gels (separating buffer, stacking buffer, and tank buffer). Hope this advice is useful Amy DeRocher From owner-proteins@net.bio.net Mon Feb 10 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!gatech!howland.erols.net!www.nntp.primenet.com!nntp.primenet.com!rill.news.pipex.net!pipex!warwick!bham!mcconvillec.can.bham.ac.uk!user From: @bham.ac.uk (room 103) Newsgroups: bionet.molbio.proteins Subject: Re: Silver Staining Reveals Contaminating Bands in Sample Buffer Date: Tue, 11 Feb 1997 17:55:30 +0000 Organization: University of Birmingham Lines: 36 Message-ID: <-1102971755300001@mcconvillec.can.bham.ac.uk> References: <5dauu6$2rdc@uvaix3e1.comp.UVic.CA> <8mzSmyi00WB_04TGk0@andrew.cmu.edu> NNTP-Posting-Host: mcconvillec.can.bham.ac.uk In article , pxpst2@vms.cis.pitt.edu (THE GREEK) wrote: > > Hi, > /> > /> I have had these bands appear also, right around 50kD or so. I think > /> there is even a reference showing that they are human skin keratin. > /> > /> Even one of the original references for silver staining (sorry I don't > /> have it here I'm writing from home) admits that "at maximum exposure" > /> these bands appear. > /> > //> I can give you refs on Monday morning (EST) if you want. > > > /> > /> Regards, > /> M. > > > > You guys are silly. I would be surprised if you did not see bands with > silver staining. Getting pure proteins without denaturing (ie > acetonitrile) is difficult to say the least. Consult a good protein > purification book and you guys could learn a lot > Peter Pediaditakis pxpst2@vms.cis.pitt.edu The answer whether these guys are silly or not might be found by looking at lanes where no sample (or only sample buffer) was applied. If bands are visible in these lanes, then the guys are maybe not so silly at all but only have to wear gloves, clean their equipment very accurately etc. (Keratins are VERY probable contaminants) Peter Weber P.weber@bham.ac.uk From owner-proteins@net.bio.net Mon Feb 10 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!news-peer.gsl.net!news.gsl.net!news-paris.gsl.net!news.gsl.net!rain.fr!jussieu.fr!univ-lyon1.fr!unice.fr!not-for-mail From: Terreux Raphael Newsgroups: bionet.molbio.proteins Subject: (no subject) Date: Tue, 11 Feb 1997 13:16:12 -0800 Organization: unsa - lartic Lines: 19 Message-ID: <3300E19C.446B@chiminfo.unice.fr> NNTP-Posting-Host: chiminfo.unice.fr Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.01S (X11; I; IRIX64 6.2 IP28) Hello, I don't find in the litterature the value of the concentration of CATECHOLAMINES in ng/mg of protein in a rat's brain. If someone can help me Best regards. TERREUX Raphael, PhD Student -- -------------------------------------- LARTIC Universite de Nice-Sophia Antipolis, Parc Valrose, F 06108, Cedex 2, NICE, FRANCE TEL : +33 (0)4 92 07 61 26 TEL : +33 (0)4 92 07 60 60 poste 27 10 e-mail : TERREUX@CHIMINFO.UNICE.FR -------------------------------------- From owner-proteins@net.bio.net Mon Feb 10 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!newsfeeds.sol.net!newspump.sol.net!howland.erols.net!cam-news-hub1.bbnplanet.com!su-news-hub1.bbnplanet.com!news.bbnplanet.com!news.alt.net!newspost1.alt.net!usenet From: lugmog96@student.umu.se (Ludvig Mortberg) Newsgroups: bionet.molbio.methds-reagnts,bionet.general,bionet.microbiology,bionet.molbio.proteins,de.sci.chemie,sci.bio.microbiology,sci.bio.misc,sci.chem Subject: How many exonucleases have been discovered? Date: Tue, 11 Feb 1997 17:39:57 GMT Organization: Altopia Corp. - Affordable Usenet Access - http://www.alt.net Lines: 22 Message-ID: <3304ae9c.3226590@news.alt.net> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Newsreader: Forte Agent .99f/32.275 Xref: