From owner-proteins@net.bio.net Fri Mar 01 22:00:00 1996 Path: biosci!agate!dog.ee.lbl.gov!news.cs.utah.edu!swen.emba.uvm.edu!hbeernin From: hbeernin@med.uvm.edu (Hans Beernink) Newsgroups: bionet.molbio.proteins Subject: Re: How can I further exploit E. Coli Date: 1 Mar 1996 14:35:29 GMT Organization: EMBA Computer Facility, The University of Vermont Lines: 40 Message-ID: <4h71vh$qov@swen.emba.uvm.edu> References: NNTP-Posting-Host: protein.med.uvm.edu X-Newsreader: TIN [version 1.2 PL1] Q. Shang (qshang@u.washington.edu) wrote: [various tried methods deleted] : Does : anybody have suggestions as of what other factors I can change to have : the bacteria work harder for me? I don't necessarily need them to express : soluble protein, I just need them to express...and express... Well, Tanya, I would take a serious look at your promoter. What vector are you using for expression, and what promoter is your gene under? Some plasmids are just not that stable, or are low copy in coli, but I assume that you're using drug selection to maintain plasmid, and that you're using a high copy expression vector. Also, as was suggested earlier, try varying the length of your induction time. Are you adding chloramphenicol to your inductions? Rifampicin? Finally, have you taken a look at the expressed protein? Is it expressed into inclusion bodies or is it soluble? It may be that your protein is toxic to coli and that's why you only get limited expression... Best regards, Hans -- _____________________________________________________________________________ "The worst monotonous drone coming from a lectern or the most eye-splitting textbook written in turgid English is nothing in comparison to the psychological Sahara that starts right in your bedroom and spurns the horizon." -Joseph Brodsky, from "In praise of Boredom" delivered as a commencement address at Dartmouth College. _____________________________________________________________________________ Hans T.H. Beernink, Department of Biochemistry, University of Vermont hbeernin@protein.med.uvm.edu hbeernin@zoo.uvm.edu FAX (802)862-8229 Tel.(802)656-8244 From owner-proteins@net.bio.net Fri Mar 01 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!elroy.jpl.nasa.gov!swrinde!newsfeed.internetmci.com!in2.uu.net!news1.digital.com!decwrl!tribune.usask.ca!rover.ucs.ualberta.ca!news.ucalgary.ca!holly.cc.uleth.ca!slip115.upanet.uleth.ca!user From: HUZIL@upanet.uleth.ca (John Torin Huzil) Newsgroups: bionet.molbio.proteins Subject: prions Date: Sat, 02 Mar 1996 23:23:56 -0700 Organization: University of Lethbridge Lines: 9 Message-ID: NNTP-Posting-Host: slip115.upanet.uleth.ca X-Newsreader: Yet Another NewsWatcher 2.0b30 I am taking a course in molecular biology and need to present a poster on the subject. My only problem is that I have not been able to find any good diagrams of them. If anyone knows of a journal or textbook that has any diagrams please let me know. Torin -- DNA neither knows nor cares. DNA just is. And we dance to its music. Richard Dawkins From owner-proteins@net.bio.net Fri Mar 01 22:00:00 1996 Path: biosci!agate!dog.ee.lbl.gov!news.cs.utah.edu!cc.usu.edu!cc.usu.edu!nntp Newsgroups: bionet.molbio.proteins Subject: recombinant thrombin Message-ID: <1996Mar2.140806.75654@cc.usu.edu> From: jmorrey@sleepy.usu.edu (jmorrey) Date: 2 Mar 96 14:08:06 MDT Organization: USU Nntp-Posting-Host: johnpm.biotec.usu.edu X-Posted-From: InterNews 1.1@johnpm.biotec.usu.edu X-Authenticated: jmorrey on VMS host sleepy.usu.edu Lines: 3 Is there a source of recombinant thrombin? I cannot use thrombin isolated from plasma since there cannot be any traces of plasma contaminants in the thrombin prep. Thanks in advance!! From owner-proteins@net.bio.net Sat Mar 02 22:00:00 1996 Path: biosci!agate!newsxfer2.itd.umich.edu!chi-news.cic.net!nntp.coast.net!fu-berlin.de!cs.tu-berlin.de!uni-erlangen.de!winx03!wpxx02.toxi.uni-wuerzburg.de!not-for-mail From: krasel@wpxx02.toxi.uni-wuerzburg.de (Cornelius Krasel) Newsgroups: bionet.molbio.proteins Subject: Re: BIOCHEMISTRY Contents ... Date: 3 Mar 1996 17:22:48 GMT Organization: University of Wuerzburg, Germany Lines: 15 Distribution: world Message-ID: <4hckh8$h1d@winx03.informatik.uni-wuerzburg.de> References: <4fpsft$7tm@mserv1.dl.ac.uk> NNTP-Posting-Host: wpxx02.toxi.uni-wuerzburg.de X-Newsreader: TIN [UNIX 1.3 950824BETA PL0] borovok ilya (borovok@POST.TAU.AC.IL) wrote: > Does anyone know the ways to Contents of BIOCHEMISTRY (American Chemical > Society) ? I mean http:// , gopher:// , etc addresses. I am interested > especially to look the latest issues. AFAIK, Biochemistry have only their supplementary material online. Look at http://pubs.acs.org/supmat/bichaw/ --Cornelius. -- /* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */ /* D-97078 Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de SP3 */ /* "Science is the game we play with God to find out what His rules are." */ From owner-proteins@net.bio.net Sat Mar 02 22:00:00 1996 Path: biosci!agate!newsxfer2.itd.umich.edu!chi-news.cic.net!nntp.coast.net!harbinger.cc.monash.edu.au!news.uwa.edu.au!newsman.murdoch.edu.au!usenet From: Jim Cummins Newsgroups: bionet.molbio.methds-reagnts,bionet.immunology,bionet.agroforestry,bionet.celegans,bionet.cellbiol,bionet.drosophila,bionet.general,bionet.microbiology,bionet.molbio.hiv,bionet.molbio.proteins,bionet.molbio.yeast,bionet.mycology,bionet.plants,bionet.virol Subject: Re: PCR Primers Database - UPDATE. Date: 4 Mar 1996 04:29:54 GMT Organization: Murdoch University Lines: 16 Message-ID: <4hdrk2$ljd@newsman.murdoch.edu.au> References: NNTP-Posting-Host: vetmac3.murdoch.edu.au Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) To: bshomer@ebi.ac.uk X-URL: news:DnEqGE.LFJ@ebi.ac.uk Xref: biosci bionet.molbio.methds-reagnts:41296 bionet.immunology:8073 bionet.agroforestry:2828 bionet.celegans:826 bionet.cellbiol:4232 bionet.drosophila:1892 bionet.general:20208 bionet.microbiology:5166 bionet.molbio.hiv:1945 bionet.molbio.proteins:7223 bionet.molbio.yeast:4871 bionet.mycology:3701 bionet.plants:10461 Wallace et al's searchable database on human mtDNA is now up on WWW page http://www.gen.emory.edu/mitomap.html Kogelnik AM, Lott MT, Brown MD, Navathe SB, Wallace DC. Mitomap - a human mitochondrial genome database. Nucleic Acids Research 1996;24(1):177-179. YOu may care to cross-reference Jim Cummins, Associate Professor in Veterinary Anatomy, Murdoch University, Western Australia 6150. TEL +61-9-360 2668 FAX +61-9-310 4144 From owner-proteins@net.bio.net Sat Mar 02 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!newsfeed.internetmci.com!newsxfer2.itd.umich.edu!news.itd.umich.edu!umich.edu!jchristn From: jchristn@umich.edu Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Help! Contract protein sequencing and phage display Date: Fri, 1 Mar 1996 15:51:28 Organization: University of Michigan Lines: 15 Distribution: world Message-ID: NNTP-Posting-Host: pm002-05.dialip.mich.net X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] Xref: biosci bionet.molbio.methds-reagnts:41284 bionet.molbio.proteins:7221 I was hoping that you people could send me information on the experiences you have had with contract protein sequencing companies. We need to get some N-terminals done from proteins transferred to PVDF and would appreciate any info on who is and isn't reliable, best deals, etc. Also, does anyone know if any decent general purpose phage display vectors (not including those from Pharmacia or Stratagene) are or ever will be made commercially available? Please E-mail me directly since I do not have regular access to Usenet. Thanks in advance, Rob Christner igglab@opus.mco.edu rchristner@opus.mco.edu From owner-proteins@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!news.unimelb.EDU.AU!news From: Roger Murphy Newsgroups: bionet.molbio.proteins Subject: Re: protein crosslinking Date: 5 Mar 1996 00:07:45 GMT Organization: University of Melbourne Lines: 8 Message-ID: <4hg0kh$eud@news.unimelb.EDU.AU> References: NNTP-Posting-Host: murphy.pharmacology.unimelb.edu.au Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.2N (Windows; I; 32bit) To: evansj@bcc.orst.edu I'd suggest you try looking through the Pierce Chemicals catalogue. Not only do they sell lots of cross-linkers, but generally they reference the use of them also. Cheers, Roger Murphy From owner-proteins@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!agate!howland.reston.ans.net!vixen.cso.uiuc.edu!uwm.edu!msunews!netnews.upenn.edu!taurus.fccc.edu!sauder From: sauder@castor.fccc.edu (John Michael Sauder) Newsgroups: bionet.molbio.proteins Subject: Re: Enzyme kinetics software Date: 4 Mar 1996 17:31:39 GMT Organization: Fox Chase Cancer Center, Philadelphia, PA Lines: 14 Message-ID: <4hf9dr$2bn@taurus.fccc.edu> References: NNTP-Posting-Host: castor.rm.fccc.edu In article j-sekiguchi@SKI.MSKCC.ORG (JoAnn Sekiguchi) writes: > I am looking for a good software package that will enable me to > plot my kinetics data and to determine rate constants (as well as Km, Kd, > etc). Does anyone have any suggestions or preferences? Is there any > shareware/freeware that may be able to carry out these functions? > Check out KINSIM. Biophys. J. (1994) 67:1260 I don't have the URL handy, but you could try e-mailing: pollard@jhuigf.med.jhu.edu -- -- Mike S. (M_Sauder@fccc.edu) www.fccc.edu/research/labs/roder/mike.html From owner-proteins@net.bio.net Sun Mar 03 22:00:00 1996 Newsgroups: bionet.molbio.proteins Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!bunyip.cc.uq.oz.au!marlin.jcu.edu.au!lionfish.jcu.edu.au!sci-baa From: Belinda Ahlers Subject: Protein silver staining. Content-Type: TEXT/PLAIN; charset=US-ASCII Message-ID: Sender: news@marlin.jcu.edu.au (USENET News System) Organization: James Cook University Mime-Version: 1.0 Date: Tue, 5 Mar 1996 14:36:13 +1000 Lines: 10 I'm just curious whether anyone has a particularly sensitive silver staining recipe thats tried and true for proteins. I have already stained my gel using Coomassie blue with unfavourable results and would like a staining method which is anywhere between 100-1000 times more sensitive if possible. Any suggestions would be most appreciated. Cheers, Belinda. From owner-proteins@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!rutgers!csn!news-1.csn.net!ub!newsstand.cit.cornell.edu!news.tc.cornell.edu!newsserver.sdsc.edu!news.cerf.net!ni1.ni.net!cyto.cytosignal.com!user From: biotech@ni.net (David J Benz) Newsgroups: bionet.molbio.proteins Subject: Constitutive Fusion Protein Expression in BL21(DE3) Cells Date: 5 Mar 1996 00:10:48 GMT Organization: CytoSignal, Inc. Lines: 27 Message-ID: NNTP-Posting-Host: cyto.cytosignal.com Hello Everyone! We're having some trouble with our protein expression system. Like almost everyone else, we use an expression vector to express a His-Tagged fusion protein, which we subsequently purify over a Qiagen Ni-NTA column. What we've noticed is that lately our proteins are beginning to show up in uninduced bacteria. The tac promoter is supposed to drive transcription, and shouldn't be active in the absence of IPTG. Why are our proteins expressing in uninduced bacteria? First of all, we do not have any IPTG contamination in our glass flasks, which are autoclaved prior to each round of preparative bacterial growth. And yes, we know about the so-called pLys variant of BL21 cells which really represses the expression of T7 polymerase, thus giving almost no expression of the his-tagged fusion protein until IPTG is added. And No, we're not using the pLys variant, and maybe we should be.... But I think we have a more fundamental problem. I think our bacteria are changing somehow so that expression becomes constitutive. This is also a problem because we can't try various levels of induction in an attempt to reduce subunit aggregation. Of course, the insoluble proteins are much harder to purify using Gu-HCL or Urea... If anyone has had this experience and knows why it happens, I would sure appreciate an email or a post!! Thanks in advance... David From owner-proteins@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!swrinde!newsfeed.internetmci.com!chi-news.cic.net!newsspool.doit.wisc.edu!news.doit.wisc.edu!martinlab From: klenchin@macc.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins Subject: Re: Enzyme kinetics software Date: Tue, 05 Mar 96 01:57:08 GMT Organization: UW-Madison Lines: 23 Message-ID: <4hg71k$95c_001@biochem.wisc.edu> References: NNTP-Posting-Host: martinlab.biochem.wisc.edu X-Newsreader: News Xpress Version 1.0 Beta #4 In article , j-sekiguchi@SKI.MSKCC.ORG (JoAnn Sekiguchi) wrote: -> I am looking for a good software package that will enable me to ->plot my kinetics date and to determine rate constants (as well as Km, Kd, ->etc). Does anyone have any suggestions or preferences? Is there any ->shareware/freeware that may be able to carry out these functions? Dear Joann, Any decent software with built-in non linear least square algorithm will do the job. I personally use SigmaPlot for DOS - great for publication quality plotting, fast and straightforward. It's expensive, though, and is getting replaced by Windows version (slow and stupid). If all your care about is finding out equilibrium constants (Michaelis-Menten + extensions), there is a freeware called "Kinetic". Since the author permits, I can send it to everyone interested via email (~ 150 K ZIP, MIME or UUencode). Drop me email if you want it. - Dima From owner-proteins@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!news.unimelb.EDU.AU!news From: Roger Murphy Newsgroups: bionet.molbio.proteins Subject: Re: Enzyme kinetics software Date: 5 Mar 1996 00:14:31 GMT Organization: University of Melbourne Lines: 8 Message-ID: <4hg118$eud@news.unimelb.EDU.AU> References: NNTP-Posting-Host: murphy.pharmacology.unimelb.edu.au Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.2N (Windows; I; 32bit) To: j-sekiguchi@SKI.MSKCC.ORG Check out GraphPad Software's Prism curve fitting and plotting software. Useful for all sorts of things, but a little pricey at around US$500. Works well though! Cheers, Roger Murphy From owner-proteins@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!agate!howland.reston.ans.net!plug.news.pipex.net!pipex!weld.news.pipex.net!pipex!tank.news.pipex.net!pipex!warwick!news.nott.ac.uk!griffin.nott.ac.uk!usenet From: Nigel Kenward Newsgroups: bionet.molbio.proteins Subject: Re: prions Date: Mon, 04 Mar 1996 11:28:40 +0000 Organization: Dept. Biochemistry, University of Nottingham, U.K. Lines: 12 Message-ID: <313AD3E8.3EF8@vax.nott.ac.uk> References: Reply-To: mbxnjk@vax.nott.ac.uk NNTP-Posting-Host: macjm2.biochem.nottingham.