From owner-proteins@net.bio.net Sun Nov 01 22:00:00 1998 Path: biosci!DIHT.COM.SG!hufacia52 From: hufacia52@DIHT.COM.SG (frezxery) Newsgroups: bionet.molbio.proteins Subject: Reach your internet clients - before competition Date: 2 Nov 1998 07:38:16 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 31 Sender: daemon@net.bio.net Distribution: world Message-ID: <19981102130VAA50171@frebrequaz.ghyis2.harryda.se> NNTP-Posting-Host: net.bio.net ___________________________________________________________ DIRECT EMAIL SERVICES - targeted & general prospects ! ___________________________________________________________ WEB SITE Submission - to the top search engines & hundreds ___________________________________________________________ Daily Lists - get FRESH NEW lists daily - great supplement ___________________________________________________________ FREE SOFTWARE DEMOS - prospect harvesters and mailers ! ___________________________________________________________ EMAIL LIST - MILLIONS: cleaned.....complete w/ software ___________________________________________________________ OPPORTUNITY $$ - software, books & internet services ___________________________________________________________ BOOKS & TAPES - Hundreds of selections - Dealers wanted ! ********************************************************* MORE INFO (24 hrs): 702-284-5074 Dist. Ref #: 254804GB This is a one time message - thank you ! ******************************************** From owner-proteins@net.bio.net Sun Nov 01 22:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!news-peer.gip.net!news.gsl.net!gip.net!newshub1.home.com!news.home.com!news.rdc1.bc.wave.home.com.POSTED!not-for-mail From: "Achim Recktenwald" Newsgroups: bionet.molbio.proteins Subject: JOB: Victoria,BC,Canada: Protein Purification/Process Development/Manufacturing Lines: 44 X-Newsreader: Microsoft Outlook Express 4.72.3155.0 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3155.0 Message-ID: Date: Tue, 03 Nov 1998 05:16:16 GMT NNTP-Posting-Host: 24.64.220.105 NNTP-Posting-Date: Mon, 02 Nov 1998 21:16:16 PDT Organization: @Home Network Canada StressGen Biotechnologies, Corp. 4243 Glanford Avenue Victoria, BC, V8Z 4B9 Canada StressGen Biotechnologies is a growing biopharmaceutical company developing medical applications of the cellular stress response. StressGen’s R&D programs focus on utilizing stress proteins as immunomodulatory agents in therapeutic applications. Research Associate / Senior Research Associate +++++++++++++++++++++++++++++++++++++++ StressGen Biotechnologies, Corp. has an immediate opening for an experienced technically qualified scientist to join our Protein Chemistry Group. The candidate should have a M.Sc. or B.Sc. with 2+ years industrial experience in the purification of biopharmaceutical proteins, preferentially up to pilot-scale. A broad knowledge of column chromatographic methods and equipment (ÄKTA/ FPLC) is essential. In addition, familiarity with standard analytical and electrophoretic methods (PAGE, Western blotting), as well as exposure to fermentation and cell disintegration procedures, is a plus. Please send C.V. c/o: Dr. Achim Recktenwald StressGen Biotechnologies, Corp. 4243 Glanford Avenue Victoria, BC, V8Z 4B9 Canada Fax: (250) 744-2877 Email: ARecktenwald@StressGen.Com We thank all candidates for applying. Only those under consideration will be contacted. Please – no phone calls or drops ins. From owner-proteins@net.bio.net Sun Nov 01 22:00:00 1998 Path: biosci!bcm.tmc.edu!news From: Brad Poland Newsgroups: bionet.molbio.proteins Subject: Thrombin cleavage in presence of detergent Date: Mon, 02 Nov 1998 11:02:55 -0800 Organization: Baylor College of Medicine, Houston, Tx Lines: 50 Message-ID: <363E01DF.895D5908@bcm.tmc.edu> NNTP-Posting-Host: elroy.qci.bioch.bcm.tmc.edu Mime-Version: 1.0 Content-Type: multipart/alternative; boundary="------------C8BD2F163B8EC7159C8BCF97" X-Mailer: Mozilla 4.07 [en] (X11; U; IRIX64 6.2 IP28) --------------C8BD2F163B8EC7159C8BCF97 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit I have a GST fusion protein that is soluble in detergent solutions, ie triton-X100, NDSB, BOG, etc. The problem I have is cutting the GST off, thrombin works well in the presence of triton-X100, but seems to be inhibited by the other detergents I tried. Does anyone have a suggestion for this problem? BTW, I am trying to crystallize this protein. Thanks, brad -- Brad Poland, PhD. __o Howard Hughes Medical Institute _ |/<_ Baylor College of Medicine (_)| (_) Houston TX bpoland@bcm.tmc.edu --------------C8BD2F163B8EC7159C8BCF97 Content-Type: text/html; charset=us-ascii Content-Transfer-Encoding: 7bit I have a GST fusion protein that is soluble in detergent solutions, ie triton-X100, NDSB, BOG, etc.
The problem I have is cutting the GST off, thrombin works well in the presence of triton-X100, but
seems to be inhibited by the other detergents I tried.  Does anyone have a suggestion for this problem?
BTW, I am trying to crystallize this protein.  Thanks,