biosci bionet.molbio.methds-reagnts:54655 bionet.general:25578 bionet.microbiology:8853 bionet.molbio.proteins:9959 sci.bio.microbiology:5258 sci.bio.misc:7307 sci.chem:75186 I'm interested in exonucleases. I'm trying to find out how many have been discovered. I know of the following six, but there are probably more; Venom phosphodiesterase Spleen phosphodiesterase E. coli exonuclease I E. coli exonuclease III E. coli exonuclease VII Phage lambda exonuclease Any clues of more exonucleases? Please provide names of them and references. Thanks in advance! Ludvig Mortberg From owner-proteins@net.bio.net Mon Feb 10 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!newsfeeds.sol.net!newspump.sol.net!howland.erols.net!rill.news.pipex.net!pipex!oleane!jussieu.fr!univ-lyon1.fr!unice.fr!not-for-mail From: Terreux Raphael Newsgroups: bionet.molbio.proteins Subject: value of concentration in [catecholamines] Date: Tue, 11 Feb 1997 13:17:47 -0800 Organization: unsa - lartic Lines: 19 Message-ID: <3300E1FB.794B@chiminfo.unice.fr> NNTP-Posting-Host: chiminfo.unice.fr Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.01S (X11; I; IRIX64 6.2 IP28) Hello, I don't find in the litterature the value of the concentration of CATECHOLAMINES in ng/mg of protein in a rat's brain. If someone can help me Best regards. TERREUX Raphael, PhD Student -- -------------------------------------- LARTIC Universite de Nice-Sophia Antipolis, Parc Valrose, F 06108, Cedex 2, NICE, FRANCE TEL : +33 (0)4 92 07 61 26 TEL : +33 (0)4 92 07 60 60 poste 27 10 e-mail : TERREUX@CHIMINFO.UNICE.FR -------------------------------------- From owner-proteins@net.bio.net Mon Feb 10 22:00:00 1997 Path: biosci!ihnp4.ucsd.edu!swrinde!howland.erols.net!cam-news-hub1.bbnplanet.com!su-news-hub1.bbnplanet.com!news.bbnplanet.com!arclight.uoregon.edu!news.bc.net!torn!resunix.sickkids.on.ca!NewsWatcher!user From: Newswatcher@sickkids.on.ca (Newswatcher) Newsgroups: bionet.molbio.proteins Subject: Room Sharing at 41st Biophysical Meeting Date: Tue, 11 Feb 1997 15:01:12 -0600 Organization: The Hospital for Sick Children Lines: 19 Message-ID: NNTP-Posting-Host: 142.20.24.101 Dear all, I am looking for a non-smoking female to share a twin room (Holiday Inn Superdome) during the 41st Biophysical Annual Meeting, from March 1st to 6th, New Orleans. If you like to save some hotel expenses and have a talkmate, please feel free to contact me at the following address: Li-Ping Liu Hospital for Sick Children Toronto, Ontario Canada M5G 1X8 Phone: (416)813-5855 Fax: (416)813-5022 E-Mail: lliu@sickkids.on.ca Thanks for your attention. -- Do not send email to Newswatcher@sickkids.on.ca. It will be bounced. From owner-proteins@net.bio.net Mon Feb 10 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!news.sgi.com!news.maxwell.syr.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!daemon From: klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins Subject: Re: Silver Staining Reveals Contaminating Bands in Sample Buffer Date: Tue, 11 Feb 97 14:38:43 GMT Organization: UW-Madison Lines: 15 Message-ID: <5dq0ck$9c2@news.doit.wisc.edu> References: <5dauu6$2rdc@uvaix3e1.comp.UVic.CA> <8mzSmyi00WB_04TGk0@andrew.cmu.edu> NNTP-Posting-Host: f182-130.net.wisc.edu Mime-Version: 1.0 Content-Type: text/plain; charset=KOI8-R Content-Transfer-Encoding: 8bit X-Newsreader: News Xpress 2.0 X-No-Archive: Yes In article , pxpst2@vms.cis.pitt.edu (THE GREEK) wrote: #/> I have had these bands appear also, right around 50kD or so. I think #/> there is even a reference showing that they are human skin keratin. # #You guys are silly. I would be surprised if you did not see bands with #silver staining. Getting pure