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0 (Macintosh; I; 68K) Try looking up reviews by S.B. Prusiner or J Hope or R Kimberlin or B Caughey. All ahve published widely (in things like Ann Rev Biochem etc etc etc) There are THOUSANDS of diagrams ! -- ____ ___ |\ | | / \ / | NIGEL KENWARD, | \ | | | ___ |__ | Dept. Biochemistry, | \ | | | | | | Nottingham University Medical School. | \| | \____/ \___ |___ Clifton Boulevard. ------------------------------- Nottingham. NG7 2UH. Tel (0115 924 9924) ext 44789 Fax (0115 942 2225) email:mbxnjk@vax.nott.ac.uk "You can tell me.......I'm a doctor" From owner-proteins@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!agate!newsxfer2.itd.umich.edu!chi-news.cic.net!uwm.edu!msunews!netnews.upenn.edu!news.tju.edu!joseph2.tjh.tju.edu!user From: boehnin1@jeflin.tju.edu (Darren Boehning) Newsgroups: bionet.molbio.proteins Subject: peptides Date: Mon, 04 Mar 1996 11:41:40 -0500 Organization: Thomas Jefferson University Lines: 7 Message-ID: NNTP-Posting-Host: joseph2.tjh.tju.edu Greetings, Does anyone know of a moderately priced/moderate quality peptide manufacturing company (for use in generating antibodies). Please e-mail me with info Thanks in advance, Darren Boehning boehnin1@jeflin.tju.edu From owner-proteins@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!chi-news.cic.net!nntp.coast.net!news00.sunet.se!sunic!news99.sunet.se!news.uni-c.dk!hteicher From: hteicher@biobase.dk (Harald Teicher) Newsgroups: bionet.molbio.proteins Subject: Re: enzyme definition - help Date: 4 Mar 1996 12:02:09 GMT Organization: News Server at UNI-C, Danish Computing Centre for Research and Education. Lines: 13 Message-ID: <4hem41$q7h@news.uni-c.dk> NNTP-Posting-Host: biobase.dk X-Newsreader: TIN [version 1.2 PL2] Why are the photosynthetic elctron mediators Fd:NADPH oxidoreductase (FNR) and the cytochrome b6-f complex (plastoquinol:plastocyanin oxidoreductase) class- fied as enzymes, while plastocyanin (cyt f:P700 oxidoreductase) and ferredoxin (Fa/Fb:FAD oxidoreductase) are not. I have posted this message to bionet.photosynthesis in a slightly longer ver- sion, and received mixed responses: some say Fd and Pc (and PSI and PSII) are enzymes, others that Fd and Pc are too small to be enzymes, others that proton (H+) transfer is an integral part of oxidoreductase activity..... Any suggestions ? Thanks in advance: Harry From owner-proteins@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!ns1.faseb.org!lamarck.sura.net!newsfeed.internetmci.com!news.inc.net!news.us.world.net!news.aus.world.net!newshost.telstra.net!news.ci.com.au!wabbit.its.uow.edu.au!seagoon.newcastle.edu.au!cc.newcastle.edu.au!mdpjr From: mdpjr@cc.newcastle.edu.au Newsgroups: bionet.molbio.proteins Subject: Re: Enzyme kinetics software Date: 4 Mar 96 18:02:07 +1100 Organization: University of Newcastle, AUSTRALIA Lines: 12 Distribution: world Message-ID: <1996Mar4.180207.1@cc.newcastle.edu.au> References: NNTP-Posting-Host: cc.newcastle.edu.au In article , j-sekiguchi@SKI.MSKCC.ORG (JoAnn Sekiguchi) writes: > I am looking for a good software package that will enable me to > plot my kinetics date and to determine rate constants (as well as Km, Kd, > etc). Does anyone have any suggestions or preferences? Is there any > shareware/freeware that may be able to carry out these functions > I use EnzFit by Robin Leatherbarrow, available from Elsevier Biosoft or Sigma. It does all that you describe very easily. Unfortunately its very expensive. I would love to know as well if you find a freeware program. Phil From owner-proteins@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!ns1.faseb.org!lamarck.sura.net!newsfeed.internetmci.com!howland.reston.ans.net!nntp.coast.net!zombie.ncsc.mil!admaix.sunydutchess.edu!ub!csn!news-1.csn.net!csn!nntp-xfer-2.csn.net!yuma!usenet From: "Stephen R. Decker" Newsgroups: bionet.molbio.proteins Subject: Re: Protein silver staining. Date: 5 Mar 1996 22:24:17 GMT Organization: CSU Lines: 31 Message-ID: <4hieuh$2248@yuma.ACNS.ColoState.EDU> References: NNTP-Posting-Host: lindenlab.vetmed.colostate.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) Belinda, The following is the silver stain protocol from the Hoefer protein electrophoresis manual (summarized, of course): All steps should be about 100 mL for a mini gel, and gently swirled at RT. 1- 40% MeOH, 7% Acetic acid 30 mins rinse with dH2O 2- 7% Acetic acid, 5% meOH 30 mins rinse 3- 10% glutaraldehyde, 30 mins 4- dH2O wash, running at least 2 hours, longer for clearer background can go O/N w/o problem 5- 5ug/mL DTT for 30 mins, do not rinse, drain well 6- 0.1%(w/v) AgNO3, 30 mins rinse quickly in dH2O, then with two small batches of developer 7- 3% Na2CO3, 0.019% formaldehyde This is the developer and should be made just prior to using. All other reagents can be stored at RT for at least a month, with the exception of glutaraldehyde which needs to be in the dark at 4oC. Add the developer and swirl until just underdeveloped. Drain and add solution from step 2 to stop rxn. Can be stored in this solution. This is the trickiest step because the developing rxn continues until the acid from the stop bath gets into the gel. If yo are in a hurry to get the gel, you can cut the first two steps back to 15 mins each and the long wash to about an hour, if you don't mind a little background. Good Luck Steve From owner-proteins@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!daresbury!nntp-trd.UNINETT.no!nntp.uio.no!news.cais.net!chi-news.cic.net!vixen.cso.uiuc.edu!ks.uiuc.edu!dalke From: dalke@ks.uiuc.edu (Andrew Dalke) Newsgroups: bionet.molbio.proteins Subject: What is the glycine sidechain? Date: 5 Mar 1996 23:25:40 GMT Organization: TB BI Lines: 24 Distribution: world Message-ID: <4hiihk$31d@vixen.cso.uiuc.edu> NNTP-Posting-Host: ophelia.ks.uiuc.edu It is a simple question, does glycine have a sidechain? If you say an amino acid base has an group "R" off the CA which is called a sidechain, then the answer is, "yes, and the sidechain is a hydrogen". I can accept that. However, as I think I've mentioned here before, I'm working on a visualization and analysis program (ObPlug: http://www.ks.uiuc.edu/Research/vmd). I want to add an atom selection term called "sidechain" which picks the sidechain atoms. It works for every case except glycine, where I have to determine which of the two hydrogens (in an all atom model, such as charmm-22) is the sidechain. Currently I choose the one with the alphabetically smallest name, so for a charmm-22 model, HC1 is the sidechain and HC2 is not. One of the researchers pointed out that I could find the hydrogen which is closest in position to where a C-beta would be. The problem is when we compute the molecular dynamics of the protein I don't know if the closest hydrogen will always be the same one. I consider it bad if the sidechain alternates between two atoms. Of course, in a reduced atom model, glycine doesn't even have hydrogen off the CA. So, does glycine really have a side chain? If so, what is the "right" (IUPAC?) way of finding it? Andrew dalke@ks.uiuc.edu From owner-proteins@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!agate!howland.reston.ans.net!quagga.ru.ac.za!uct.ac.za!pc14.mic.uct.ac.za!murray From: murray@molbiol.uct.ac.za Newsgroups: bionet.molbio.proteins Subject: Bacterial Rhodanese (Thiosulphate:cyanide sulfertransferase) Date: Tue, 5 Mar 1996 11:45:03 Organization: University of Cape Town Lines: 11 Distribution: world Message-ID: NNTP-Posting-Host: pc14.mic.uct.ac.za Keywords: help I've just started working on a bacterial rhodanese and am having a lot of trouble finding literature in this field. I have found masses of info regarding mammalian rhodanese but very little on the bacterial enzyme. If anyone has any info for me or where I can get hold of it please e-mail me. Thanks in advance Murray From owner-proteins@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!ns1.faseb.org!lamarck.sura.net!ra.nrl.navy.mil!news.math.psu.edu!chi-news.cic.net!nntp.coast.net!swidir.switch.ch!scsing.switch.ch!rzunews.unizh.ch!NewsWatcher!user From: zjons@vetbio.unizh.ch (Zophonias O. Jonsson) Newsgroups: bionet.molbio.proteins Subject: Re: Constitutive Fusion Protein Expression in BL21(DE3) Cells Date: 5 Mar 1996 08:21:27 GMT Organization: Universitat Zurich-Irchel Lines: 30 Message-ID: References: NNTP-Posting-Host: 130.60.120.12 In article , biotech@ni.net (David J Benz) wrote: [snip!] > But I think we have a more fundamental problem. I think our bacteria are > changing somehow so that expression becomes constitutive. [snip!] > David There is a very easy way to test you hypothesis. Get some fresh BL21(DE3) cells and compare to the ones you are using. If it is really important that there is no leakage expression I would recommend pLysS or pLysE strains, but to me it would seem that you could turn the constitutive expression to your advantage. You could grow the bugs slowly (e.g. at low temp) and thus giving the expressed protein a better chance of folding correctly. Zophonias _____________________________________________________________________ Zophonias O. Jonsson Institut fur Veterinarbiochemie Tel: (41-1)-257-54-75 Universitat Zurich-Irchel Fax: (41-1)-362-05-01 Winterthurerstrasse 190 CH-8057 Zurich Switzerland _____________________________________________________________________ From owner-proteins@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!agate!howland.reston.ans.net!tank.news.pipex.net!pipex!dispatch.news.demon.net!demon!mchlhnln.demon.co.uk!michael From: Michael Hanlon Newsgroups: bionet.molbio.proteins Subject: dehydration of protein environments Date: Tue, 5 Mar 1996 23:09:19 +0000 Organization: nolnah inc. Lines: 6 Distribution: world Message-ID: NNTP-Posting-Host: mchlhnln.demon.co.uk X-NNTP-Posting-Host: mchlhnln.demon.co.uk MIME-Version: 1.0 X-Newsreader: Turnpike Version 1.09 Does anyone know of any references regarding the effects of dehydration of the solvent environment on the structure of water soluble proteins, particularly myoglobin. -- Michael Hanlon From owner-proteins@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!vixen.cso.uiuc.edu!newsfeed.internetmci.com!in2.uu.net!ftpbox!newsfeed.acns.nwu.edu!news.cc.uic.edu!usenet From: Keld Sorensen Newsgroups: bionet.molbio.proteins Subject: Re: Protein silver staining. Date: 5 Mar 1996 15:26:02 GMT Organization: Univ. Illinois / College of Med. Lines: 10 Message-ID: <4hhmea$fvc@piglet.cc.uic.edu> References: NNTP-Posting-Host: sliprock-03.dialin.uic.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; PPC) To: sci-baa@jcu.edu.au X-URL: news:Pine.OSF.3.91.960305143053.28160A-100000@lionfish.jcu.edu.au Most companies will sell silver stains that work well. In addition, it is good to remember that the sensitivity of silver staining is enhanced if you FIRST do a Coomassie stain - even if you don't see any bands with the coomassie, you are depsositing coomassie dye that is then the nucleus for the silver. And it can't hurt, so it is a win-win. Keld. From owner-proteins@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!natinst.com!news-relay.us.dell.com!swrinde!howland.reston.ans.net!Germany.EU.net!wizard.pn.com!brighton.openmarket.com!decwrl!tribune.usask.ca!rover.ucs.ualberta.ca!news From: wklimke@gpu.srv.ualberta.ca (William Klimke) Newsgroups: bionet.molbio.proteins Subject: Re: What is "N-terminal blockage"? Date: 5 Mar 1996 21:52:53 GMT Organization: University of Alberta, Edmonton, Canada Lines: 5 Message-ID: <4hid3l$v0e@pulp.ucs.ualberta.ca> NNTP-Posting-Host: pili.biology.ualberta.ca Mime-Version: 1.0 Content-Type: Text/Plain; charset=US-ASCII X-Newsreader: WinVN 0.99.6 Certain proteins in prokaryotes are blocked at the N-terminus via N-methylpheylalanine. Bill Klimke From owner-proteins@net.bio.net Mon Mar 04 22:00:00 1996 Newsgroups: bionet.molbio.proteins Path: biosci!agate!howland.reston.ans.net!torn!utnut!oci!news From: Flip Hoedemaeker Subject: Re: Native PAGE @ pH < 6.5 Content-Type: text/plain; charset=us-ascii Message-ID: <313CEC17.2DB4@oci.utoronto.ca> Sender: news@oci.utoronto.ca Content-Transfer-Encoding: 7bit Organization: The Holistic Detective Agency References: <4hhheo$afa@lyra.csx.cam.ac.uk> Mime-Version: 1.0 Date: Wed, 6 Mar 1996 01:36:23 GMT X-Mailer: Mozilla 2.0 (Win16; I) Lines: 4 Just a thought.... Use a buffer below your pI (glycine pH 2.5?) and reverse the polarity of your electrodes! From owner-proteins@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!agate!dog.ee.lbl.gov!news.cs.utah.edu!cc.usu.edu!cc.usu.edu!nntp Newsgroups: bionet.molbio.proteins Subject: recombinant thrombin source Message-ID: <1996Mar5.100017.75908@cc.usu.edu> From: jmorrey@sleepy.usu.edu (jmorrey) Date: 5 Mar 96 10:00:17 MDT Organization: USU Nntp-Posting-Host: johnpm.biotec.usu.edu X-Posted-From: InterNews 1.1@johnpm.biotec.usu.edu X-Authenticated: jmorrey on VMS host sleepy.usu.edu Lines: 3 Is there a source of recombinant thrombin? I cannot use thrombin isolated from plasma since there cannot be any traces of plasma contaminants in the thrombin prep. Thanks in advance!! From owner-proteins@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!agate!dog.ee.lbl.gov!news.cs.utah.edu!cc.usu.edu!cc.usu.edu!nntp Newsgroups: bionet.molbio.proteins Subject: recombinant thrombin source Message-ID: <1996Mar5.095949.75907@cc.usu.edu> From: jmorrey@sleepy.usu.edu (jmorrey) Date: 5 Mar 96 09:59:49 MDT Organization: USU Nntp-Posting-Host: johnpm.biotec.usu.edu X-Posted-From: InterNews 1.1@johnpm.biotec.usu.edu X-Authenticated: jmorrey on VMS host sleepy.usu.edu Lines: 3 Is there a source of recombinant thrombin? I cannot use thrombin isolated from plasma since there cannot be any traces of plasma contaminants in the thrombin prep. Thanks in advance!! From owner-proteins@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!ns1.faseb.org!lamarck.sura.net!newsfeed.internetmci.com!in1.uu.net!brighton.openmarket.com!decwrl!tribune.usask.ca!rover.ucs.ualberta.ca!gallin.biology.ualberta.ca!wgallin From: wgallin@gpu.srv.ualberta.ca (Warren Gallin) Newsgroups: bionet.molbio.proteins Subject: Re: Native PAGE @ pH < 6.5 Date: Tue, 5 Mar 96 19:02:31 GMT Organization: University of Alberta Lines: 28 Message-ID: References: <4hhheo$afa@lyra.csx.cam.ac.uk> NNTP-Posting-Host: gallin.biology.ualberta.ca X-Newsreader: VersaTerm Link v1.1.4 In Article <4hhheo$afa@lyra.csx.cam.ac.uk>, cdw1000@mole.bio.cam.ac.uk (Christopher Walentas (Bioc)) wrote: >I was wondering if anyone had a buffering system that they could >recommend for the resolution of two proteins using native PAGE at >a pH at or below 6.5? I've got a mixture of two species which >differ by a series of histidines, and need to utilise their charge >differences (which are only relevant below their pKa), but cannot >go too acidic due their pI is around 4. I've tried citric acid >at pH 5.5 (100mM) but with not much luck-- the proteins are 100kDa >and hardly made it into the resolving gel by the time the BPBlue >had scooted off the bottom. Two points tha might be helpful. 