brad 

-- 

Brad Poland, PhD.                                __o   
Howard Hughes Medical Institute               _ |/<_  
Baylor College of Medicine                   (_)| (_)   
Houston TX                                              
bpoland@bcm.tmc.edu
  --------------C8BD2F163B8EC7159C8BCF97-- From owner-proteins@net.bio.net Sun Nov 01 22:00:00 1998 Newsgroups: bionet.molbio.proteins Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!news.gtei.net!cpk-news-hub1.bbnplanet.com!news.news.gtei.net!newsfeed.cwix.com!194.72.7.126!btnet-peer!btnet!nntp.news.xara.net!xara.net!server6.netnews.ja.net!leeds.ac.uk!news From: bmbjmm@bmb.leeds.ac.uk (jeremy murray) Subject: inclusion bodies X-Accept-Language: en Message-ID: <363E3E45.A01D50ED@bmb.leeds.ac.uk> NNTP-Posting-Host: bmbsgi15.leeds.ac.uk X-Mailer: Mozilla 4.5 [en] (X11; I; IRIX 6.2 IP22) Content-Type: text/plain; charset=us-ascii Organization: University of Leeds MIME-Version: 1.0 Date: Mon, 2 Nov 1998 23:24:37 +0000 (GMT) Lines: 27 Content-Transfer-Encoding: 7bit hi y'all just a quick survey into methods for dealing with inclusion bodies... has anybody had any experience good, bad or indifferent, when growing cells and over-expressing proteins prone to inclusion body formation in minimal media? TIA j -- . .-. .-. .-. .-. .-. .-. .-. .-. .-. .-. .-. .-. .-. .- |\ /|||X|||\ /|||X|||\ /|||X|||\ /|||X|||\ /|||X|||\ /|||X|||\ /|||X|| ||X|||/ \|||X|||/ \|||X|||/ \|||X|||/ \|||X|||/ \|||X|||/ \|||X|||/ \| -' `-' `-' `-' `-' `-' `-' `-' `-' `-' `-' `-' `-' `-' ` Jeremy Murray MAIL: bmbjmm@bmb.nospam.leeds.ac.uk Biochemistry & Molecular Biology TEL: +44 113 233 2591 Leeds University, LEEDS LS2 9JT FAX: +44 113 233 3167 http://www.smb.leeds.ac.uk/wwwsb/jez.html . .-. .-. .-. .-. .-. .-. .-. .-. .-. .-. .-. .-. .-. .- |\ /|||X|||\ /|||X|||\ /|||X|||\ /|||X|||\ /|||X|||\ /|||X|||\ /|||X|| ||X|||/ \|||X|||/ \|||X|||/ \|||X|||/ \|||X|||/ \|||X|||/ \|||X|||/ \| -' `-' `-' `-' `-' `-' `-' `-' `-' `-' `-' `-' `-' `-' ` From owner-proteins@net.bio.net Mon Nov 02 22:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!newshub.northeast.verio.net!newsserver.jvnc.net!newsfeed.metronet.de!RRZ.Uni-Koeln.DE!news-koe1.dfn.de!news-han1.dfn.de!news-ham1.dfn.de!news.mu-luebeck.de!not-for-mail From: "Lars Komorowski" Newsgroups: bionet.molbio.proteins Subject: Re: Thrombin cleavage in presence of detergent Date: Tue, 3 Nov 1998 12:29:56 +0100 Organization: Med. Universitaet zu Luebeck Lines: 85 Message-ID: <71mppc$mtq$1@gwsun.medinf.mu-luebeck.de> References: <363E01DF.895D5908@bcm.tmc.edu> NNTP-Posting-Host: 141.83.167.171 Mime-Version: 1.0 Content-Type: multipart/alternative; boundary="----=_NextPart_000_0017_01BE0725.AA45D440" X-Newsreader: Microsoft Outlook Express 4.72.3110.5 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3110.3 This is a multi-part message in MIME format. ------=_NextPart_000_0017_01BE0725.AA45D440 Content-Type: text/plain; charset="iso-8859-1" Content-Transfer-Encoding: quoted-printable How is the concentration of the detergents especially Triton ? Lars Brad Poland schrieb in Nachricht <363E01DF.895D5908@bcm.tmc.edu>... I have a GST fusion protein that is soluble in detergent solutions, = ie triton-X100, NDSB, BOG, etc.=20 The problem I have is cutting the GST off, thrombin works well in = the presence of triton-X100, but=20 seems to be inhibited by the other detergents I tried. Does anyone = have a suggestion for this problem?=20 BTW, I am trying to crystallize this protein. Thanks,=20 brad=20 --=20 Brad Poland, PhD. __o =20 Howard Hughes Medical Institute _ |/<_ =20 Baylor College of Medicine (_)| (_) =20 Houston TX =20 bpoland@bcm.tmc.edu =20 ------=_NextPart_000_0017_01BE0725.AA45D440 Content-Type: text/html; charset="iso-8859-1" Content-Transfer-Encoding: quoted-printable
How is the concentration of the detergents = especially Triton=20 ?
Lars
Brad Poland schrieb in Nachricht <363E01DF.895D5908@bcm.tmc.e= du>...
I=20 have a GST fusion protein that is soluble in detergent solutions, ie = triton-X100, NDSB, BOG, etc.
The problem I have is cutting the = GST off,=20 thrombin works well in the presence of triton-X100, but
seems to = be=20 inhibited by the other detergents I tried.  Does anyone have a=20 suggestion for this problem?
BTW, I am trying to crystallize = this=20 protein.  Thanks,=20