proteins without denaturing (ie #acetonitrile) is difficult to say the least. Consult a good protein #purification book and you guys could learn a lot #Peter Pediaditakis pxpst2@vms.cis.pitt.edu Obviously, you don't understand what you are talking about. FYI, the bands appear in empty lanes too. From owner-proteins@net.bio.net Mon Feb 10 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!gatech!csulb.edu!hammer.uoregon.edu!arclight.uoregon.edu!worldnet.att.net!howland.erols.net!newsfeed.internetmci.com!news.westnet.com!not-for-mail From: Jil Tardiff Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Anyone have a source for bovine cTnT? Date: Tue, 11 Feb 1997 21:43:39 -0500 Organization: WestNet Internet Services Lines: 24 Message-ID: <33012E5B.30A3@westnet.com> NNTP-Posting-Host: jtardiff.dialup.westnet.com Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.02E (OS/2; I) Xref: biosci bionet.molbio.methds-reagnts:54703 bionet.molbio.proteins:9966 OK, This is admittedly a long-shot, but I thought I would give it a try.... I'm interested in doing some troponin exchange experiments. As my "donor" I would like to use bovine cardiac Troponin T. Now I know that there are protocols I could use to purify it myself, but because this is for a very limited number of experiments I would like to avoid the long and somewhat difficult purification process. Does anyone know of a commercial source? Any and all suggestions would be appreciated! regards, Jil Tardiff Albert Einstein College of Medicine and the Columbia-Presbyterian Medical Center NY, NY jtardiff@westnet.com From owner-proteins@net.bio.net Tue Feb 11 22:00:00 1997 Path: biosci!agate!howland.erols.net!news.nacamar.de!uunet!in2.uu.net!205.173.251.8!news1.iamerica.net!iax-nachtx-ppp0015.iamerica.net!thomasmw From: thomasmw@iamerica.net (Myron W. Thomas) Newsgroups: bionet.molbio.proteins Subject: Polyketide Biosynthesis Pathway Help Date: Wed, 12 Feb 1997 20:04:20 -0500 Organization: iAmerica, Inc. Lines: 12 Message-ID: NNTP-Posting-Host: 207.101.125.24 X-Newsreader: Trumpet for Windows [Version 1.0 Rev B] Does anybody know if this pathway goes under another name as I can find no references in Biochem Texts. I am working on the synthesis of Amphotericin B. If anybody could possibly help it would be greatly appreciated. thanx in advance Myron Thomas Stephen F. Austin State University Biotechnology Program thomasm@iamerica.net From owner-proteins@net.bio.net Tue Feb 11 22:00:00 1997 Path: biosci!ihnp4.ucsd.edu!swrinde!howland.erols.net!cam-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news.ums.edu!news.umbc.edu!kcarter From: kcarter@umbc.edu (Mr. Ken Carter) Newsgroups: bionet.molbio.proteins Subject: rDNA Techniques Workshop Date: 12 Feb 1997 20:33:08 GMT Organization: University of Maryland, Baltimore County Lines: 59 Message-ID: <5dt9e4$t21@news.umbc.edu> NNTP-Posting-Host: f-umbc7.umbc.edu NNTP-Posting-User: kcarter X-Newsreader: TIN [version 1.2 PL2] >>>>>>>RECOMBINANT DNA: TECHNIQUES AND APPLICATIONS<<<<<<< American Type Culture Collection (ATCC) April 14-18, 1997, Rockville, MD This five-day, laboratory-intensive, course covers up-to-date recombinant DNA procedures and methodologies both in theory and in practice. This workshop provides introductory and intermediate level instruction in recombinant DNA laboratory techniques. A basic knowledge of nucleic acids is helpful but not necessary. Lecture topics will include Molecular Cloning, Enzymes, Vectors, E. coli Host Genotypes, Library Construction, Nucleic Acid Sequencing, Restriction Mapping, Probes and PCR, Synthesis and Cloning of cDNA. Laboratory sessions will include three simultaneous experiments; characterizing and subcloning a DNA fragment and constructing a eukaryotic genomic library in lambda phage. 