1) If possible, lower your acrylamide concentration (but no lower than 5%) to increase the protein's mobility in the gel. 2) There is nothing magic about BPBlue going off the end of the gel. Just run longer (use a timer). You already know how far your bands migrate into the gel in the time it takes for the BPBlue to run off, just multiply that time by gel length divided by the migration distance of the fastest migrating band. These two points will help you maximize the separation using the citric acid system. Warren Gallin, Department of Biological Sciences, University of Alberta wgallin@gpu.srv.ualberta.ca From owner-proteins@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!agate!howland.reston.ans.net!vixen.cso.uiuc.edu!uwm.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!martinlab From: klenchin@macc.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins Subject: Re: Constitutive Fusion Protein Expression in BL21(DE3) Cells Date: Tue, 05 Mar 96 15:58:34 GMT Organization: UW-Madison Lines: 21 Message-ID: <4hhoba$8rs_002@biochem.wisc.edu> References: NNTP-Posting-Host: martinlab.biochem.wisc.edu X-Newsreader: News Xpress Version 1.0 Beta #4 In article , biotech@ni.net (David J Benz) wrote: ->Hello Everyone! We're having some trouble with our protein expression ->system. Like almost everyone else, we use an expression vector to express ->a His-Tagged fusion protein, which we subsequently purify over a Qiagen ->Ni-NTA column. What we've noticed is that lately our proteins are ->beginning to show up in uninduced bacteria. The tac promoter is supposed ->to drive transcription, and shouldn't be active in the absence of IPTG. ->Why are our proteins expressing in uninduced bacteria? I have exactly the samr with GST fusions (pGEX-2T in JM109). Up to the point where in dense culture there is no difference between induced nad uninduced cultures. Would really love to know how I can prevent this. Please, if you get any useful info, post summary or email copy to me. Thanks, - Dima From owner-proteins@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!ns1.faseb.org!lamarck.sura.net!ra.nrl.navy.mil!news.math.psu.edu!psuvax1!uwm.edu!chi-news.cic.net!newsxfer2.itd.umich.edu!tank.news.pipex.net!pipex!swrinde!news.uh.edu!news.uth.tmc.edu!bmb155.med.uth.tmc.edu!user From: tliang@utmmg.med.uth.tmc.edu (T. Chyau Liang) Newsgroups: bionet.molbio.proteins Subject: Re: posttranslational mod site? Date: 5 Mar 1996 15:36:38 GMT Organization: UT Medical School at Houston Lines: 22 Message-ID: References: <4h2q3g$7eh@thorn.cc.usm.edu> NNTP-Posting-Host: bmb155.med.uth.tmc.edu Mime-Version: 1.0 Content-Type: text/plain;charset=US-ASCII Content-Transfer-Encoding: 7bit In article <4h2q3g$7eh@thorn.cc.usm.edu>, rbateman@ocean.st.usm.edu (Robert Bateman) wrote: > I am teaching a course on proteins and have asked my students to find web > sites at which they could submit a protein sequence and it would predict > posttranslational modifications (phosphorylation & glycosylation sites, > etc). They (and I) are having a hard time finding such sites. Are there > any? Any suggestions would be appreciated. > > Bob Bateman Try Baylor Search Launcher at http://kiwi.imgen.bcm.tmc.edu:8080/search-launcher/launcher.html use the PROSITE search from the menu. Good luck. T. Chyau Liang, U Texas-Houston From owner-proteins@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!tank.news.pipex.net!pipex!peer-news.britain.eu.net!lyra.csx.cam.ac.uk!mole.bio.cam.ac.uk!cdw1000 From: cdw1000@mole.bio.cam.ac.uk (Christopher Walentas (Bioc)) Newsgroups: bionet.molbio.proteins Subject: Native PAGE @ pH < 6.5 Date: 5 Mar 1996 14:00:56 GMT Organization: University of Cambridge, England Lines: 17 Message-ID: <4hhheo$afa@lyra.csx.cam.ac.uk> NNTP-Posting-Host: mole.bio.cam.ac.uk Keywords: Nativ PAGE X-Newsreader: NN version 6.5.0 #3 (NOV) I was wondering if anyone had a buffering system that they could recommend for the resolution of two proteins using native PAGE at a pH at or below 6.5? I've got a mixture of two species which differ by a series of histidines, and need to utilise their charge differences (which are only relevant below their pKa), but cannot go too acidic due their pI is around 4. I've tried citric acid at pH 5.5 (100mM) but with not much luck-- the proteins are 100kDa and hardly made it into the resolving gel by the time the BPBlue had scooted off the bottom. I've looked into a number of Practical Approach-type reference manuals, but for the most part they are too general and of little or no help. Any advice in this matter would be most appreciated. Cheers, tripp From owner-proteins@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!bcm.tmc.edu!pendragon!news.msfc.nasa.gov!elroy.jpl.nasa.gov!swrinde!cs.utexas.edu!howland.reston.ans.net!Germany.EU.net!Dortmund.Germany.EU.net!gsf.de!bartok.gsf.de!atkinson From: Mike Newsgroups: bionet.molbio.methds-reagnts,bionet.cellbiol,bionet.molbio.proteins Subject: Re: inducible eukaryotic expression Date: 5 Mar 1996 11:20:41 GMT Organization: a Lines: 11 Distribution: world Message-ID: <4hh829$a77@cony.gsf.de> References: NNTP-Posting-Host: bartok.gsf.de X-Newsreader: Nuntius Version 1.2 X-XXMessage-ID: X-XXDate: Tue, 5 Mar 1996 11:24:47 GMT Xref: biosci bionet.molbio.methds-reagnts:41344 bionet.cellbiol:4238 bionet.molbio.proteins:7236 In article Annette, vertino_bell@cber.cber.fda.gov writes: ...Problems with inducible expression. Like many I was seduced by the Stratagene protocol on lac induction. We made the lines and found that the presence of a reporter AND a test plasmid in our repressor lines causes leaky repression.I guess the problem is that we dont get enough lac repressor expression. I still cant get Stratagene to send me a list of papers citing sucessful use of the system. From owner-proteins@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!rutgers!gatech!newsfeed.internetmci.com!howland.reston.ans.net!news-e2a.gnn.com!newstf01.news.aol.com!newsbf02.news.aol.com!not-for-mail From: cpnintt@aol.com (CPN INTt) Newsgroups: bionet.molbio.proteins Subject: Looking For Experienced People to conduct Cloning and Expressing of Purified Fungal Proteins Date: 5 Mar 1996 20:56:40 -0500 Organization: America Online, Inc. (1-800-827-6364) Lines: 8 Sender: root@newsbf02.news.aol.com Message-ID: <4hirco$opr@newsbf02.news.aol.com> Reply-To: cpnintt@aol.com (CPN INTt) NNTP-Posting-Host: newsbf02.mail.aol.com We are looking for individuals or research groups to clone and express purifed proteins from fungi into industrial host. If you have such expereince please fax us your information to 1-407-743-8343 or e.mail to this address rgds MAE From owner-proteins@net.bio.net Mon Mar 04 22:00:00 1996 Newsgroups: bionet.molbio.proteins Path: biosci!daresbury!nntp-trd.UNINETT.no!nntp.uio.no!news.cais.net!nntp.coast.net!news.sprintlink.net!news1!vanfrank From: vanfrank@iquest.net (richard van frank) Subject: Re: Protein silver staining. X-Nntp-Posting-Host: ind-000-236-41.iquest.net Message-ID: <4hig5h$1fs_002@vanfrank.iquest.net> Keywords: Protein stain Sender: news@iquest.net (News Admin) Organization: IQuest Network Services X-Newsreader: News Xpress Version 1.0 Beta #4 References: Date: Tue, 5 Mar 1996 22:45:32 GMT Lines: 15 In article , Belinda Ahlers wrote: > >I'm just curious whether anyone has a particularly sensitive silver >staining recipe thats tried and true for proteins. I have already stained my >gel using Coomassie blue with unfavourable results and would like a >staining method which is anywhere between 100-1000 times more sensitive >if possible. Any suggestions would be most appreciated. > >Cheers, > >Belinda. See Slisz and Van Frank, Electrophoresis 1985,6,405-407. This really works. Good luck RMVF From owner-proteins@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!CCMAILGW.MCGAWPARK.BAXTER.COM!Tony_Hong_at_HYL101 From: Tony_Hong_at_HYL101@CCMAILGW.MCGAWPARK.BAXTER.COM Newsgroups: bionet.molbio.proteins Subject: Measuring DNA using fluorescence plate reader Date: 5 Mar 1996 12:36:16 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 9 Sender: daemon@net.bio.net Distribution: world Message-ID: <13c9e4f0@ccmailgw.mcgawpark.baxter.com> NNTP-Posting-Host: net.bio.net Hi, I'm developing an assay to measure DNA contents in CCS and purified materials using a fluorescence plate reader. So far I've used Hoechst 33258 and PicoGreen as the dye and found that they have limited range of sensitivity. Does anyone know of any other dye with a wider range? Any comment will be appreciated. Thanks. T Hong From owner-proteins@net.bio.net Tue Mar 05 22:00:00 1996 Path: biosci!daresbury!nntp-trd.UNINETT.no!Norway.EU.net!nntp.uio.no!news.cais.net!chi-news.cic.net!cs.utexas.edu!news.tamu.edu!news From: mab7690@zeus.tamu.edu (Michail Belyavskyi) Newsgroups: bionet.molbio.methds-reagnts,bionet.cellbiol,bionet.molbio.proteins Subject: Re: inducible eukaryotic expression Date: 5 Mar 1996 17:28:26 GMT Organization: Texas A&M University Lines: 18 Message-ID: <4hhtjq$4i3@news.tamu.edu> References: NNTP-Posting-Host: medpath-leibowitz-lab.tamu.edu X-Newsreader: WinVN version 0.82 Xref: biosci bionet.molbio.methds-reagnts:41383 bionet.cellbiol:4245 bionet.molbio.proteins:7252 In article , vertino_bell@cber.cber.fda.gov (Annette) says: > >Hi Folks. > >I am interested in hearing others' experience with inducible eukaryotic >expression. > >Specifically, my problem is this. I would like to over-express my protein >in CHO cells. Hi Annette In my case I was using MMTV inducible system. I constructed plasmid that have MMTV promotor and NEO resistance gene simultaneously. For some reasons, when I done cotransfection with two vectors - G418 selection will result in obtaining lines without protein of interest. I used C127 cells. It takes some time to work dexamethasone concentration in my case the best was 10 -7M. best luck and let me know if I can be of any help Michail Belyavskyi, Ph D From owner-proteins@net.bio.net Tue Mar 05 22:00:00 1996 Path: biosci!daresbury!nntp-trd.UNINETT.no!nntp.uio.no!biotek19 From: damartin@bioslave.uio.no (David Martin) Newsgroups: bionet.molbio.proteins Subject: Re: Protein silver staining. Date: Wed, 06 Mar 96 10:41:44 GMT Organization: Biotechnology Centre of Oslo Lines: 42 Message-ID: <4hjq5d$2me@ratatosk.uio.no> References: <4hieuh$2248@yuma.ACNS.ColoState.EDU> NNTP-Posting-Host: biotek19.uio.no X-Newsreader: News Xpress Version 1.0 Beta #3 In article <4hieuh$2248@yuma.ACNS.ColoState.EDU>, "Stephen R. Decker" wrote: -->Belinda, --> -->If yo are in a hurry to get the gel, you can cut the first two steps back to 15 mins -->each and the long wash to about an hour, if you don't mind a little background. --> If you are in a _real_ hurry and want a result in 10 mins (and don't mind a bit of background) then you can speed things up a little: Use a clean glass lasanga dish about 8-9 inches (20-25cm) square Place gel on the dish without touching it (split the plates and drop the gel carefully) 1. 10% Acetic acid 40% Methanol, 100 ml, 1 min full power in the microwave. 2. Pour off. rinse briefly with dH20 (30 secs). 3. Pour off. add 100ml dH20 containing 100ul 5mg/ml DTT. Microwave 1 min. 4. Pour off. add 100ml 0.1% AgNO3. Microwave 1 min. agitate 2 min. 5. Pour off. Add 100 ml developer (3% Na2CO3 0.04% formaldehyde), swirl till the liquid is brown then pour off. 6. Add 100ml developer and develop the bands. 7. stop by addition of citric acid Its most definitely not the prettiest silver stain but if you want to cast, run, and stain a mini gel in 90 mins........ ....d ========================================================= * David Martin, PhD - Post-Doctoral Research Fellow * * Atherosclerosis and Thrombosis research group * * Biotechnologisenteret i Oslo * * Gaustadalleen 21 (Postboks 1125 Blindern) * * N-0316 Oslo * * Norway * * Tel: +47 22 95 84 54 Fax: +47 22 69 41 30 * * email: david.martin@biotek.uio.no * ========================================================= From owner-proteins@net.bio.net Tue Mar 05 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!elroy.jpl.nasa.gov!swrinde!howland.reston.ans.net!surfnet.nl!ruu.nl!cble46.chem.ruu.nl!user From: f.s.wouters@chem.ruu.nl (fred wouters) Newsgroups: bionet.molbio.proteins Subject: GFP fusion problems Date: Wed, 06 Mar 1996 12:41:23 +0100 Organization: ruu Lines: 16 Message-ID: NNTP-Posting-Host: cble46.chem.ruu.nl X-Newsreader: Yet Another NewsWatcher 2.1.8 I am desperately seeking for users of the pGFP-N3 N-terminal Protein Fusion Vector (Clontech). Our lab ordered this vector and it doesn't work at all, and Clontech is not very willing to help us with our problem (it doesn't give any fluorescence, not even the uncloned vector). We spoke to another user with the same problem. So now I am looking for users of this vector, or for people who work with GFP-fusion proteins and constructed them in another way (GFP on the N-terminal site). I would appreciate any reaction! (everything!!) Petra de Graaf E-mail: petra@emsaserv.biol.ruu.nl From owner-proteins@net.bio.net Tue Mar 05 22:00:00 1996 Path: biosci!daresbury!yama.mcc.ac.uk!peer-news.britain.eu.net!warwick!leicester!mac57.bioc.le.ac.uk!user From: owe1@le.ac.uk (Daidalos) Newsgroups: bionet.molbio.proteins Subject: Re: Nickel agarose for His tag Date: 6 Mar 1996 09:24:51 GMT Organization: Personal Lines: 22 Message-ID: References: <4h05hf$seg@larry.rice.edu> NNTP-Posting-Host: mac57.bioc.le.ac.uk In article <4h05hf$seg@larry.rice.edu>, "Eugenio L. de Hostos" wrote: > Hi, > anybody out there know how to make Ni2+-agarose for affinity > chromatography of His-tagged proteins? > > Thanks, > Eugenio L. de Hostos, Ph.D. > Department of Biochemistry and Cell Biology, MS 140 > Rice University > email hostos@bioc.rice.edu > URL http://bioc.rice.edu/~hostos Pharmacia make a chelating sepharose support, haven't heard of Chelating agarose for his-tagging though. Orhan. -- --- From the gates of hell I spit my last at thee. From owner-proteins@net.bio.net Tue Mar 05 22:00:00 1996 Path: biosci!ns1.faseb.org!lamarck.sura.net!ra.nrl.navy.mil!news.math.psu.edu!chi-news.cic.net!vixen.cso.uiuc.edu!newsfeed.internetmci.com!in1.uu.net!newsflash.concordia.ca!news.nstn.ca!news.cs.indiana.edu!lynx.unm.edu!bubba.NMSU.Edu!dkim From: dkim@nmsu.edu (DANIEL Y KIM) Newsgroups: bionet.molbio.proteins Subject: Re: Removing Phenol from Protein Fraction Date: 1 Mar 1996 19:44:10 GMT Organization: New Mexico State University, Las Cruces, NM Lines: 6 Message-ID: <4h7k2a$ce@bubba.NMSU.Edu> References: <4gih35$2fq@taco.cc.ncsu.edu> <4gtt4d$857@news.orst.edu> NNTP-Posting-Host: verdi.nmsu.edu Say. . . isn't this is feature of the TRIZOL DNA-RNA-Protein extraction method? You might want to see what they do with their Phenol phase. Daniel Kim From owner-proteins@net.bio.net Tue Mar 05 22:00:00 1996 Newsgroups: bionet.molbio.proteins Path: biosci!bcm.tmc.edu!cs.utexas.edu!natinst.com!news-relay.us.dell.com!swrinde!newsfeed.internetmci.com!tank.news.pipex.net!pipex!peer-news.britain.eu.net!bcc.ac.uk!rmiller From: rmiller@bsm.bioc.ucl.ac.uk (Rob Miller) Subject: Re: What is the glycine sidechain? Message-ID: <1996Mar6.162518.25181@ucl.ac.uk> Date: Wed, 6 Mar 1996 16:25:18 GMT References: <4hiihk$31d@vixen.cso.uiuc.edu> Organization: University College London X-Newsreader: TIN [version 1.2 PL2] Lines: 63 Andrew Dalke (dalke@ks.uiuc.edu) wrote: : It is a simple question, does glycine have a sidechain? If you say an amino : acid base has an group "R" off the CA which is called a sidechain, then the : answer is, "yes, and the sidechain is a hydrogen". : Currently I choose the one with the alphabetically smallest name, so for a : charmm-22 model, HC1 is the sidechain and HC2 is not. One of the researchers : pointed out that I could find the hydrogen which is closest in position to where : a C-beta would be. The problem is when we compute the molecular dynamics of the : protein I don't know if the closest hydrogen will always be the same one. I : consider it bad if the sidechain alternates between two atoms. : So, does glycine really have a side chain? If so, what is the "right" : (IUPAC?) way of finding it? Normally occurring amino acids have L-chirality, so I'd base it on that. Jane Richardson's `Anatomy and Taxonomy of Protein Structure' (think that's the name) paper gives a `corn crib' model for correct L-amino acid chirality; looking along the H-Calpha bond for an amino acid with an R group, you would see: C=O R \ / H ( the C-alpha is behind the H; read `corn' clockwise ) | N This is also how we build `virtual' Glycine C-betas for various modelling and analysis tasks here. rob. -- ----------------------------------------------------- Rob Miller, Ph.D. "...life too closely scrutinized will lead to madness or suicide." Mihaly Csikszentmihalyi Biomolecular Structure and Modelling Unit (BSM), Department of Biochemistry and Molecular Biology, University College / Gower Street / London WC1E 6BT. United Kingdom. Tel: +44 171419 3890 Fax: +44 171380 7193 Internet: rmiller@bsm.bioc.ucl.ac.uk http://www.biochem.ucl.ac.uk/~rmiller ----------------------------------------------------- From owner-proteins@net.bio.net Tue Mar 05 22:00:00 1996 Path: biosci!rutgers!gatech!newsfeed.internetmci.com!newsxfer2.itd.umich.edu!uunet!in2.uu.net!vol.net!news.glink.net.hk!hpg30a.csc.cuhk.hk!news.cuhk.edu.hk!newsfeeder.ust.hk!nntp.hk.super.net!news.duke.edu!galactose.mc.duke.edu.uucp!tschantz From: tschantz@galactose.mc.duke.edu (William P. Tschantz) Newsgroups: bionet.molbio.proteins Subject: Equilibrium dialysis Date: 5 Mar 1996 21:33:48 GMT Organization: Duke University; Durham, N.C. Lines: 14 Distribution: world Message-ID: <4hibvs$jqj@news.duke.edu> NNTP-Posting-Host: galactose.mc.duke.edu Hi I have a question about equilibrium dialysis to find a binding constant for a product to an enzyme. I only plan this to be a one time thing and was wondering if there was a reference out there that explains the set up and maybe how to do this simply with things in the lab instead of buying an equilibrium dialysis setup. I expect the Kd to be in the submicromolar range and the enzyme is soluble. Any help appreciated. Thanks in advance. Bill From owner-proteins@net.bio.net Tue Mar 05 22:00:00 1996 Path: biosci!agate!howland.reston.ans.net!newsjunkie.ans.net!newsfeeds.ans.net!news.interlink.net!Vir.com!not-for-mail From: demeler@selway.umt.edu (Borries Demeler) Newsgroups: bionet.immunology,bionet.microbiology,bionet.molbio.yeast,bionet.molecules.peptides,sci.bio.microbiology,sci.bio.misc,bionet.cellbiol,bionet.general,bionet.molbio.methds-reagnts,bionet.molbio.proteins,sci.bio.technology,bionet.biophysics,comp.infosystems.www.announce Subject: Announce WWW: Analytical Ultracentrifugation/XL-A Followup-To: comp.infosystems.www.misc Date: 7 Mar 1996 01:50:38 -0500 Organization: University of Montana, Missoula Lines: 36 Sender: root@Vir.com Approved: www-announce@boutell.com Distribution: inet Message-ID: <4hkvfd$208@selway.umt.edu> NNTP-Posting-Host: news.vir.com Xref: biosci bionet.immunology:8113 bionet.microbiology:5215 bionet.molbio.yeast:4897 bionet.molecules.peptides:212 sci.bio.microbiology:2844 sci.bio.misc:2407 bionet.cellbiol:4248 bionet.general:20265 bionet.molbio.methds-reagnts:41443 bionet.molbio.proteins:7263 sci.bio.technology:4974 bionet.biophysics:1769 comp.infosystems.www.announce:14143 ANNOUNCING: Analytical Ultracentrifugation WWW Page We are pleased to announce the first WWW page dedicated to analytical ultracentrifugation with the Beckman XL-A analytical ultracentrifuge. The page has been developed at the University of Texas Health Science Center at San Antonio, Dept. of Biochemistry, and can be found at: Contents include: -A searchable reference database -XL-A Sedimentation Analysis Software archive (PC/MAC/DEC/UNIX) -Searchable RASMB Mailing List Archives (RASMB: Reversibel Associations in Structural and Molecular Biology) -Bulletin Board with relevant conference announcements -Data Analysis Tutorial and Guide -Sample Data Analysis for several representative systems -Sample Preparation Guide In addition, information is provided for investigators desiring to perform analytical ultracentrifugation runs at the Departmental Center for Analytical Ultracentrifugation of Macromolecular Assemblies through a collaborative or fee for service arrangement. Please visit often! Regards, -Borries Demeler ******************************************************************************* * Borries Demeler, Ph.D. * * The University of Texas Health Science Center at San Antonio * * Dept. of Biochemistry, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7760 * * Voice: (210) 567-6592 Fax: (210) 567-6595 Email: demeler@bioc02.uthscsa.edu * ******************************************************************************* From owner-proteins@net.bio.net Tue Mar 05 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!elroy.jpl.nasa.gov!swrinde!howland.reston.ans.net!nntp.coast.net!torn!uunet.ca!news.uunet.ca!rcogate.rco.qc.ca!altitude!usenet From: Achim Recktenwald Newsgroups: bionet.molbio.proteins Subject: Re: Enzyme kinetics software Date: Wed, 06 Mar 1996 09:59:23 -0500 Organization: IBEX Technologies, Inc. Lines: 25 Message-ID: <313DA84B.F82@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 2.0 (Macintosh; I; PPC) Hi JoAnn, If you are looking for a Windows-program, I can recommend GraFit 3.0, Erithacus Software. It is very powerful and easy to use. For the Mac I have not found anything really convincing, yet. Since my company is all Mac, I bought UltraFit from Biosoft. On paper it should be able to do everything GraFit can do, but I do not like it. The menus are complicated and I have found some bugs. I prefer to run GraFit under Softwindows on my Mac, though it is a bit slow. GraFit is available from SIGMA Chemical Company, though the price is a bit high. You might get it cheaper from other companies, like SciTech. So far, I have not seen any shareware/freeware I could recommend. Achim _____________________________ Achim Recktenwald, PhD IBEX Technologies, Inc. 5485 rue Pare Montreal, PQ, H4P 1P7 achim@ibex.ca From owner-proteins@net.bio.net Tue Mar 05 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!natinst.com!news-relay.us.dell.com!swrinde!cssun.mathcs.emory.edu!gatech!newsfeed.internetmci.com!in1.uu.net!news.maz.net!unlisys!usenet From: kopatsch@berlin.snafu.de (Jens Kopatsch) Newsgroups: bionet.molbio.proteins Subject: Question about Invertase Date: 6 Mar 1996 23:01:59 GMT Organization: T! Berlin Lines: 6 Message-ID: <4hl5h7$doo@unlisys.unlisys.net> NNTP-Posting-Host: pppx33.berlin.snafu.de X-Newsreader: WinVN 0.92.6+ Can anybody please tell me about the usage of invertase in the field of biotechnology and food industry ? Jens From owner-proteins@net.bio.net Tue Mar 05 22:00:00 1996 Path: biosci!daresbury!bioftp.unibas.ch!infobiogen.fr!jussieu.fr!oleane!tank.news.pipex.net!pipex!newsfeed.internetmci.com!newsxfer2.itd.umich.edu!agate!hpg30a.csc.cuhk.hk!news.cuhk.edu.hk!usenet From: "He, Xianhui" Newsgroups: bionet.molbio.proteins Subject: Re: Protein silver staining. Date: 6 Mar 1996 13:27:16 GMT Organization: Department of Physiology, The Chinese University of Hong Kong Lines: 8 Message-ID: <4hk3rk$j6e@hpg30a.csc.cuhk.hk> References: <4hieuh$2248@yuma.ACNS.ColoState.EDU> NNTP-Posting-Host: @137.189.46.135 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) I have use this protocol to stain SDS-gel. "But why do you use so high concentration of glutaraldehyde(10%)", my colleage in once asked me. So I reduce the concentration to 1% or even 0.5% of glutaraldehyde and get the same result. By using a shaker and raising temperature, the time for each step may be reduced to half of the original and the stain may be finished in 2 hour. From owner-proteins@net.bio.net Tue Mar 05 22:00:00 1996 Path: biosci!agate!howland.reston.ans.net!news-e2a.gnn.com!newstf01.news.aol.com!newsbf02.news.aol.com!not-for-mail From: dvchem@aol.com (DVCHEM) Newsgroups: bionet.molbio.proteins Subject: What should students know? Date: 6 Mar 1996 07:47:45 -0500 Organization: America Online, Inc. (1-800-827-6364) Lines: 31 Sender: root@newsbf02.news.aol.com Message-ID: <4hk1hh$6qc@newsbf02.news.aol.com> Reply-To: dvchem@aol.com (DVCHEM) NNTP-Posting-Host: newsbf02.mail.aol.com The Departments of Biology and Chemistry-Physics of Kean College are considering offering a collaborative graduate program designed to train students for employment in the pharmaceutical and biotechnology industries. However, we are aware that a program designed for the private sector by academicians runs the risk of missing the target. We are therefore asking for input from those who know what background is needed for a student to become an effective employee in these industries: What specific experimental techniques from molecular biology/biochemistry are most valuable in industry? What methods of instrumental analysis are most important in the pharmaceutical and biotechnology industries? What should students know about tissue culture? What should students know about genetics? What should students know about synthetic organic chemistry and separation science? What should students know about pharmacology? Should students have experience with online literature searching? Please send your response(s), via email, to any or all of these questions to: dvitale@turbo.kean.edu Thanks for your help! From owner-proteins@net.bio.net Wed Mar 06 22:00:00 1996 Path: biosci!daresbury!yama.mcc.ac.uk!peer-news.britain.eu.net!warwick!bham!usenet From: bcm4jah2@bham.ac.uk (Jim) Newsgroups: bionet.molbio.proteins Subject: Help! How can I look at the conformational changes in proteins? Date: 7 Mar 1996 11:09:07 GMT Organization: University of Birmingham Lines: 4 Message-ID: <4hmg4j$n7l@sun4.bham.ac.uk> NNTP-Posting-Host: bcs126.bham.ac.uk Mime-Version: 1.0 Content-Type: Text/Plain; charset=ISO-8859-1 X-Newsreader: WinVN 0.99.5 Can anybody please tell me the best way to look at the conformational changes in proteins as they are bound by substrates, inhibitors, etc. Thanks very much James Harper. From owner-proteins@net.bio.net Wed Mar 06 22:00:00 1996 Path: biosci!CS.Arizona.EDU!math.arizona.edu!noao!ennfs.eas.asu.edu!gatech!newsfeed.internetmci.com!news.compuserve.com!news.production.compuserve.com!news From: Geoffrey D. Wheelock <74710.2416@CompuServe.COM> Newsgroups: bionet.molbio.proteins Subject: Re: Protein silver staining. Date: 7 Mar 1996 16:31:36 GMT Organization: Paracelsian, Inc. Lines: 4 Message-ID: <4hn318$66j$1@mhafn.production.compuserve.com> References: Silver staining method: Try Blum et al. Electrophoresis vol 8 pg 93-99, 1987. If you can find it or I messed up the reference, I can send the method. GL, GD Wheelock From owner-proteins@net.bio.net Wed Mar 06 22:00:00 1996 Path: biosci!proteome.com!jg From: jg@proteome.com (James I. Garrels) Newsgroups: bionet.molbio.proteins Subject: Yeast Protein Database (YPD) Date: 7 Mar 1996 04:03:56 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 43 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net To Protein Researchers who need information on yeast proteins: YPD is a WWW resource for the proteins of budding yeast. YPD provides tabulated properties and annotations with a powerful search form: One can search for: 1) specific yeast proteins, searchable by name or by keywords. 