brad 

-- 

Brad Poland, =
PhD.           &nb=
sp;           &nbs=
p;        __o   
Howard Hughes Medical =
Institute          &nbs=
p;    _ |/<_  
Baylor College of =
Medicine           =
;        (_)| (_)   
Houston =
TX            =
;            =
            &=
nbsp;         
bpoland@bcm.tmc.edu
  ------=_NextPart_000_0017_01BE0725.AA45D440-- From owner-proteins@net.bio.net Mon Nov 02 22:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!newsfeed.enteract.com!newsfeed.ecrc.net!newshub2.home.com!newshub1.home.com!news.home.com!news.rdc1.bc.wave.home.com.POSTED!not-for-mail From: "Achim Recktenwald" Newsgroups: bionet.molbio.proteins Subject: JOB: Victoria,BC,Canada: Protein Purification/Process Development/Manufacturing Lines: 44 X-Newsreader: Microsoft Outlook Express 4.72.3155.0 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3155.0 Message-ID: Date: Wed, 04 Nov 1998 01:52:37 GMT NNTP-Posting-Host: 24.64.220.105 NNTP-Posting-Date: Tue, 03 Nov 1998 17:52:37 PDT Organization: @Home Network Canada StressGen Biotechnologies, Corp. 4243 Glanford Avenue Victoria, BC, V8Z 4B9 Canada StressGen Biotechnologies is a growing biopharmaceutical company developing medical applications of the cellular stress response. StressGen’s R&D programs focus on utilizing stress proteins as immunomodulatory agents in therapeutic applications. Research Associate / Senior Research Associate +++++++++++++++++++++++++++++++++++++++ StressGen Biotechnologies, Corp. has an immediate opening for an experienced technically qualified scientist to join our Protein Chemistry Group. The candidate should have a M.Sc. or B.Sc. with 2+ years industrial experience in the purification of biopharmaceutical proteins, preferentially up to pilot-scale. A broad knowledge of column chromatographic methods and equipment (ÄKTA/ FPLC) is essential. In addition, familiarity with standard analytical and electrophoretic methods (PAGE, Western blotting), as well as exposure to fermentation and cell disintegration procedures, is a plus. Please send C.V. c/o: Dr. Achim Recktenwald StressGen Biotechnologies, Corp. 4243 Glanford Avenue Victoria, BC, V8Z 4B9 Canada Fax: (250) 744-2877 Email: ARecktenwald@StressGen.Com We thank all candidates for applying. Only those under consideration will be contacted. Please – no phone calls or drops ins. From owner-proteins@net.bio.net Mon Nov 02 22:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!news.maxwell.syr.edu!news.mel.connect.com.au!news.unimelb.edu.au!ludwignt-4 From: murphy_r@licre.ludwig.edu.au (Roger Murphy) Newsgroups: bionet.molbio.proteins Subject: Re: Ni-NTA Regeneration Date: Wed, 04 Nov 98 02:15:33 GMT Organization: Ludwig Institute for Cancer Research Lines: 40 Message-ID: <71o9ef$8b4$1@izvestia.its.unimelb.edu.au> References: <363F2A73.728E51FF@gmx.de> NNTP-Posting-Host: ludwignt-4.austin.unimelb.edu.au X-Trace: izvestia.its.unimelb.edu.au 910141711 8548 128.250.186.193 (4 Nov 1998 01:08:31 GMT) X-Complaints-To: news@news.unimelb.edu.au NNTP-Posting-Date: 4 Nov 1998 01:08:31 GMT X-Newsreader: News Xpress 2.0 Beta #0 We've used both Qiagen Ni-NTA and Pharmacia Chelating Sepharose FF and had no problems after regeneration. Indeed, our standard approach now is to use the column only once and then regenrate - ever since we found out the "lifetime" of the columns was limited to only 3-4 runs before very little adsorption occurred. Hope this helps, Roger In article <363F2A73.728E51FF@gmx.de>, Andreas Savelsbergh wrote: >Hi netters, > >Qiagen and other companies give protocols how to regenerate the matrices >for affinity purification with a His-tag. > >My question: has anybody ever tried that succesfully? > >I once re-used Ni-NTA after a preparative application for analytical >purpose with the result that much of the OTHER protein appeared, too. > >Thanks a lot, > >Andreas > Roger Murphy, Ph.D. Biological Production Facility Ludwig Institute for Cancer Research Austin & Repatriation Medical Centre Studley Road, Heidelberg, Vic. 3084 Australia. Tel 61-3-94965463 Fax 61-3-94965436 Email murphy_r@licre.ludwig.edu.au From owner-proteins@net.bio.net Mon Nov 02 22:00:00 1998 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!news.gtei.net!cpk-news-hub1.bbnplanet.com!news.news.gtei.net!fu-berlin.de!we27pc04.ukbf.fu-berlin.DE!not-for-mail From: "Oliver Politz" Newsgroups: bionet.cellbiol,bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: PVDF contra gel extraction for protein sequencing ? Date: Tue, 3 Nov 1998 13:00:57 +0100 Organization: Freie Universitaet Berlin Lines: 12 Message-ID: <71mnkl$9ro$1@fu-berlin.de> NNTP-Posting-Host: we27pc04.ukbf.fu-berlin.de (160.45.184.205) Mime-Version: 1.0 Content-Type: text/plain; charset="iso-8859-1" Content-Transfer-Encoding: quoted-printable X-Access: 16 17 19 X-Trace: fu-berlin.de 910090709 10104 (none) 