1.DNA Characterization: restriction digest/mapping of a selected plasmid, fragment size and number determined by horizontal agarose gel-electrophoresis, DNA transfer to a nylon transfer matrix, probe selection, nick translation of probe with labelled dNTP and characterization of DNA fragments by Southern blot analysis. 2.Subcloning of a DNA Fragment: vector digestion to completion, isolation of DNA fragment by preparative restriction digest, fragment size determination by horizontal gel-electrophoresis, DNA extraction from agarose gel, vector insert ligation, screening for transformants by colony hybridization and analysis of plasmid DNA (miniscreens). 3.Construction of a Genomic Library; isolation of eukaryotic genomic DNA, digestion, ligation, in vitro packaging, plating bacteria, assays of packaged phage and screening by plaque hybridization. Approximately 75% of the course is devoted to laboratory instruction while 25% is lecture-oriented. An intensive manual with protocols and procedures is provided for each participant. Faculty: William Nierman, Ph.D. (Workshop Director), Director, ATCC Program in Molecular Biology and Virology; Donna Maglott, Ph.D., Assoc. Staff Scientist; Tamara Feldblyum, M.S., Collection Scientist. Also, Ramon Jordan, Ph.D., Research Plant Pathologist/Virologist, United States Department of Agriculture. Limited to 25 participants FEE: $1,395.00 **For a full schedule and on-line registration, please visit our Web site: http://www.atcc.org/workshops/workshop.html **Or request a brochure, which includes a full schedule and registration form from: kcarter@atcc.org -- ****************************************************************************** Ken Carter ^ American Type Culture Collection / l \ (301) 231-5525 12301 Parklawn Drive /___l___\__ fax:(301) 816-4364 Rockville, Maryland 20852 \______/ kcarter@atcc.org ~~~~~~~~~~~~~~ From owner-proteins@net.bio.net Tue Feb 11 22:00:00 1997 Path: biosci!ihnp4.ucsd.edu!swrinde!howland.erols.net!cam-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!cpk-news-feed4.bbnplanet.com!europa.chnt.gtegsc.com!news.umbc.edu!kcarter From: kcarter@umbc.edu (Mr. Ken Carter) Newsgroups: bionet.molbio.proteins Subject: PCR Techniques Workshop Date: 12 Feb 1997 20:34:12 GMT Organization: University of Maryland, Baltimore County Lines: 62 Message-ID: <5dt9g4$t21@news.umbc.edu> NNTP-Posting-Host: f-umbc7.umbc.edu NNTP-Posting-User: kcarter X-Newsreader: TIN [version 1.2 PL2] >>>>>>>POLYMERSE CHAIN REACTION (PCR) APPLICATIONS/ CYCLE DNA SEQUENCING<<<<<<< American Type Culture Collection (ATCC) April 21-24, 1997, Rockville, MD A four-day, laboratory-intensive, course covering a broad range of applications of the polymerase chain reaction (PCR) for addressing biological problems. Included will be a DNA sequencing experiment using a thermostable DNA polymerase and a temperature cycling protocol (cycle sequencing). The workshop will provide introductory and intermediate level instruction in the application, procedures and trouble-shooting for this extremely useful and versatile technology. A basic knowledge of nucleic acid laboratory procedures is helpful, but not necessary. Approximately 75% of the course is devoted to laboratory instruction while 25% is lecture-oriented. An extensive manual with protocols and procedures is provided for each participant. Lecture topics will include PCR Reaction Basics, Optimizing and Troubleshooting the PCR Reaction, DNA Sequencing, PCR Analysis of Ribosomal DNA to Determine Taxonomic Relatedness, PCR Fingerprinting, PCR in the Diagnosis of Infectious and