2) proteins of specific function, such as protein kinases, transcription factors, cyclins, etc. 3) proteins with particular post-translational modifications. 4) proteins by subcellular localization, including integral vs peripheral membrane, DNA-binding, and RNA-binding. 5) proteins with particular functional motifs. YPD contains an extensive literature survey. More than 8000 references are cited and 20000 lines of annotations are presented for more than 5000 yeast proteins. YPD includes all proteins of known sequence. When the genome sequence is completed later this year, YPD will contain information on all proteins of S. cerevisiae. YPD can be found at: http://www.proteome.com/YPDhome.html . It is a public resource. Comments and suggestions are always welcome. Jim Garrels --------------------------------------------------------------- James I. Garrels, Ph.D. Tel (508) 922-1643 PROTEOME INC. FAX (508) 922-3971 181 Elliott St., Suite 909 Email jg@proteome.com Beverly, MA 01915 WWW http://www.proteome.com --------------------------------------------------------------- From owner-proteins@net.bio.net Wed Mar 06 22:00:00 1996 Path: biosci!daresbury!yama.mcc.ac.uk!peer-news.britain.eu.net!uknet!tank.news.pipex.net!pipex!swrinde!newsfeed.internetmci.com!in2.uu.net!world!zilker.net!newshost.convex.com!news.duke.edu!soprano.cellbio.duke.edu!user From: ChunLin_Lu@cellbio.duke.edu (C.L.L.) Newsgroups: bionet.molbio.proteins Subject: Re: Constitutive Fusion Protein Expression in BL21(DE3) Cells Date: 7 Mar 1996 04:10:59 GMT Organization: Duke University Lines: 14 Message-ID: References: NNTP-Posting-Host: soprano.cellbio.duke.edu In article , biotech@ni.net (David J Benz) wrote: What we've noticed is that lately our proteins are > beginning to show up in uninduced bacteria. The tac promoter is supposed > to drive transcription, and shouldn't be active in the absence of IPTG. > Why are our proteins expressing in uninduced bacteria? > It is true in most cases, not in all. I expressed six different kinds of proteins in BL21 system and found that some of them did make protein without IPTG induction, and got same amount of it. After sveral passages, it disappeared. One protein which is very toxic to bacteria, failed to make in plain BL21, but did in BL21/plys S, no activity. I dont know what cause your expression constitutively, it was once disscussed in this group on this matte, I can not remmember what they posted. From owner-proteins@net.bio.net Wed Mar 06 22:00:00 1996 Newsgroups: bionet.molbio.proteins Path: biosci!CS.Arizona.EDU!noao!ennfs.eas.asu.edu!gatech!newsfeed.internetmci.com!howland.reston.ans.net!surfnet.nl!sun4nl!news From: Gerard Dijkstra Subject: Looking for human cloned NEP X-Nntp-Posting-Host: asp96-0.amsterdam.nl.net Content-Type: text/plain; charset=us-ascii Message-ID: <313F70F1.5B1@duphar.nl> Sender: news@solair1.inter.NL.net (News at news) Content-Transfer-Encoding: 7bit Organization: Solvay Duphar B.V. Mime-Version: 1.0 Date: Thu, 7 Mar 1996 23:27:45 GMT X-Mailer: Mozilla 2.0 (Win16; I) Lines: 4 We are looking for reseacher who have cloned (human) NEP (Neutral endopeptidase-24.11). Please react dijkstr@duphar.nl From owner-proteins@net.bio.net Wed Mar 06 22:00:00 1996 Path: biosci!biosci!not-for-mail From: Eugene A Kapp <100277.2367@CompuServe.COM> Newsgroups: bionet.molbio.proteins,bionet.software Subject: Isoelectric Point calculations Date: 7 Mar 1996 22:31:35 -0800 Organization: MicroMass Lines: 11 Sender: daemon@net.bio.net Distribution: world Message-ID: <4hmra1$c71$1@mhadf.production.compuserve.com> NNTP-Posting-Host: net.bio.net Xref: biosci bionet.molbio.proteins:7273 bionet.software:14911 I have found that the predicted pI calculations from two WWW sites differ by approximately 0.4 for a given protein sequence. Expasy (http://expasy.hcuge.ch/ch2d/pi_tool.html) and the genome sequencing site at (http://genome1.bio.bnl.gov/bbq.html) are therefore using slightly different pKR values. Could someone suggest the correct pKR values or add some light to the situation. Many thanks. From owner-proteins@net.bio.net Wed Mar 06 22:00:00 1996 Path: biosci!agate!dog.ee.lbl.gov!news.cs.utah.edu!cc.usu.edu!cc.usu.edu!nntp Newsgroups: bionet.molbio.proteins Subject: recombinant thrombin source? Message-ID: <1996Mar6.123422.76010@cc.usu.edu> From: jmorrey@sleepy.usu.edu (jmorrey) Date: 6 Mar 96 12:34:22 MDT Organization: USU Nntp-Posting-Host: johnpm.biotec.usu.edu X-Posted-From: InterNews 1.1@johnpm.biotec.usu.edu X-Authenticated: jmorrey on VMS host sleepy.usu.edu Lines: 3 Is there a source of recombinant thrombin? I cannot use thrombin isolated from plasma since there cannot be any traces of plasma contaminants in the thrombin prep. Thanks in advance!! From owner-proteins@net.bio.net Wed Mar 06 22:00:00 1996 Path: biosci!agate!dog.ee.lbl.gov!news.cs.utah.edu!cc.usu.edu!cc.usu.edu!nntp Newsgroups: bionet.molbio.proteins Subject: recombinant thrombin source? Message-ID: <1996Mar6.123338.76008@cc.usu.edu> From: jmorrey@sleepy.usu.edu (jmorrey) Date: 6 Mar 96 12:33:37 MDT Organization: USU Nntp-Posting-Host: johnpm.biotec.usu.edu X-Posted-From: InterNews 1.1@johnpm.biotec.usu.edu X-Authenticated: jmorrey on VMS host sleepy.usu.edu Lines: 3 Is there a source of recombinant thrombin? I cannot use thrombin isolated from plasma since there cannot be any traces of plasma contaminants in the thrombin prep. Thanks in advance!! From owner-proteins@net.bio.net Wed Mar 06 22:00:00 1996 Path: biosci!bloom-beacon.mit.edu!senator-bedfellow.mit.edu!cancer-ctr-mac-38.mit.edu!user From: ege@mit.edu (ege) Newsgroups: bionet.molbio.methds-reagnts,bionet.cellbiol,bionet.molbio.proteins Subject: Re: inducible eukaryotic expression Date: 7 Mar 1996 21:56:29 GMT Organization: mit Lines: 23 Message-ID: References: NNTP-Posting-Host: cancer-ctr-mac-38.mit.edu Xref: biosci bionet.molbio.methds-reagnts:41476 bionet.cellbiol:4253 bionet.molbio.proteins:7270 In article , vertino_bell@cber.cber.fda.gov (Annette) wrote: > Hi Folks. > > I am interested in hearing others' experience with inducible eukaryotic > expression. > Hi Annette Wow RPCI alumni on the net. I've been beating my head against the wall with Bujard's original Tet system for conditional expression of E2Fs. Much heartache and frustration. It seems that you get leaky expression of the E2Fs even in the presence of tet which is sufficent to induce apoptosis (At least in some cell lines). A recent report in MCB from a Danish group showed this could be suppressed by coexpressing Bcl2 allowing for the isolation of inducible lines. The modified version from the Yale lab presumablly wouldn't help with this problem and besides with the autoregulatory loop you make so much tTA in the absence of tet that you probablly induce your gene to such gross levels that it's no longer physiological. As for the Stratagene system I have no clue. Good luck Scott Estes From owner-proteins@net.bio.net Wed Mar 06 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!natinst.com!news-relay.us.dell.com!swrinde!news.uh.edu!news.uth.tmc.edu!bmb155.med.uth.tmc.edu!user From: tliang@utmmg.med.uth.tmc.edu (T. Chyau Liang) Newsgroups: bionet.molbio.proteins Subject: Re: Help! How can I look at the conformational changes in proteins? Date: 7 Mar 1996 19:04:12 GMT Organization: UT Medical School at Houston Lines: 29 Message-ID: References: <4hmg4j$n7l@sun4.bham.ac.uk> NNTP-Posting-Host: bmb155.med.uth.tmc.edu Mime-Version: 1.0 Content-Type: text/plain;charset=US-ASCII Content-Transfer-Encoding: 7bit In article <4hmg4j$n7l@sun4.bham.ac.uk>, bcm4jah2@bham.ac.uk (Jim) wrote: > Can anybody please tell me the best way to look at the conformational changes > in proteins as they are bound by substrates, inhibitors, etc. > Thanks very much James Harper. Hi Jim, You have several possibilities: (1) Fluorescence spectroscopy: very sentitive and can tell you whether there is conformational change or not. However, it does not tell what kind of conformational changes. Often, you can monitor the intrinsic fluorophore on the protein or the substrate/inhibitor. (2) Circular dichroism (CD): sensitivity comparable to UV/VIS spectroscopy and can tell you more info on conformational changes (e.g., extent of alpha-helix or beta sheet changes). (3) NMR: a very insensitive method, but can potentially provide more info. The same can be said about X-ray. These are just a few mthods come to my mind. Others may be able to provide you alternate methods. hope this helps. T. Chyau Liang, Ph.D. U. Texas-Houston From owner-proteins@net.bio.net Thu Mar 07 22:00:00 1996 Path: biosci!daresbury!sunsite.doc.ic.ac.uk!news.cc.ic.ac.uk!usenet From: non-authenticated Newsgroups: bionet.molbio.proteins Subject: Overexpressed E.Coli Protein sticks DNA - help needed! Date: 8 Mar 1996 09:14:01 GMT Organization: Imperial College, London, UK Lines: 26 Message-ID: <4hotop$rh3@oban.cc.ic.ac.uk> NNTP-Posting-Host: bc-57.sm.ic.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Windows; I; 16bit) Hi I am trying to purify a his-tagged fusion protein in E.coli. It expresses well, although comes out in inclusion bodies. After solubilisation in 6M urea we can dilute and dialyse away the denaturant without precipitating the protein - so far so good...! Anyway we tried to put the protein over a s-sepharose column and hardly anything would stick. We put a sample in the spec and did a 250nm-350nm scan and saw lots of DNA. So we thought adding DNase/RNase would help - added the two enzymes and finally the magnesium (MgCl2) and the whole lot precipitated.We tried the components on their own and it is the magnesium which is precipitating the mixture - we checked the pellet and it is all protein. My questions are - why does the magnesium precipitate the proteins (even at 1mM), how can we avoid having the DNA in our prep, is there anything we can try with the resulting pellet? By the way, we have tried taking the pH down to about 5 and this precipitates the protein as well. Thanks in advance for any help / suggestions. Andy From owner-proteins@net.bio.net Thu Mar 07 22:00:00 1996 Path: biosci!agate!usenet.kornet.nm.kr!xpat.postech.ac.kr!news.kreonet.re.kr!news.dacom.co.kr!nntp.coast.net!torn!resunix.ri.sickkids.on.ca!news From: Randall Willis Newsgroups: bionet.molbio.proteins Subject: Re: Concentrating Small Protein Date: 8 Mar 1996 14:38:35 GMT Organization: The Hospital for Sick Children Lines: 16 Message-ID: <4hpgpb$4iu@resunix.ri.sickkids.on.ca> References: <8MAR199609015232@watson.bms.com> NNTP-Posting-Host: resunix.ri.sickkids.on.ca Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) To: curtis@watson.bms.com X-URL: news:8MAR199609015232@watson.bms.com Mike, Another method to concentrate your protein would be to place it in a dialysis bag of ~1kD cutoff and place this into dry PEG. This will serve to draw out the buffer solution while keeping back your protein. Good luck...Randall C Willis, Publisher, Aliquotes Press "ALIQUOTES: A Journal of Molecular and Biochemical Humour" 58 Balfour Ave. Toronto, ON M4C 1T6 CANADA 416-813-5933(ph) willis@gandalf.psf.sickkids.on.ca 416-813-5022(fax) rogerb@microsoft.com From owner-proteins@net.bio.net Thu Mar 07 22:00:00 1996 Path: biosci!agate!usenet.kornet.nm.kr!xpat.postech.ac.kr!news.kreonet.re.kr!news.dacom.co.kr!nntp.coast.net!torn!resunix.ri.sickkids.on.ca!news From: Randall Willis Newsgroups: bionet.molbio.proteins Subject: Re: Overexpressed E.Coli Protein sticks DNA - help needed! Date: 8 Mar 1996 14:33:52 GMT Organization: The Hospital for Sick Children Lines: 20 Message-ID: <4hpggg$4iu@resunix.ri.sickkids.on.ca> References: <4hotop$rh3@oban.cc.ic.ac.uk> NNTP-Posting-Host: resunix.ri.sickkids.on.ca Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) To: sm-postmaster@ic.ac.uk X-URL: news:4hotop$rh3@oban.cc.ic.ac.uk Andy, While I don't have a protocol, I remeber in the old days, we used to perform a polymine P precipitation of nucleic acids (has the side effect of taking down any DNA-bound proteins as well). Also, you could try pre-running your material over a DEAE or DE52 column to which the nucleic acids will stick gang busters (usually elutes at ~1M NaCl). Good luck...Randall C Willis, Publisher, Aliquotes Press "ALIQUOTES: A Journal of Molecular and Biochemical Humour" 58 Balfour Ave. Toronto, ON M4C 1T6 CANADA 416-813-5933 (ph) willis@gandalf.psf.sickkids.on.ca 416-813-5022 (fax) rogerb@microsoft.com From owner-proteins@net.bio.net Thu Mar 07 22:00:00 1996 Path: biosci!CS.Arizona.EDU!noao!ennfs.eas.asu.edu!gatech!newsfeed.internetmci.com!uwm.edu!vixen.cso.uiuc.edu!howland.reston.ans.net!newsserver.jvnc.net!crick.bms.com!watson.bms.com!curtis From: curtis@watson.bms.com (Michael S Curtis) Newsgroups: bionet.molbio.proteins Subject: Concentrating Small Protein Date: 8 Mar 1996 09:01 EST Organization: Bristol Myers Squibb Pharmaceutical Research Institute Lines: 14 Distribution: world Message-ID: <8MAR199609015232@watson.bms.com> NNTP-Posting-Host: watson.bms.com News-Software: VAX/VMS VNEWS 1.41 I am trying to concentrate a small protein (~6KD). I am currently using a Cetricon-3 (3000 MWCO), however I am getting alot of the protein going through the membrane. Does anyone know of a supplier who has a smaller MWCO centrifugation/ultrfiltration apparatus, the smallest Centricon I know of is the 3000 MWCO. Does anyone know of a better way to concentrate such a small protein? Any suggestions would be greatly appreciated. Thanks in Advance. Mike Curtis curtis@bms.com From owner-proteins@net.bio.net Thu Mar 07 22:00:00 1996 Path: biosci!daresbury!nntp-trd.UNINETT.no!Norway.EU.net!EU.net!newsfeed.internetmci.com!in1.uu.net!ftpbox!newsfeed.acns.nwu.edu!news.cc.uic.edu!usenet From: Keld Sorensen Newsgroups: bionet.molbio.proteins Subject: Re: Help! How can I look at the conformational changes in proteins? Date: 7 Mar 1996 17:44:25 GMT Organization: Univ. Illinois / College of Med. Lines: 4 Message-ID: <4hn79p$rue@piglet.cc.uic.edu> References: <4hmg4j$n7l@sun4.bham.ac.uk> NNTP-Posting-Host: sliprock-03.dialin.uic.