160.45.184.205 X-Newsreader: Microsoft Outlook Express 4.72.3110.5 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3110.3 Xref: biosci bionet.cellbiol:10751 bionet.molbio.methds-reagnts:71927 bionet.molbio.proteins:13510 Dear netters, I would like to get some opinions about advantages / disadvantages to = use either gel extraction of proteins or blotting them on PVDF membrane = to perform tryptic digests for further HPLC separation and = microsequencing. My problem is that I have to get the protein of = interest out of a number of faint coomassie bands. I am thinking of = using the electro-eluter module for the Biorad Miniprotean cell (any = experience out there). Please email to politz@gmx.de Thanks in advance oliver From owner-proteins@net.bio.net Mon Nov 02 22:00:00 1998 From: James Fethiere Newsgroups: bionet.molbio.proteins Subject: Thermolysin Date: Tue, 03 Nov 1998 09:54:23 +0100 Organization: MPI-Med Forschung Lines: 22 Message-ID: <363EC4BF.41C6@mpimf-heidelberg.mpg.de> NNTP-Posting-Host: ile.mpimf-heidelberg.mpg.de Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01SGoldC-SGI (X11; I; IRIX 6.3 IP32) Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!newsfeed.cwix.com!193.190.198.38!news.belnet.be!news-zh.switch.ch!news.belwue.de!news.uni-ulm.de!rz.uni-karlsruhe.de!news.urz.uni-heidelberg.de!usenet Hi everyone, I just got some thermolysin from Boehringer. Does it ever go into solution? I tried 2mg/ml in water like most of the other proteases, and the solution is cloudy. Then I added sequentially 5mM CaCl2, 100mM NaCl, 50mM Tris pH 7.5 but it still stayed cloudy. It this the way it should be, should I heat it? Thank you James -- ************************************************************************ * James Fethiere * * MPI-Medical Research Int'l Tel: +49-6221-486154 * * Ion Channel Structure Group Int'l Fax: +49-6221-486437 * * Jahnstrasse 29 email: james@mpimf-heidelberg.mpg.de * * 69120, Heidelberg * * Germany * ************************************************************************ From owner-proteins@net.bio.net Mon Nov 02 22:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!dca1-hub1.news.digex.net!digex!europa.clark.net!194.162.162.196!newsfeed.nacamar.de!wuff.mayn.de!fu-berlin.de!molbio4.nawi.uni-wh.DE!not-for-mail From: Andreas Savelsbergh Newsgroups: bionet.molbio.proteins Subject: Ni-NTA Regeneration Date: Tue, 03 Nov 1998 17:08:19 +0100 Lines: 14 Message-ID: <363F2A73.728E51FF@gmx.de> NNTP-Posting-Host: molbio4.nawi.uni-wh.de (193.175.78.33) Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Access: 16 1597 1598 X-Trace: fu-berlin.de 910109352 10623 (none) 193.175.78.33 X-Mailer: Mozilla 4.03 [en] (Win95; I) Hi netters, Qiagen and other companies give protocols how to regenerate the matrices for affinity purification with a His-tag. My question: has anybody ever tried that succesfully? I once re-used Ni-NTA after a preparative application for analytical purpose with the result that much of the OTHER protein appeared, too. Thanks a lot, Andreas From owner-proteins@net.bio.net Mon Nov 02 22:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!news.msfc.nasa.gov!bcm.tmc.edu!news From: Brad Poland Newsgroups: bionet.molbio.proteins Subject: Re: Thrombin cleavage in presence of detergent Date: Tue, 03 Nov 1998 09:39:28 -0800 Organization: Baylor College of Medicine, Houston, Tx Lines: 90 Message-ID: <363F3FD0.58D7CA3D@bcm.tmc.edu> References: <363E01DF.895D5908@bcm.tmc.edu> <71mppc$mtq$1@gwsun.medinf.mu-luebeck.de> NNTP-Posting-Host: elroy.qci.bioch.bcm.tmc.edu Mime-Version: 1.0 Content-Type: multipart/alternative; boundary="------------1C5ACE446639E8764411770B" X-Mailer: Mozilla 4.07 [en] (X11; U; IRIX64 6.2 IP28) --------------1C5ACE446639E8764411770B Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit The concentration of detergents is ~1%. brad Lars Komorowski wrote: > How is the concentration of the detergents especially Triton ?Lars > > Brad Poland schrieb in Nachricht > <363E01DF.895D5908@bcm.tmc.edu>...I have a GST fusion > protein that is soluble in detergent solutions, ie > triton-X100, NDSB, BOG, etc. > The problem I have is cutting the GST off, thrombin works > well in the presence of triton-X100, but > seems to be inhibited by the other detergents I tried. Does > anyone have a suggestion for this problem? > BTW, I am trying to crystallize this protein. Thanks, > > brad > > -- > > Brad Poland, PhD. __o > Howard Hughes Medical Institute _ |/<_ > Baylor College of Medicine (_)| (_) > Houston TX > bpoland@bcm.tmc.edu > > > -- Brad Poland, PhD. __o Howard Hughes Medical Institute _ |/<_ Baylor College of Medicine (_)| (_) Houston TX bpoland@bcm.tmc.edu --------------1C5ACE446639E8764411770B Content-Type: text/html; charset=us-ascii Content-Transfer-Encoding: 7bit The concentration of detergents is ~1%.
brad