Genetic Disease, and PCR and Recombinant DNA Methodology. Time will be structured for presentation and discussion of workshop participants' PCR applications/problems. The workshop is intended to be a laboratory-intensive course. Laboratory exercises will include: 1) PCR Diagnosis of Sickle Cell Disease; 2) Fingerprint Analysis of the DNA of Human Individuals; 3) Relatedness of Organisms by Analysis of Amplified Ribosomal DNA; 4) Amplification from mRNA; 5) Optimization of PCR Reactions; 6) Labeling of Hybridization Probes by PCR; 7) Cycle DNA Sequencing; and 8) Fingerprinting of Microorganisms by RAPD and Inter-repeat PCR. Results will be analyzed and discussed. Faculty: William Nierman, Ph.D. (Workshop Director), Director, ATCC Program in Molecular Biology and Virology; Donna Maglott, Ph.D., Research Scientist; Yvonne Reid, Ph.D., Collection Scientist; Francis Molina, Ph.D., Research Scientist; Tamara Feldblyum, M.S., Collection Scientist. * The polymerase chain reaction (PCR) process is covered by patents owned by Hoffmann-La Roche. Use of the PCR process requires a license. Limited to 25 participants FEE: $1,195.00 2.8 CEUs **For a full schedule and on-line registration, please visit our Web site: http://www.atcc.org/workshops/workshop.html **Or request a brochure, which includes a full schedule and registration form from: kcarter@atcc.org -- ****************************************************************************** Ken Carter ^ American Type Culture Collection / l \ (301) 231-5525 12301 Parklawn Drive /___l___\__ fax:(301) 816-4364 Rockville, Maryland 20852 \______/ kcarter@atcc.org ~~~~~~~~~~~~~~ From owner-proteins@net.bio.net Tue Feb 11 22:00:00 1997 Path: biosci!bloom-beacon.mit.edu!spool.mu.edu!uwm.edu!www.nntp.primenet.com!nntp.primenet.com!arclight.uoregon.edu!hammer.uoregon.edu!zephyr.texoma.net!uunet!in1.uu.net!138.95.18.2!wilbur.sequent.com!newsfeed.orst.edu!news.orst.edu!lignin.FSL.ORST.EDU!caldwelb From: caldwelb@fsl.orst.edu (Bruce A. Caldwell) Newsgroups: bionet.molbio.proteins Subject: acid phosphatase inhibitors Date: Wed, 12 Feb 1997 21:53:18 GMT Organization: Forest_Science_Department,_Oregon_State_University Lines: 11 Message-ID: NNTP-Posting-Host: lignin.fsl.orst.edu I am looking for information on selective inhibitors for acid phosphomonoesterase. I am trying to distinguish phospholipases C and D in fungal cultures. I know vanadium salts inhibit PME, but I don't know their effects on the phospholipases and phosphodiesterases Any help will be greatly appreciated. Bruce Caldwell Department of Forest Science caldwelb@ccmail.orst.edu Oregon State University 541-737-3674 phone Corvallis, OR 97331-7501 541-737-1393 fax USA From owner-proteins@net.bio.net Tue Feb 11 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!gatech!www.nntp.primenet.com!nntp.primenet.com!news.maxwell.syr.edu!europa.clark.net!newsfeeds.sol.net!hammer.uoregon.edu!zephyr.texoma.net!uunet!in1.uu.net!136.142.185.26!newsfeed.pitt.edu!mv29.pathology.pitt.edu!user From: pxpst2@vms.cis.pitt.edu (THE GREEK) Newsgroups: bionet.molbio.proteins Subject: Re: solubilization of polymerized proteins Date: 12 Feb 1997 17:55:40 GMT Organization: USA Lines: 34 Message-ID: References: <3301efc6.0@wau.nl> NNTP-Posting-Host: mv29.pathology.pitt.edu /> hello everyone, > I am looking for solubilization methods for material strongly polymerized. Are these biopolymers? If so which? > This material contains some enzymes that I want to identify by > electrophoresis. This is tricky and filled with possible pitfalls esp. if sequencing is involved. The more you treat a protein with something the more lickly you are to block the N-terminal. C-terminal is possible