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; PPC) To: bcm4jah2@bham.ac.uk X-URL: news:4hmg4j$n7l@sun4.bham.ac.uk Try circular dichroism Keld. From owner-proteins@net.bio.net Thu Mar 07 22:00:00 1996 Path: biosci!CS.Arizona.EDU!noao!ennfs.eas.asu.edu!gatech!newsxfer2.itd.umich.edu!chi-news.cic.net!mr.net!umn.edu!newsstand.tc.umn.edu!hiv.med.umn.edu!selby From: selby@hiv.med.umn.edu (Scott Selby (Med-Hem)) Newsgroups: bionet.cellbiol,bionet.molbio.ageing,bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: single-strand DNA break detection Date: 6 Mar 1996 18:17:03 GMT Organization: University of Minnesota Lines: 26 Message-ID: <4hkkqv$c97@epx.cis.umn.edu> NNTP-Posting-Host: hiv.med.umn.edu Keywords: DNA damage, p53 X-Newsreader: TIN [version 1.2 PL2] Xref: biosci bionet.cellbiol:4258 bionet.molbio.ageing:2549 bionet.molbio.methds-reagnts:41507 bionet.molbio.proteins:7275 I'm wondering if anyone could direct me toward proteins that might be involved in recognition of single-stranded DNA breaks (SSB). The cell line I'm working with has a decrease response to single-stranded DNA damage, as evidenced by Western blottig, of p53. The p53 is wild-type, and I'm curious as to whether the defect is in a regulatory region of p53 upstream somewhere, or whether the actual detection of single-stranded breaks is faulty. Does anyone have knowledge as to what proteins are specifically involved in detection SSBs? I'd love to hear any discussion about other possible causes of this muted p53 response. Thanks, in advance. ______________________________________________________________________________ || Scott A. Selby 6098)o%:::%o(860 ==== Univ. of Minnesota 098)o%:::%o(8609 | |__ Dept. of Med/Hematology 6o%:%o(86098) | |-.\ Box 480 UMHC (86098)o |__| \\ Minneapolis, MN 55455 6098)o%::%o9 || || 098)o%::::::%o9 ======__| selby@lenti.med.umn.edu 6o%::::::%o(860 ________||__ 6o%::%o(8609 /____________\ o(86098) ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ From owner-proteins@net.bio.net Thu Mar 07 22:00:00 1996 Newsgroups: bionet.molbio.proteins Path: biosci!agate!howland.reston.ans.net!gatech!newsfeed.internetmci.com!in1.uu.net!nih-csl!loglady.ninds.nih.gov!johnk From: johnk@spasm.niddk.nih.gov (John Kuszewski) Subject: Re: Help! How can I look at the conformational changes in proteins? Message-ID: <1996Mar8.183826.27204@alw.nih.gov> Sender: postman@alw.nih.gov (AMDS Postmaster) Nntp-Posting-Host: spasm.niddk.nih.gov Reply-To: johnk@spasm.niddk.nih.gov (John Kuszewski) Organization: National Insts. of Health References: <4hmg4j$n7l@sun4.bham.ac.uk> Date: Fri, 8 Mar 1996 18:38:26 GMT Lines: 82 In article , tliang@utmmg.med.uth.tmc.edu (T. Chyau Liang) writes: |> In article <4hmg4j$n7l@sun4.bham.ac.uk>, bcm4jah2@bham.ac.uk (Jim) wrote: |> |> > Can anybody please tell me the best way to look at the conformational changes |> > in proteins as they are bound by substrates, inhibitors, etc. |> > Thanks very much James Harper. Exactly what do you want to know about these conformational changes? Just that something happened? Or do you need precise information about the changes in atomic positions? |> |> Hi Jim, |> |> You have several possibilities: |> |> (1) Fluorescence spectroscopy: very sentitive and can tell you whether |> there is conformational change or not. However, it does not tell what |> kind of conformational changes. Often, you can monitor the intrinsic |> fluorophore on the protein or the substrate/inhibitor. Or it could seem to indicate a conformational change when none occurs. For example, imagine a protein that has a hydrophobic pocket. Imagine that fluorophore can bind into it without causing any changes in its conformation or that of the protein. Simply because of the lack of solvent quenching, the fluorophore's fluorescence will change dramatically. I'd call this a false-positive indication of conformational change upon ligand binding. |> |> (2) Circular dichroism (CD): sensitivity comparable to UV/VIS spectroscopy |> and can tell you more info on conformational changes (e.g., extent of |> alpha-helix or beta sheet changes). This isn't a bad suggestion if the original poster just wants to know that something happened upon ligand binding, but CD can't really be interpreted beyond gross changes in structure. |> |> (3) NMR: a very insensitive method, but can potentially provide more info. |> The same can be said about X-ray. NMR can give atomic-level information about the changes in conformation and dynamics that occur in proteins upon ligand binding. Its sensitivity is lower than fluorescence (you need ~0.5 ml of ~1 mM protein solution to get decent spectra, and if you want to do a structure, you'll probably have to make a sample that's labeled with 13C and 15N), but it usually produces vastly more information about protein structure and dynamics than fluorescence or CD. |> |> These are just a few mthods come to my mind. Others may be able to provide |> you alternate methods. Analytical ultracentrifugation can tell you whether ligand binding induces gross changes in the protein's conformation or oligomerization state. Boy, this sounds like an answer to the final in my methods in physical biochemistry class. 8-) |> |> hope this helps. |> |> T. Chyau Liang, Ph.D. |> U. Texas-Houston -- _____________ | ___/_ | |/ / -- /\ // /-- || || / /|| || || / / || || ||/ / || John Kuszewski || |/ /| || johnk@spasm.niddk.nih.gov || / /|| || \/ / / || \/ that's MISTER protein G to you! |/__/| | /_________| "Biophysics has driven me to an attitude of apocalyptic doom" --Frank Delaglio From owner-proteins@net.bio.net Thu Mar 07 22:00:00 1996 Path: biosci!CS.Arizona.EDU!noao!ennfs.eas.asu.edu!gatech!newsxfer2.itd.umich.edu!chi-news.cic.net!news1.io.org!news From: khaddad@io.org (keith haddad) Newsgroups: bionet.molbio.proteins Subject: Hexadecylrimethylammonium bromide substitute?? Date: Fri, 08 Mar 1996 21:14:45 GMT Organization: Internex Online (io.org), Toronto, Ontario, Canada Lines: 15 Message-ID: <4hptd4$4c4@news1.io.org> NNTP-Posting-Host: dyna-122.net7b.io.org X-Newsreader: Forte Free Agent 1.0.82 Hexadecylrimethylammonium bromide substitute?? I am using 1% Hexadecylrimethylammonium bromide as precipitant to detect microbes producing pectinase. Is there any substitute reagents?? Is it the bromide that reacts with the pectinase?? So can I use other bromide compounds?? Thanx khaddad@io.org http://www.io.org/~khaddad/ From owner-proteins@net.bio.net Thu Mar 07 22:00:00 1996 Path: biosci!daresbury!bioftp.unibas.ch!news.vub.ac.be!news.belnet.be!news.rediris.es!scsing.switch.ch!swsbe6.switch.ch!swidir.switch.ch!in2p3.fr!univ-lyon1.fr!pasteur.fr!jussieu.fr!oleane!tank.news.pipex.net!pipex!howland.reston.ans.net!torn!newshub.ccs.yorku.ca!laurel.yorku.ca!yu108044 From: Lauren Brown Newsgroups: bionet.molbio.proteins Subject: DNA rearrangement in ciliate Date: Fri, 8 Mar 1996 14:33:39 -0500 Organization: York University, Ontario, Canada Lines: 20 Message-ID: NNTP-Posting-Host: laurel.yorku.ca Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII Hello, I am an undergrad working on my honours thesis at York U TO. I am trying to identify a protein - DNA binding interaction thought to be invovled in a developmentally programmed DNA rearrangement, occuring in the holotrichous ciliate Tetrahymena thermophila. This rearrangement involves the deletion of an intron element(arbitraly termed mse2.9) by chromosome breakage and rejoining. It occurs 12-14hrs after conjugation (the sexual cycle of the organism), during macronuclear formation. To my knowledge, no protein has yet been determined to have a specific role in DNA deletion events in the ciliates(there are many). However, my search for current information on this subject is limited to mid 1995 publications. If anyone has come across any recent articles on this subject I would be extremely interested to know. Thanks for your time, Lauren e-mail: 108044@yorku.ca From owner-proteins@net.bio.net Thu Mar 07 22:00:00 1996 Path: biosci!CS.Arizona.EDU!noao!ennfs.eas.asu.edu!gatech!newsfeed.internetmci.com!chi-news.cic.net!nntp.coast.net!fu-berlin.de!cs.tu-berlin.de!uni-erlangen.de!news.th-darmstadt.de!news.uni-mainz.de!usenet From: Jan Schroeder Newsgroups: bionet.molbio.proteins Subject: HELP!--Immunoaffinity column doesn't work Date: 6 Mar 1996 11:33:55 GMT Organization: Johannes Gutenberg-Universitaet Mainz Lines: 10 Message-ID: <4hjt73$huf@kralle.zdv.Uni-Mainz.DE> NNTP-Posting-Host: iabwes.biologie.uni-mainz.de Mime-Version: 1.0 Content-Type: text/plain Content-Transfer-Encoding: quoted-printable X-Mailer: Mozilla 1.22 (Windows; I; 16bit) Hi there, I am quite new to handle with Immunoaffinity Chromatography and I want to purify a protein from Hordeum vulgare with this subject. Before I start to purify the protein. I have to clean the polyclonal mouse IgG with a protein A column (BioRad, Affi-Gel Protein A G= el). The antibody did not bound to the protein A and I found the complete IgG in the first wash step. Then I tried a different way: I tried to couple the IgG via caboanhydrate moities to hydrazide activated agarose beats (BioRad, Affi-Gel Hz Immunoaffinity Column)= In the first step I detected again the complete IgG so it did not bind to the gel. Maybe there are eome hints behind the productinformations? Please contact Jan Schroeder Thanks, Tanja From owner-proteins@net.bio.net Thu Mar 07 22:00:00 1996 Path: biosci!CS.Arizona.EDU!noao!ennfs.eas.asu.edu!gatech!swrinde!howland.reston.ans.net!nntp.coast.net!fu-berlin.de!zrz.TU-Berlin.DE!cs.tu-berlin.de!uni-erlangen.de!news.th-darmstadt.de!news.uni-mainz.de!usenet From: Tanja Newsgroups: bionet.molbio.proteins Subject: HELP!--Immunoaffinity column doesn't work Date: 6 Mar 1996 16:15:49 GMT Organization: Johannes Gutenberg-Universitaet Mainz Lines: 10 Message-ID: <4hkdnl$i1m@kralle.zdv.Uni-Mainz.DE> NNTP-Posting-Host: iabwes.biologie.uni-mainz.de Mime-Version: 1.0 Content-Type: text/plain Content-Transfer-Encoding: quoted-printable X-Mailer: Mozilla 1.22 (Windows; I; 16bit) Hi there, I am quite new to handle with Immunoaffinity Chromatography and I want to purify a protein from Hordeum vulgare with this subject. Before I start to purify the protein. I have to clean the polyclonal mouse IgG with a protein A column (BioRad, Affi-Gel Protein A G= el). The antibody did not bound to the protein A and I found the complete IgG in the first wash step. Then I tried a different way: I tried to couple the IgG via caboanhydrate moities to hydrazide activated agarose beats (BioRad, Affi-Gel Hz Immunoaffinity Column)= In the first step I detected again the complete IgG so it did not bind to the gel. Maybe there are eome hints behind the productinformations? Please contact Jan Schroeder Thanks, Tanja From owner-proteins@net.bio.net Thu Mar 07 22:00:00 1996 Path: biosci!agate!cocoa.brown.edu!news From: "Alan_Rosmarin@brown.edu" Newsgroups: bionet.molbio.proteins Subject: Sp1 dimerization Date: 8 Mar 1996 21:20:57 GMT Organization: Brown University Lines: 12 Message-ID: <4hq8bp$i0b@cocoa.brown.edu> NNTP-Posting-Host: cis-ts2-slip2.cis.brown.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Windows; I; 16bit) Does anyone have experience with dimerization of purified Sp1 protein in EMSA gels? -- __________________________________________________ Alan G. Rosmarin, MD Internet: Rosmarin@Brown.edu Fax: 401-751-2398 Telephone: 401-331-8500 X4648 From owner-proteins@net.bio.net Thu Mar 07 22:00:00 1996 Path: biosci!daresbury!nntp-trd.UNINETT.no!nntp.uio.no!news.cais.net!chi-news.cic.net!newsspool.doit.wisc.edu!news.doit.wisc.edu!martinlab From: klenchin@macc.