Lars Komorowski wrote:

 How is the concentration of the detergents especially Triton ?Lars
Brad Poland schrieb in Nachricht <363E01DF.895D5908@bcm.tmc.edu>...I have a GST fusion protein that is soluble in detergent solutions, ie triton-X100, NDSB, BOG, etc.
The problem I have is cutting the GST off, thrombin works well in the presence of triton-X100, but
seems to be inhibited by the other detergents I tried.  Does anyone have a suggestion for this problem?
BTW, I am trying to crystallize this protein.  Thanks,

brad 

-- 

Brad Poland, PhD.                                __o   
Howard Hughes Medical Institute               _ |/<_  
Baylor College of Medicine                   (_)| (_)   
Houston TX                                              
bpoland@bcm.tmc.edu
 
-- 

Brad Poland, PhD.                                __o   
Howard Hughes Medical Institute               _ |/<_  
Baylor College of Medicine                   (_)| (_)   
Houston TX                                              
bpoland@bcm.tmc.edu
  --------------1C5ACE446639E8764411770B-- From owner-proteins@net.bio.net Tue Nov 03 22:00:00 1998 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!news.gtei.net!cpk-news-hub1.bbnplanet.com!news.news.gtei.net!newshub.northeast.verio.net!nntp.newengland.verio.net!news.pn.com!mozo.cc.purdue.edu!news From: Lena Zaitseva Newsgroups: bionet.molbio.proteins Subject: Re: Thrombin cleavage in presence of detergent Date: Wed, 04 Nov 1998 09:31:24 -0500 Organization: Purdue University Lines: 104 Message-ID: <3640653C.2EA5A99F@biochem.purdue.edu> References: <363E01DF.895D5908@bcm.tmc.edu> <71mppc$mtq$1@gwsun.medinf.mu-luebeck.de> <363F3FD0.58D7CA3D@bcm.tmc.edu> NNTP-Posting-Host: hermod323a.biochem.purdue.edu Mime-Version: 1.0 Content-Type: multipart/alternative; boundary="------------73107965758B3DA92F6AD637" X-Mailer: Mozilla 4.04 [en] (Win95; I) --------------73107965758B3DA92F6AD637 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: quoted-printable =A0 Brad Poland wrote: > =A0The concentration of detergents is ~1%. > brad > > Lars Komorowski wrote: > >> =A0How is the concentration of the detergents especially Triton ?Lars >> >> Brad Poland schrieb in Nachricht >> <363E01DF.895D5908@bcm.tmc.edu>...I have a GST fusion >> protein that is soluble in detergent solutions, ie >> triton-X100, NDSB, BOG, etc. >> The problem I have is cutting the GST off, thrombin works >> well in the presence of triton-X100, but >> seems to be inhibited by the other detergents I tried.=A0 >> Does anyone have a suggestion for this problem? >> BTW, I am trying to crystallize this protein.=A0 Thanks, >> >> brad >> =A0=A0=A0=A0 Two different factors can be involved:1. Detergent concentra= tion: 1% for Triton X-100 is much higher than its CMC (at low salt concentrations CMC=3D0.018%); for BOG CMC=3D0.7-0.8% at the same conditio= ns, so 1% is just slightly higher of CMC. Unfortunately, I don't know what is NDSB? So, in this case it might be not enough BOG to slightly loosen up the protein folding (to allow cleavage with protease). 2. Some detergents might cause the protein aggregation. So, it's possible that BOG is not a appropriate detergent for your protein, and protein starts to aggregate. Ultracentrifugation is one of the ways to check the presence of protein aggregates. If aggregates are big enough than you'll see them in a pellet after centrifugation (30 min-1hr. at 300,000-400,00 g). =A0=A0=A0=A0 Actually it is hard to predict the behavior of=A0 membrane p= rotein. It is important to know what detergent you use for membrane solubilization, what buffer, do you exchange detergents after solubilization (purification) and how, if yes? How hydrophobic is your protein? What conditions are you using for protease cleavage? And so on. =A0=A0=A0=A0 Lena. =A0 --------------73107965758B3DA92F6AD637 Content-Type: text/html; charset=us-ascii Content-Transfer-Encoding: 7bit  