but requires lots of material It could be done only if I could solubilize it and recover > the activities. I have used several methods, including, Urea, guanidine > chloride, sonication, combination with soft detergent. > this material can be observed by light microscopy after staining with > coomassie blue. It may be possible to electroelute the protein from the polymer if the protein is not covalently linked to the polmer and this would be very easy. The electrolution buffer should be amoninia carbonate and can be lyophilized away leaving you pure protein. > any help or referencies would be appreciate. Tell me how it goes. I could give you more references if needed and if you are dealing with biopolmers I can give you list of enzyms to use. > > my e-mail address is : anas cherqui@medew.ento.wau.nl Peter Pediaditakis pxpst2@vms.cis.pitt.edu From owner-proteins@net.bio.net Tue Feb 11 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!gatech!csulb.edu!hammer.uoregon.edu!zephyr.texoma.net!uunet!in1.uu.net!136.142.185.26!newsfeed.pitt.edu!mv29.pathology.pitt.edu!user From: pxpst2@vms.cis.pitt.edu (THE GREEK) Newsgroups: bionet.molbio.proteins Subject: Re: Silver Staining Reveals Contaminating Bands in Sample Buffer Date: 12 Feb 1997 17:43:48 GMT Organization: USA Lines: 32 Message-ID: References: <5dauu6$2rdc@uvaix3e1.comp.UVic.CA> <8mzSmyi00WB_04TGk0@andrew.cmu.edu> <-1102971755300001@mcconvillec.can.bham.ac.uk> NNTP-Posting-Host: mv29.pathology.pitt.edu In article , Mustafa Unlu wrote: /> > You guys are silly. I would be surprised if you did not see bands with /> > silver staining. Getting pure proteins without denaturing (ie /> > acetonitrile) is difficult to say the least. Consult a good protein /> > purification book and you guys could learn a lot /> > Peter Pediaditakis pxpst2@vms.cis.pitt.edu /> /> /> Dear Peter, /> /> In Morissey's paper (Anal Biochem 117:307-310 (1988)), he clearly /> states that there is a contaminant "in the sample buffer" that appears /> no matter what you load in a gel. Presumably this contaminant is low /> enough of a conecetration that it only appears when you "stain to /> maximum sensitivity", which I think is a nice way of saying /> "overstaining". In the future, I would suggest doing some background /> search or having an idea of the topic being discussed before jumping /> into a thread. /> /> Regards, /> M. To all: I missed the earlier post regarding the exact problem and to that I appoligize. But when dealing with silver stain which is capable of seeing trace impurities(< 2ng), I would assume that you guys would use HPLC grade reagents and wear gloves. If your problem band is coming from your reagents then that should have been controled for in one of the lanes of your gels. Peter From owner-proteins@net.bio.net Tue Feb 11 22:00:00 1997 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.erols.net!cam-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!europa.clark.net!arclight.uoregon.edu!hammer.uoregon.edu!zephyr.texoma.net!uunet!in3.uu.net!136.142.185.26!newsfeed.pitt.edu!bb3.andrew.cmu.edu!andrew.cmu.edu!mustafa+ From: Mustafa Unlu Newsgroups: bionet.molbio.proteins Subject: Re: Silver Staining Reveals Contaminating Bands in Sample Buffer Date: Wed, 12 Feb 1997 12:21:00 -0500 Organization: Doctoral student, Chemistry, Carnegie Mellon, Pittsburgh, PA Lines: 23 Message-ID: References: <5dauu6$2rdc@uvaix3e1.comp.UVic.CA> <8mzSmyi00WB_04TGk0@andrew.cmu.edu> <-1102971755300001@mcconvillec.can.bham.ac.uk> NNTP-Posting-Host: po9.andrew.cmu.edu In-Reply-To: <-1102971755300001@mcconvillec.can.bham.ac.uk> > You guys are silly. I would be surprised if you did not see bands with > silver st