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins,bionet.software Subject: Re: Isoelectric Point calculations Date: Fri, 08 Mar 96 19:09:38 GMT Organization: UW-Madison Lines: 21 Message-ID: <4hq0li$a4o_003@biochem.wisc.edu> References: <4hmra1$c71$1@mhadf.production.compuserve.com> NNTP-Posting-Host: martinlab.biochem.wisc.edu X-Newsreader: News Xpress Version 1.0 Beta #4 Xref: biosci bionet.molbio.proteins:7283 bionet.software:14919 In article <4hmra1$c71$1@mhadf.production.compuserve.com>, Eugene A Kapp <100277.2367@CompuServe.COM> wrote: ->I have found that the predicted pI calculations from two WWW ->sites differ by approximately 0.4 for a given protein sequence. ->Expasy (http://expasy.hcuge.ch/ch2d/pi_tool.html) and the genome ->sequencing site at (http://genome1.bio.bnl.gov/bbq.html) are ->therefore using slightly different pKR values. -> ->Could someone suggest the correct pKR values or add some light to ->the situation. -> ->Many thanks. Very simple. Ignore both. Predicted pI values are meaningless because they treat all AA as if the protein were random coil. It could be OK for small proteins and sometimes is OK with some proteins. Real pI of the folded protein could be entirely different. So, IMHO, 0.4 unit is not a big deal of difference. - Dima From owner-proteins@net.bio.net Thu Mar 07 22:00:00 1996 Path: biosci!CS.Arizona.EDU!noao!ennfs.eas.asu.edu!gatech!newsjunkie.ans.net!newsfeeds.ans.net!inet.d48.lilly.com!dallas.d50.lilly.com!user Newsgroups: bionet.molbio.proteins Subject: Re: What is the glycine sidechain? Message-ID: From: jrm@lilly.com (James R. Miller) Date: 8 Mar 96 16:39:10 EST References: <4hiihk$31d@vixen.cso.uiuc.edu> Distribution: world Organization: Lilly Research Labs Nntp-Posting-Host: dallas.d50.lilly.com Lines: 51 In article <4hiihk$31d@vixen.cso.uiuc.edu>, dalke@ks.uiuc.edu (Andrew Dalke) wrote: > It is a simple question, does glycine have a sidechain? If you say an amino > acid base has an group "R" off the CA which is called a sidechain, then the > answer is, "yes, and the sidechain is a hydrogen". > I can accept that. However, as I think I've mentioned here before, I'm > working on a visualization and analysis program (ObPlug: > http://www.ks.uiuc.edu/Research/vmd). I want to add an atom selection term > called "sidechain" which picks the sidechain atoms. It works for every case > except glycine, where I have to determine which of the two hydrogens (in an all > atom model, such as charmm-22) is the sidechain. > > Currently I choose the one with the alphabetically smallest name, so for a > charmm-22 model, HC1 is the sidechain and HC2 is not. One of the researchers > pointed out that I could find the hydrogen which is closest in position to where > a C-beta would be. The problem is when we compute the molecular dynamics of the > protein I don't know if the closest hydrogen will always be the same one. I > consider it bad if the sidechain alternates between two atoms. > Of course, in a reduced atom model, glycine doesn't even have hydrogen off the > CA. > > So, does glycine really have a side chain? If so, what is the "right" > (IUPAC?) way of finding it? Probably the biggest problem (and a trick question when taking my exams for doctoral degree) is that glycine is not optically active. That is, without the "sidechain", glycine is an amino acid, but not L- or R-. When translated into protein the Hydrogens at the alpha carbon have no identity, except by an NMR signal based on what those hydrogens are nearest to. Even then the glycine may rotate freely if in the proper environment and that signal may be obscured. The amino acids in protein are L-amino acids except for glycine. Now, have I mudded the waters even more?! :-) > > Andrew > dalke@ks.uiuc.edu Jim Miller Lilly Research Labs Indianapolis, IN jrm@lilly.com --------------------------------------------------------------------- From owner-proteins@net.bio.net Fri Mar 08 22:00:00 1996 Path: biosci!agate!howland.reston.ans.net!nntp.coast.net!zombie.ncsc.mil!cs.umd.edu!haven.umd.edu!purdue!oitnews.harvard.edu!das-news2.harvard.edu!fas-news.harvard.edu!mito!robison From: robison@mito.harvard.edu (Keith Robison) Newsgroups: bionet.molbio.proteins,bionet.software Subject: Re: Isoelectric Point calculations Followup-To: bionet.molbio.proteins,bionet.software Date: 9 Mar 1996 17:42:25 GMT Organization: Harvard University, Cambridge, Massachusetts Lines: 36 Message-ID: <4hsfu1$103@decaxp.harvard.edu> References: <4hmra1$c71$1@mhadf.production.compuserve.com> <4hq0li$a4o_003@biochem.wisc.edu> NNTP-Posting-Host: mito.harvard.edu X-Newsreader: TIN [version 1.2 PL2] Xref: biosci bionet.molbio.proteins:7289 bionet.software:14928 Dima Klenchin (klenchin@macc.wisc.edu) wrote: : In article <4hmra1$c71$1@mhadf.production.compuserve.com>, : Eugene A Kapp <100277.2367@CompuServe.COM> wrote: : ->I have found that the predicted pI calculations from two WWW : ->sites differ by approximately 0.4 for a given protein sequence. : ->Expasy (http://expasy.hcuge.ch/ch2d/pi_tool.html) and the genome : ->sequencing site at (http://genome1.bio.bnl.gov/bbq.html) are : ->therefore using slightly different pKR values. : -> : ->Could someone suggest the correct pKR values or add some light to : ->the situation. : -> : ->Many thanks. : Very simple. Ignore both. Predicted pI values are meaningless because : they treat all AA as if the protein were random coil. It could be OK for : small proteins and sometimes is OK with some proteins. Real pI of the folded : protein could be entirely different. So, IMHO, 0.4 unit is not a big deal : of difference. It all depends on what you are using the pI value for. If you are trying to predict some sort of in vivo behaviour, then Dr. Klenchin's advice is well taken. On the other hand, if you are trying to predict the behaviour of the protein in an isoelectric focusing gel, the computer-predicted values are generally on-target. Keith Robison Harvard University Department of Molecular & Cellular Biology Department of Genetics robison@mito.harvard.edu From owner-proteins@net.bio.net Fri Mar 08 22:00:00 1996 Newsgroups: bionet.molbio.proteins Path: biosci!agate!howland.reston.ans.net!torn!nott!cunews!freenet.carleton.ca!FreeNet.Carleton.CA!at332 From: at332@FreeNet.Carleton.CA (Matt Parker) Subject: Evaluating a program called "LOOK" Message-ID: Sender: at332@freenet.carleton.ca (Matt Parker) Reply-To: at332@FreeNet.Carleton.CA (Matt Parker) Organization: The National Capital FreeNet Date: Sat, 9 Mar 1996 20:28:09 GMT Lines: 6 Hi! Our group is considering whether to buy a program called "LOOK," from Molecular Applications Group, Palo Alto, CA. It displays PDB files, etc., does minimizations, that kind of thing. Has anybody out there used it, and are there any comments you'd like to make? Thanks! From owner-proteins@net.bio.net Fri Mar 08 22:00:00 1996 Path: biosci!agate!howland.reston.ans.net!gatech!newsxfer2.itd.umich.edu!chi-news.cic.net!mr.net!umn.edu!newsstand.tc.umn.edu!maroon.tc.umn.edu!mcnei002 From: mcnei002@maroon.tc.umn.edu (Kenneth J McNeil) Newsgroups: bionet.molbio.proteins Subject: (Q) Problems w/silver enhancement of gold particles Date: 8 Mar 1996 15:05:31 -0600 Organization: University of Minnesota Lines: 32 Message-ID: NNTP-Posting-Host: maroon.tc.umn.edu I have been attempting to localize floral-specific gene products in tomato flowers using silver enhancement of gold particles. The problem is I have been getting a relatively high background (browning of tissue and black particle formation all over the slide). I had heard of photosynthetic tissue reacting with the silver-enhancing solution, but haven't been able to find any references listing possible solutions to the problem. Conditions: 10 micron cryosections of tomato flowers fixed in HistoChoice (Amresco) and mounted in OCT. Sections placed on gelatin subbed slides. Tissue blocked in 5% nonfat dry milk in Tris buffered saline, pH 7.5, with 0.1% Tween-20 (TBST) for 1 hour. Rinsed 3x5' TBST. Primary Ab, 1:200 dilution (Ab raised in rabbits against bacterial fusion protein [pET28a fused to a floral specific gene]), 1 hour in TBST. Rinse 3x5' TBST. Secondary Ab, 1:80 dilution, goat antirabbit with 4 nM gold conjugate in TBST, 1 hour. Rinse 3x5' TBST. Rinse PBS 2x5'. Fix with 2% glutaraldehyde in PBS, 15'. Rinse 2x5' PBS. Rinse 2x5' di H2O. Silver enhancement solution (silver acetate solution mixed with hydroquinone in citrate buffer at a pH of 3.8). Detection time is approximately 18 minutes at 23C. Slides kept in darkness during silver enhancement. Slides rinsed di H20, placed in fixative 2', rinsed di H20 5'. Viewed under light microscope. Thank you for your help, Ken McNeil Horticultural Science Department University of Minnesota mncei002@maroon.tc.umn.edu From owner-proteins@net.bio.net Sat Mar 09 22:00:00 1996 Path: biosci!agate!howland.reston.ans.net!gatech!newsfeed.internetmci.com!chi-news.cic.net!nntp.coast.net!fu-berlin.de!zrz.TU-Berlin.DE!cs.tu-berlin.de!uni-erlangen.de!news.th-darmstadt.de!news.uni-mainz.de!usenet From: Tanja Newsgroups: bionet.molbio.proteins Subject: IMMUNOAFFINITY COLUMN DOESN'T WORK Date: 6 Mar 1996 18:28:48 GMT Organization: Johannes Gutenberg-Universitaet Mainz Lines: 10 Message-ID: <4hklh0$nts@kralle.zdv.Uni-Mainz.DE> NNTP-Posting-Host: iabwes.biologie.uni-mainz.de Mime-Version: 1.0 Content-Type: text/plain Content-Transfer-Encoding: quoted-printable X-Mailer: Mozilla 1.22 (Windows; I; 16bit) Hi there, I am quite new to handle with Immunoaffinity Chromatography and I want to purify a protein from Hordeum vulgare with this subject. Before I start to purify the protein. I have to clean the polyclonal mouse IgG with a protein A column (BioRad, Affi-Gel Protein A G= el). The antibody did not bound to the protein A and I found the complete IgG in the first wash step. Then I tried a different way: I tried to couple the IgG via caboanhydrate moities to hydrazide activated agarose beats (BioRad, Affi-Gel Hz Immunoaffinity Column)= In the first step I detected again the complete IgG so it did not bind to the gel. Maybe there are eome hints behind the productinformations? Please contact Jan Schroeder Thanks, Tanja From owner-proteins@net.bio.net Sat Mar 09 22:00:00 1996 Path: biosci!CS.Arizona.EDU!noao!ennfs.eas.asu.edu!gatech!newsfeed.internetmci.com!in1.uu.net!vol.net!news.glink.net.hk!hpg30a.csc.cuhk.hk!news.cuhk.edu.hk!newsfeeder.ust.hk!nntp.hk.super.net!news.duke.edu!mcdon016.mc.duke.edu!user From: diehl001@mc.duke.edu (Eddie Diehl) Newsgroups: bionet.molbio.proteins Subject: Looking for RXR Antibodies Date: Sat, 09 Mar 1996 13:08:05 -0500 Organization: Duke University Lines: 8 Message-ID: NNTP-Posting-Host: mcdon016.mc.duke.edu I'm looking for antibodies directed against human RXR alpha and gamma, better yet, Abs which will recognize one or the other. Any help would be greatly appreciated! Eddie Diehl Pharmacology Duke University diehl001@mc.duke.edu From owner-proteins@net.bio.net Sun Mar 10 22:00:00 1996 Path: biosci!agate!howland.reston.ans.net!gatech!newsfeed.internetmci.com!swrinde!tank.news.pipex.net!pipex!warwick!bham!bhamcs!news.ox.ac.uk!news From: "Simon M. Brocklehurst" Newsgroups: bionet.molbio.proteins Subject: ANNOUNCE NAOMI Upgrade Date: Mon, 11 Mar 1996 13:34:41 +0000 Organization: University of Oxford Lines: 84 Message-ID: <31442BF1.2781E494@bioch.ox.ac.uk> NNTP-Posting-Host: nmrv.ocms.ox.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0 (X11; I; SunOS 4.1.3_U1 sun4m) NAOMI - Version Upgrade Announcement (Please note, NAOMI is provided at zero charge for academic use) (e-mail contact smb@bioch.ox.ac.uk) _____________________________________________________________________________ The computer program NAOMI Version 2.4 is available as of now from the NAOMI Web site at: http://www.ocms.ox.ac.uk/~smb/Software/N_details/naomi.html or via anonymous ftp ftp://nmrz.ocms.ox.ac.uk/pub/smb/naomi i.e. at nmrz.ocms.ox.ac.uk in directory pub/smb/naomi/ Users of versions older than 2.10 will need new license keys to allow the upgrade to work (please contact the author in this case). _____________________________________________________________________________ New features in upgrade: In main module: New fully automatic interface to RASMOL Wild-card selection of residues Automated identification of interior and exterior residues new "protwrite" command for outputing protein-level calculated properties In the NMR module: New prediction of NOEs from structure command _____________________________________________________________________________ What is NAOMI? NAOMI is an easy-to-use, state-of-the-art computer program which is aimed at both specialist and non-specialist researchers who make use of three-dimensional structures of proteins in their work. It has hundreds of users Worldwide. Some facilities offered by the program for working with structure include: automatic 'key' residue identification automatic hydrophobic core/packing analysis automatic hydrogen bonds main-chain and side-chain identification (including high quality energy calculations) automatic secondary structure (helix, strand and turn) classification using fuzzy logic automatic supersecondary structure classification (beta-hairpin loops) conformational parameters: phi,psi,chi1,chi2,chi3,chi4,chi5 etc solvent accessibility (both absolute and percentage) calculations automatic identification of disulphide bonds, salt bridges, chain-breaks side-chain modelling and manipulation applying symmetry operators automatic structure repair (building in missing atoms) NMR structure refinement module interfaces to graphics programs (MOLSCRIPT (and thus Raster3D), RASMOL, INSIGHT and QUANTA to allow automatic preparation of figures and time-efficient visualization of structures. More details are available on the Web site. NB NAOMI currently works only on Silicon Graphics workstations running IRIX 5.* or IRIX 6.