Brad Poland wrote:

 The concentration of detergents is ~1%.
brad

Lars Komorowski wrote:

 How is the concentration of the detergents especially Triton ?Lars
Brad Poland schrieb in Nachricht <363E01DF.895D5908@bcm.tmc.edu>...I have a GST fusion protein that is soluble in detergent solutions, ie triton-X100, NDSB, BOG, etc.
The problem I have is cutting the GST off, thrombin works well in the presence of triton-X100, but
seems to be inhibited by the other detergents I tried.  Does anyone have a suggestion for this problem?
BTW, I am trying to crystallize this protein.  Thanks,

brad

     Two different factors can be involved:1. Detergent concentration: 1% for Triton X-100 is much higher than its CMC (at low salt concentrations CMC=0.018%); for BOG CMC=0.7-0.8% at the same conditions, so 1% is just slightly higher of CMC. Unfortunately, I don't know what is NDSB? So, in this case it might be not enough BOG to slightly loosen up the protein folding (to allow cleavage with protease).
2. Some detergents might cause the protein aggregation. So, it's possible that BOG is not a appropriate detergent for your protein, and protein starts to aggregate. Ultracentrifugation is one of the ways to check the presence of protein aggregates. If aggregates are big enough than you'll see them in a pellet after centrifugation (30 min-1hr. at 300,000-400,00 g).
     Actually it is hard to predict the behavior of  membrane protein. It is important to know what detergent you use for membrane solubilization, what buffer, do you exchange detergents after solubilization (purification) and how, if yes? How hydrophobic is your protein? What conditions are you using for protease cleavage? And so on.
     Lena.
  --------------73107965758B3DA92F6AD637-- From owner-proteins@net.bio.net Tue Nov 03 22:00:00 1998 Path: biosci!SINGNET.COM.SG!pueinam From: pueinam@SINGNET.COM.SG (Tay Puei Nam) Newsgroups: bionet.molbio.proteins Subject: Purification of His-Tag Proteins Date: 4 Nov 1998 06:52:58 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 7 Sender: daemon@net.bio.net Distribution: world Message-ID: <36414E09.1C2A@singnet.com.sg> NNTP-Posting-Host: net.bio.net Hi! Has anyone tried purifying secreted His-Tag proteins (using metal chelate system) from culture medium of transiently transfected Cos cells? I need some advice regarding this. Thanks! Pueinam (Please email to: pueinam@singnet.com.sg) From owner-proteins@net.bio.net Tue Nov 03 22:00:00 1998 Path: biosci!ihnp4.ucsd.edu!not-for-mail From: "Antonin Tutter" Newsgroups: bionet.molbio.proteins Subject: Re: Protein purification by affinity tags Date: Wed, 04 Nov 1998 22:40:21 -0800 Organization: University of California, San Deigo Lines: 30 Distribution: world Message-ID: <910248024.925769@news1.ucsd.edu> References: <363A5867.AD5D5051@student.mahidol.ac.th> NNTP-Posting-Host: news1.ucsd.edu Mime-Version: 1.0 Content-Type: text/plain; charset="US-ASCII" Content-Transfer-Encoding: 7bit X-Trace: ihnp4.ucsd.edu 910248029 4400 132.239.1.221 (5 Nov 1998 06:40:29 GMT) X-Complaints-To: Abuse@UCSD.Edu NNTP-Posting-Date: 5 Nov 1998 06:40:29 GMT >> This is a general question about making fusion protein for affinity >> purification. Several protein purification systems offer a choice of N- >> or C- terminal fusion of affinity tags, such as CBD, His6 etc while >> other systems have only N-terminal tags, such as GST, MBP. It is >> obvious that if the tag is at the N-terminus, one can get truncated >> protein products bound onto the affinity column. If the tag is >> C-terminal, only full-length fusion protein will be retented. With such >> consideration, why would so many affinity tags be made to the >> N-terminus? It seems to me that it offers no advantage at all... >> Actually, I had this discussion with some colleagues and the general consensus was that it is better to have a C-terminal tag if the tag is 6His, because protein secondary structure begins to form during translation starting with the N-terminus of the nascent peptide. An N-terminal tag may interfere with proper folding. GST tags are preferentially fused to the N-terminus for the same reason -- the GST must fold correctly for it to bind glutathione, and once the protein is correctly folded as its own domain, the c-terminal fusion will follow on with its own secondary structure formation _______________________________________ Antonin Tutter Salk Institute for Biological Studies RBIO-J 10010 N. Torrey Pines Rd. La Jolla, CA 92037 email: atutter@aim.salk.edu web: http://www-biology.ucsd.edu/~atutter/ From owner-proteins@net.bio.net Tue Nov 03 22:00:00 1998 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!news.gtei.net!newsfeed.berkeley.edu!nntp2.dejanews.com!nnrp1.dejanews.com!not-for-mail From: lichu_liu@my-dejanews.com Newsgroups: bionet.molbio.proteins Subject: Looking for a postdoctoral position on molecular biology Date: Wed, 04 Nov 1998 14:21:38 GMT Organization: Deja News - The Leader in Internet Discussion Lines: 131 Message-ID: <71pntj$pdg$1@nnrp1.dejanews.com> NNTP-Posting-Host: 202.130.229.252 X-Article-Creation-Date: Wed Nov 04 12:17:10 1998 GMT X-Http-User-Agent: Mozilla/4.05 [en] (Win95; I) X-Http-Proxy: 1.0 x7.dejanews.com:80 (Squid/1.1.22) for client 202.130.229.252 I am looking for a postdoctoral position or research position in a laboratory in the middle of 1999. In the past two years, I have been pursuing my Ph.D degree in the National Laboratory of Molecular Virology and Genetic Engineering,one of the 2 top biomedicine labs in China. In my university study, I have received strict researsh training and am experienced in many experimental techniques, such as cDNA cloning, sequencing, RT-PCR, molecular hybridization, DNA recombination,mutagenesis cell culture, gene expression in prokaryotic and eukaryotic system, puri-fication and assay of protein, immunological methods etc. Moreover, I am especially skilled in animal experiment techniques. So I am confident in my ability to make a contribution, though small, to a coming research project. CURRICULUM VITAE Name: Lichu Liu gender: Male Age: 33 Health: Excellent Status: Married Nationality: Chinese Present Address: 411 National Laboratory of Molecular Virology and Genetic Engineering, Institute of Virology, Chinese Academy of Proventive Medicine(CAPM). 100 Ying Xin St., Xuan Wu Qu, Beijing, 100052, P.R. China. Tel: 8610-63528481 Fax: 8610-63532053 E-mail: Lichuliu@public.bta.net.cn EDUCATION 1996-present: National Laboratory of Molecular Virology and Genetic Engineering, Institute of Viyology, Chinese Academy of Proventive Medicine CAPM. Ph.D degree to be granted in July of 1999. 1994-1996: TongJi Medical University, Wuhan, China. M.S. Orthopaedic Medicine I won the prize of outstanding postgraduate student and was recommend for Ph.D candidate. 1982-1987: Hunan Medical University, Changsha, China. M.D. clinical medicine. WORK EXPERIENCE 1990-1994: Clinical Orthopaedist, Department of Orthopadic Surgery, The First Hospital of TongJi Medical University, Wuhan, China. 1989-1990: Clinical Orthopadist, Dept of Orthopaedic Surgery, China-Japan Friendship Hospital, Beijing, China. 1987-1989: Clinical Surgeon, Dept of Surgery, The Fourth Hospital of Shihezi Medical College, Xingjiang province, China. PROFESSIONAL EXPERIENCE 1996-present(Work for Ph.D): 1. Cloning and sequencing of BMP-7 and BMP-2 cDNA from U2OS cells. In this work, I did cell culture, RNA isolation, RT-PCR, construction of cDNA library, followed by screening of the library and sequecing the positive clones. 2. Construction and expression of rhBMP-2/7 a fusion gene encoding BMP-2 and BMP-7 mature peptide in Ecoli.. The fusion protein was purified and verified by west blotting. 3. Expression and purification of BMP-2/7 heretodimer and BMP-2/7 fusion protein in CHO cells. Biological activity of them was assayed and compared with BMP-7 using MC3T3 cells mouse or rabbits model respectively. 4. As a collaborator, I participate in the study of il-12 targeted gene therapy for hepatocellar carcinoma HCC. 1994-1996(Work for M.S.): 1. Immunohistochemical observation on BMPs, IGF-I/II, bFGF, TGF in callus and osteosacoma. 2. Study on bone and cartilagious defect repaired with BMPs. 1982-1987(an undergraduate student of clinical medicine): Systematically trained in biology, immunology, biochemistry, genetics, pathology, microbiology, and clinical medicine. SKILLS: 1. Diagnosis and treatment of common orthopaedic diease. 2. DNA recombination, cDNA cloning, sequencing, mutagenesis and vector construction. 3. Gene expression in prokaryotic and eukaryotic system. 4. Purification and assay of protein. 5. Isolation of RNA, mRNA, RT-PCR, and molecular hybridization, southern blot, northern blot, west blot. 6. Tissue and cell culture, immunological methods ELISA, PAP etc. 7. Animal experiment techniques, especially in surgerical operation. MAIN: 1. Lichu Liu et al:Treatment of comminuted patellar fracture with cross band wire(in Chinese) vol2(4):236,1995.The Orthp. J. of China. 2. Lichu Liu et al:Immunohistochemical observation on bone morpho- genetic proteins during fracture healing(in Chinese). vol 6(1):12,1996 Chinese J. of Orthop. and Traumatology. 3. Lichu Liu et al: The use of bone morphogenetic protein/demineralized bone matrix for repair of articular cartilagous defects(in Chinese) vol 7(2):56,1997. Chinese J. of Orthop and Traumatology. 4. Lichu Liu et al: Distribution and effect of bone morphogenetic protein(BMP) and osteprogenitors during bone repair(in Chinese) The Orthop. J. of China. in press. 