* _____________________________________________________________________________ | | ,_ o Simon M. Brocklehurst, | / //\, Oxford Centre for Molecular Sciences, Department of Biochemistry, | \>> | University of Oxford, Oxford, UK. | \\, E-mail: smb@bioch.ox.ac.uk | WWW: http://www.ocms.ox.ac.uk/~smb/ |____________________________________________________________________________ From owner-proteins@net.bio.net Sun Mar 10 22:00:00 1996 Path: biosci!news.Stanford.EDU!agate!howland.reston.ans.net!newsfeed.internetmci.com!in1.uu.net!news.tufts.edu!opal.tufts.edu!jstrassw From: jstrassw@opal.tufts.edu Newsgroups: bionet.molbio.proteins Subject: Any cDNA for Src, Fyn,...? Date: 11 Mar 96 09:36:28 -0500 Organization: Tufts University - Medford, MA Lines: 6 Distribution: world Message-ID: <1996Mar11.093628@opal.tufts.edu> NNTP-Posting-Host: opal.tufts.edu Hello! If anyone has any cDNA for Src, Fyn, Yes, or Lck, I would be very grateful to obtain them for in vitro binding studies. Thanks, John From owner-proteins@net.bio.net Sun Mar 10 22:00:00 1996 Path: biosci!ns1.faseb.org!lamarck.sura.net!newsfeed.internetmci.com!swrinde!cs.utexas.edu!news.tamu.edu!news From: Dilip Dias Newsgroups: bionet.molbio.proteins Subject: Re: Constitutive Fusion Protein Expression in BL21(DE3) Cells Date: 11 Mar 1996 21:34:07 GMT Organization: Texas A&M University Lines: 11 Message-ID: <4i268f$bn3@news.tamu.edu> References: NNTP-Posting-Host: 128.194.184.226 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) X-URL: news:biotech-0403961716060001@cyto.cytosignal.com David Do you know that BL21 DE3 is not recA deficient and that any plasmid that you put in can undergo recombination. I had that problem (Actually the opposite-no expression) and when I checked the plasmids isolated from BL21 DE3 the insert was gone. May be your plasmid had DNA rearrangment. Check plasmid, if this is true then switch over to another host like JM109 DE3, NovaBlue DE3 etc. Hope this helps. Dilip From owner-proteins@net.bio.net Sun Mar 10 22:00:00 1996 Path: biosci!agate!howland.reston.ans.net!vixen.cso.uiuc.edu!uwm.edu!msunews!harbinger.cc.monash.edu.au!bunyip.cc.uq.oz.au!news From: Mark Williams Newsgroups: bionet.molbio.proteins Subject: Re: GFP fusion problems Date: 11 Mar 1996 10:10:16 GMT Organization: University of Queensland Lines: 25 Message-ID: <4i0u68$q0o@hobyah.cc.uq.oz.au> References: NNTP-Posting-Host: 130.102.98.10 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) f.s.wouters@chem.ruu.nl (fred wouters) wrote: >I am desperately seeking for users of the pGFP-N3 N-terminal Protein >Fusion Vector (Clontech). >Our lab ordered this vector and it doesn't work at all, and Clontech is >not very willing to help us with our problem (it doesn't give any >fluorescence, not even the uncloned vector). We spoke to another user with >the same problem. Petra, I purchased the N3 vector in the middle of last year, but after cloning my gene into it, Clontech (well, the suppliers at least) called to tell me that the N3 vector was defective: a frameshift mutation was apparently introduced during construction of the multiple cloning site. They promised to send the new, improved (and hopefully correct) vector when it's available: firstly, mid-January, then February, but it still hasn't arrived. Mark PS When I first ordered the N3 vector, I was told that Clontech hadn't finished making it yet!! Maybe I rushed them and caused the defect :) From owner-proteins@net.bio.net Sun Mar 10 22:00:00 1996 Path: biosci!bloom-beacon.mit.edu!tribune.meitca.com!ukma!news.cuny.edu!NewsWatcher!user From: Laurette@msvax.mssm.edu (GLUXLAB) Newsgroups: bionet.molbio.proteins Subject: thrombin for GST prot cleav Followup-To: bionet.molbio.proteins Date: Mon, 11 Mar 1996 14:25:59 -0400 Organization: Structural Neurobiology- Mt Sinai Med Cent Lines: 12 Message-ID: NNTP-Posting-Host: 146.203.5.185 Protein People: We are currently using highly purified human thrombin 4,000 Units/mg [sigma #T-3010] for cleaving our fused protein from glutathione S transferase. the price is becoming prohibitive ~$300/1000 units. is there a better source for thrombin, and not neccessarily human that will work as well. our end product id being crystallized for structure determination as well as for nmr characterization. thanks, laurette From owner-proteins@net.bio.net Sun Mar 10 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!news.mel.connect.com.au!harbinger.cc.monash.edu.au!lugb.latrobe.edu.au!newsmgr From: "R.K. Scopes" Newsgroups: bionet.molbio.proteins Subject: Re: GroEL structure Date: 12 Mar 1996 07:46:49 GMT Organization: La Trobe University Lines: 10 Message-ID: <4i3a59$50s@lugb.latrobe.edu.au> References: <199603011835.MAA20154@biosci.cbs.umn.edu> <4i3914$4ft@lugb.latrobe.edu.au> NNTP-Posting-Host: bcrs1.biochem.latrobe.edu.au "R.K. Scopes" wrote: about the structure of GroEL. Specifically, does it contain disulfide bonds. I was wrong: E. coli GroEL does has 6 cysteines; it is a thermophilic GroEL that has none. Nevertheless, as an intracellular protein, all cysteines should be free and reduced. Bob Scopes From owner-proteins@net.bio.net Sun Mar 10 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!news.mel.connect.com.au!harbinger.cc.monash.edu.au!lugb.latrobe.edu.au!newsmgr From: "R.K. Scopes" Newsgroups: bionet.molbio.proteins Subject: Re: GroEL structure Date: 12 Mar 1996 07:27:32 GMT Organization: La Trobe University Lines: 12 Message-ID: <4i3914$4ft@lugb.latrobe.edu.au> References: <199603011835.MAA20154@biosci.cbs.umn.edu> NNTP-Posting-Host: bcrs1.biochem.latrobe.edu.au gardner@BIOSCI.CBS.UMN.EDU ("Nancy Gardner") wrote: > > Could anyone comment on the structure of GroEL. Specifically, does it contain > disulfide bonds. > Thank you, > Nancy > GroEL does not have any disulfide bonds because (a) almost no intracellular protein has any disulfide bonds and (b) GroEL is particularly unlikley to have any because it has no cysteine at all ! Bob Scopes From owner-proteins@net.bio.net Sun Mar 10 22:00:00 1996 Path: biosci!rutgers!gatech!newsfeed.internetmci.com!in2.uu.net!brighton.openmarket.com!decwrl!waikato!comp.vuw.ac.nz!pc155 From: StentV@agresearch.cri.nz (Vicki Stent) Newsgroups: bionet.molbio.proteins Subject: Novagen Date: Tue, 12 Mar 96 02:38:02 GMT Organization: AgResearch, Wallaceville Lines: 15 Message-ID: <4i2kdi$hpa@st-james.comp.vuw.ac.nz> NNTP-Posting-Host: pc155.warc.cri.nz X-Newsreader: News Xpress Version 1.0 Beta #4 Does someone know the email address for Novagen? If so, could you please send it to stentv@agresearch.cri.nz Thanks, Vicki Vicki Stent Internet: StentV@agresearch.cri.nz Researcher Phone: 64-4-5286089 P.O.Box 40063, Upper Hutt Fax: 64-4-5281380 NEW ZEALAND ---------------------------------------------------------------------- Disclaimer: The above is a personal opinion and does not reflect the official view of AgResearch Ltd. ---------------------------------------------------------------------- From owner-proteins@net.bio.net Sun Mar 10 22:00:00 1996 Path: biosci!bcm.tmc.edu!NewsWatcher!user From: rs690935@condor.mbcr.bcm.tmc.edu (ramiro salas) Newsgroups: bionet.molbio.proteins Subject: b-gal transgenics Followup-To: bionet.molbio.proteins Date: Mon, 11 Mar 1996 13:41:00 -0600 Organization: Baylor College of Medicine Lines: 12 Message-ID: NNTP-Posting-Host: cb.cellb.bcm.tmc.edu Hi all, I have a question. I hope somebody knows this. My name is Ramiro Salas and I'm a graduate student at Baylor, Houston. My question is, what is the half life of b-gal in a living transgenic animal? I have these mice expressing b-gal in the brain. I'm trying to block the promoter so the b-gal expression should be much smaller, but I don't know exactly how much should I wait for the old protein (synthesized before I block the promoter) to be degraded. If there is any insight on this, I would very much appreciate it. Thatnks in advance, Ramiro Salas RS690935@condor.mbcr.bcm.tmc.edu From owner-proteins@net.bio.net Sun Mar 10 22:00:00 1996 Path: biosci!rutgers!gatech!newsfeed.internetmci.com!swrinde!cs.utexas.edu!news.tamu.edu!news From: Dilip Dias Newsgroups: bionet.molbio.proteins Subject: Help! Protein Expression in E. coli Date: 11 Mar 1996 21:20:04 GMT Organization: Texas A&M University Lines: 17 Message-ID: <4i25e4$add@news.tamu.edu> NNTP-Posting-Host: 128.194.184.226 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) X-URL: news:bionet.molbio.proteins I am trying to express two cDNA clones we isolated, in E. coli BL21 DE3 to make antibodies. Both these cDNA clones (proposed to) code for highly glycosylated proteins in plants (both have N terminal signal sequence which has been deleted by PCR amplification of the rest of the cDNA and cloning in pET30 expression vector) and have a hydrophobic C terminal. I seem to get very low level induction (as per S tag western) but the molecular weight of the product is about 10 kd higher than expected (The expression vector with the cloned gene cannot have a longer reading frame because of stop codons in other reading frames). I need help in two areas. 1. Does any of you have any suggestions to increase yield. 2. Is it possible for proteins to run at higher than expected molecular weight level in SDS-PAGE? I am new to protein chemistry and would appreciate all the help I can get. Thanks Dilip From owner-proteins@net.bio.net Sun Mar 10 22:00:00 1996 Path: biosci!bcm.tmc.edu!pendragon!news.msfc.nasa.gov!newsfeed.internetmci.com!gatech!newsjunkie.ans.net!newsfeeds.ans.net!rcogate.rco.qc.ca!altitude!usenet From: Achim Recktenwald Newsgroups: bionet.molbio.proteins Subject: Re: Native PAGE @ pH < 6.5 Date: Mon, 11 Mar 1996 09:48:49 -0500 Organization: IBEX Technologies, Inc. Lines: 45 Message-ID: <31443D51.1C0D@ibex.ca> References: <4hhheo$afa@lyra.csx.cam.ac.uk> 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 2.0 (Macintosh; I; PPC) Christopher Walentas (Bioc) wrote: > > I was wondering if anyone had a buffering system that they could > recommend for the resolution of two proteins using native PAGE at > a pH at or below 6.5? I've got a mixture of two species which > differ by a series of histidines, and need to utilise their charge > differences (which are only relevant below their pKa), but cannot > go too acidic due their pI is around 4. I've tried citric acid > at pH 5.5 (100mM) but with not much luck-- the proteins are 100kDa > and hardly made it into the resolving gel by the time the BPBlue > had scooted off the bottom. I've looked into a number of Practical > Approach-type reference manuals, but for the most part they are too > general and of little or no help. Any advice in this matter would > be most appreciated. > > Cheers, > > tripp I just found in a copy of an old thesis the following recipe: Electrodebuffer: acetic acid/beta-alanine pH 4.5 a 10x solution: 312g beta-alanine + 80mL acetic acid, add water to 1L Separation gel: acetic acid/KOH pH 4.3 26.93g KOH + 172mL acetic acid, add water to 1L Stacking gel: acetic acid/KOH pH 6.8 26.93g KOH + 29mL acetic acid. add water to 1L I never tried it myself, so don't sue me, if it doesn't work. Achim _______________________ Achim Recktenwald IBEX Technologies, Inc 5485 rue Pare Montreal, PQ, H4P 1P7 Canada From owner-proteins@net.bio.net Sun Mar 10 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!scripps.edu!usenet From: beutler@scripps.edu (Ernest Beutler, M.D.) Newsgroups: bionet.molbio.proteins Subject: Free internet subscription to BCMD - bcmd.msg [1/1] Date: 9 Mar 1996 20:12:19 GMT Organization: The Scripps Research Institute Lines: 17 Message-ID: <4hson3$hc8@riscsm.scripps.edu> NNTP-Posting-Host: beutler_pc.scripps.edu Mime-Version: 1.0 X-Newsreader: WinVN 0.93.11 Blood Cells, Molecules and Diseases has initiated a series of tabulations of hematologically important mutations. The first of these appeared in the Feb 29 issue, and like all articles in BCMD is available on the web: http://www.scripps.edu/bcmd Please feel free to sign on to our listserve by sending the following e-mail message to majordomo@scripps.edu: subscribe bloodcell There is no charge for being on the listserve or viewing and/or printing out full articles from our web site. Ernest Beutler, M.D. Editor From owner-proteins@net.bio.net Mon Mar 11 22:00:00 1996 Path: biosci!daresbury!nntp-trd.UNINETT.no!nntp.uio.no!news.cais.net!news.sprintlink.net!news.smartlink.net!usenet From: doug@smartdocs.com (Doug) Newsgroups: bionet.molbio.proteins Subject: MAKE $50,000 IN 1 MONTH TO BYE LOTS OF PROTIEN - $$$$.txt (0/1) Date: Wed, 13 Mar 1996 21:40:58 GMT Organization: SmartLink.net Premier ISP 805-294-1273 Lines: 2 Message-ID: <4i4r5n$g0@frodo.smartlink.net> NNTP-Posting-Host: pm229-52.smartlink.net X-Newsreader: Forte Free Agent 1.0.82 OPEN THIS TXT FILE WITH ANY TEXT EDITOR From owner-proteins@net.bio.net Mon Mar 11 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!elroy.jpl.nasa.gov!swrinde!sgigate.sgi.com!nntp.coast.net!swidir.switch.ch!swsbe6.switch.ch!news.unige.ch!news From: philippe lalle Newsgroups: bionet.molbio.proteins Subject: Re: Help! Protein Expression in E. coli Date: 12 Mar 1996 09:42:17 GMT Organization: génétique & microbio, centre médical universitaire, genève Lines: 11 Message-ID: <4i3gtp$ta@uni2f.unige.ch> References: <4i25e4$add@news.tamu.edu> NNTP-Posting-Host: 129.194.99.33 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) To: MAD2631@rsgis6.tamu.edu Of course it's possible that a protein migrates at an higher molecular weight than expected. The most wellknown example is p53 for which the theorical molecular weight is something around 43 kD. It depends mainly of the amino acid structure: with a lot of prolines the migration is slower than expected. I'm now working on a protein that migartes at 190 kD but its calculated molecular weight is 135kD. Be optimistic with your data. Good luck. Phil From owner-proteins@net.bio.net Mon Mar 11 22:00:00 1996 Path: biosci!daresbury!nntp-trd.UNINETT.no!nntp.uio.no!news.cais.net!news.sprintlink.net!news.smartlink.net!usenet From: doug@smartdocs.com (Doug) Newsgroups: bionet.molbio.proteins Subject: MAKE $50,000 IN 1 MONTH TO BYE LOTS OF PROTIEN - $$$$.txt (1/1) Date: Wed, 13 Mar 1996 21:40:59 GMT Organization: SmartLink.net Premier ISP 805-294-1273 Lines: 181 Message-ID: <4i4r5v$g0@frodo.smartlink.net> NNTP-Posting-Host: pm229-52.smartlink.net X-Newsreader: Forte Free Agent 1.0.82 begin 644 $$$$.txt M36%K92`D)"0D)"0D)"0D)"0D)"0D)"0D)"0D)"0D)"0D)"0D)"0D)"0D)"0D M)"0D)"0D)"0D)"0D)"0D)"0@9F%S=`T*#0I,:71E7-T96T@8V]N2UT;R!F;VQL;W<@2!N M;R!R:7-K+B`@#0H-"B0D)"0D)"0D)"0D)"0D)"0D)"0D)"0D)"0D)"0D)"0D M)"0D)"0D)"0D)"0D)"0D)"0D)"0D)"0D)"0D)"0D#0H-"D%.1"P@4T\L(%=) M5$A/550@1E525$A%4B!!1$\N+BX-"@T*5&AE(%-Y2X@(%1H90T*2!T:&4@=F5R>2!B87-I8W,@87)E(&EN8VQU9&5D+B`@1F%I M;&EN9R!T;R!F;VQL;W<-"F%L;"!F;W5R('-T97!S('1O('1H90T*;&5T=&5R M('-I9VYI9FEC86YT;'D@2!O9B!T:&ES('!O6]U6]U(')E860-"F]N+"!Y;W4G;&P@0T*9F]L M;&]W:6YG(&5V97)Y('-T97`@97AA8W1L>2!I2!D;V5S('!A>0T* M;6]R92$-"@T*0GD@=&%K:6YG('!A7-T96TL('EO=2!A M2!A8F]U="!K965P:6YG(&%N M>2!I;G9E;G1O2!C86X@8V]M92!I;B!I;B`R('=A>7,N("!&:7)S="P@9G)O;2!P M96]P;&4@=VAO('=I;&P@<&%Y('EO=2!T;PT*:F]I;B!Y;W5R(&UA:6QI;F<- M"FQI2!F2!Y;W4@=&\@7-T96T@<')I;6%R:6QY(&-O=F5R M2`M('1H90T*6]U<@T* M;6%I;&EN9R!L:7-T('1O+@T*#0HH4U1%4"`Q+BD@("`@($QO8V%T92!T:&4@ M;&ES="!O9B!T96X@;F%M97,@86YD(&%D9')E2!B92!M86EL:6YG(&$- M"FQE='1E2!O6]U2X@($1O M(&YO="!C:&%N9V4@=&AE;2!O"!S=69F M:6-I96YT('!O6]U'!L86EN#0IT:&4@6]U(&AA=F4-"G)E M<')O9'5C960@979E6]U(')E8V5I=F5D(&ET+@T*06QS;R!B92!S=7)E('1H97)E#0IA'!L86EN M('1H92!R96%S;VX@9F]R#0IP;W-T:6YG(&%T(&QE87-T(#$P('1I;65S(&]R M#0IM;W)E+BXN#0H-"BA35$50(#0N*2`@("!(97)E)W,@=&AE(&AA2!3="X-"B`@("`@0F%Y2!G M970-"FQA'!L86EN(&AO=R!T:&ES('=O M'!O6]U6]U('1H90T*;G5M8F5R(&]F(&QE='1E M6]U2X-"@T*1F]R(&5X86UP;&4L('5S:6YG(&5A2P-"C0U,"!P96]P;&4@87)E#0IR96%D:6YG('1H92!L971T M97(L(#(E(&]F('=H:6-H(')E2!T:&4-"G1I;64@37(N($$-"FUO=F5S M(&EN=&\@<&]S:71I;VX@(S4L('=H97)E(&AE('=I;&P@8F5G:6X@2!N=6UB97(@;V8@=&AI M;F=S(&-A;B!H87!P96X@=&\-"FME97`@=&AE(&UE2!W:'D@:70@ M:7,@82!G;V]D(&ED96$@=&\-"F)L86YK970@87,@;6%N>0T*<&]S=&EN9W,@ M:6X@87,@;6%N>2!P;&%C97,@87,@<&]S