5. Lichu liu et al: Cloning and sequencing the full-length cDNA encoding human osteogenic protein-1. Chinese J. Orthop. in press. 6. Lichu Liu et al: Construction and expression of BMP-2/7 fusion gene in Ecoli. Chinese J. of Experimental and Clinical Virology. in press. 7. Lichu Liu et al: Expression and identification of BMP-2/7 hereto- dimeric in CHO cells. in preparation. 8. Lichu Liu et al: Cloning and expression of the gene encoding for recombination human BMP-2/7 fusion protein in CHO cells. in preparation. REFERENCES: 1. Guodong Liang, Prof. Vice Director of Institute Virology, National Laboratory of Molecular Virology and Genetic Engineering, Institute of Virology, Chinese Academy of Proventive Medicine(CAPM). 100 Ying Xin St. Xuan Wu Qu, Beijing, 100052, P.R. China. Tel: 8610-63528481 Fax: 8610-63532053 2. Zhiqing Zhang, Prof. and Ph.D Vice Director of Institute Virology, National Laboratory of Molecular Virology and Genetic Engineering, Institute of Virology, CAMP. 100 Ying Xin St. Xuan Wu Qu. Beijing, 100052, P.R. China. Tel: 8610-63519655 Fax: 8610-63532053 3. XingKe Yang, Prof. and Ph.D National Laboratory of Molecular Virology and Genetic Engineering, Institute of Virology, CAMP. 100 Ying Xin St. Xuan Wu Qu. Beijing, 100052, China. Tel: 8610-63531511 Fax: 8610-63532053  -----------== Posted via Deja News, The Discussion Network ==---------- http://www.dejanews.com/ Search, Read, Discuss, or Start Your Own From owner-proteins@net.bio.net Wed Nov 04 22:00:00 1998 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!news.gtei.net!cpk-news-hub1.bbnplanet.com!news.news.gtei.net!rill.news.pipex.net!pipex!server1.netnews.ja.net!hgmp.mrc.ac.uk!pegasus.csx.cam.ac.uk!not-for-mail From: Yu Wai Chen Newsgroups: bionet.molbio.proteins,bionet.molbio.methds-reagents Subject: Protein purification - getting rid of DNA in lysate Date: Thu, 05 Nov 1998 09:29:33 +0000 Organization: MRC Centre for Protein Engineering Lines: 19 Message-ID: <36416FFD.60D5FF31@mrc-lmb.cam.ac.uk> NNTP-Posting-Host: ind4.mrc-lmb.cam.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.5 [en] (X11; I; IRIX64 6.4 IP30) X-Accept-Language: zh-TW,zh-CN Dear all, As a first step in purifying my protein, I sonicated my culture and got a very thick lysate. This material was too thick to be filtered (e.g. only < 5 ml can go through each 0.45um syringe filter). I tried loading onto a column without filtering and it clogged the column. What is the best way of clearing the DNA in the lysate? I came across reports that one can use a reagent called "PEI", does anybody know what this is? Is it good? Some other people use DNAse. Can somebody share with me their experiences of which method is the best and cleanest? Thanks. -- =================================================================== Yu Wai CHEN, Ph.D. .................. email: ywc@mrc-lmb.cam.ac.uk Centre for Protein Engineering, tel: 44-(1223) 402148 MRC Centre, Cambridge CB2 2QH, U.K. fax: 44-(1223) 402140 WWW homepage: http://www.mrc-cpe.cam.ac.uk/people/wai.html From owner-proteins@net.bio.net Wed Nov 04 22:00:00 1998 From: Cornelius Krasel Subject: Re: Protein purification - getting rid of DNA in lysate Newsgroups: bionet.molbio.proteins,bionet.molbio.methds-reagents Followup-To: bionet.molbio.proteins References: <36416FFD.60D5FF31@mrc-lmb.cam.ac.uk> User-Agent: tin/pre-1.4-980514 (UNIX) (Linux/2.0.34 (i486)) Date: Thu, 5 Nov 1998 17:50:58 +0100 Message-ID: NNTP-Posting-Host: wpxx02.toxi.uni-wuerzburg.de Lines: 21 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!news.gtei.net!newsfeed.corridex.com!btnet-peer!btnet!newsfeed.ecrc.net!newsfeed.metronet.de!uni-erlangen.de!uni-wuerzburg.de!not-for-mail Yu Wai Chen wrote: > As a first step in purifying my protein, I sonicated my culture and got > a very thick lysate. There are basically two possibilities: either you did not sonicate enough (i.e. the DNA was not sufficiently sheared) or you used too many cells per buffer volume. Of course, after lysis you have to centrifuge or filter your lysate to get rid of membranes, unlysed cells and other aggregates. Dependent on your starting material, you may also want to include a filtering step through gaze to get rid of lipids (probably not so much with cells). Other ways of getting rid of DNA include digestion with DNAse or precipitation with streptomycin. --Cornelius. -- /* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */ /* D-97078 Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de SP4 */ /* "Science is the game we play with God to find out what His rules are." */ From owner-proteins@net.bio.net Wed Nov 04 22:00:00 1998 Path: biosci!TC3NET.COM!cola From: cola@TC3NET.COM (cola) Newsgroups: bionet.molbio.proteins Subject: protein extraction Date: 5 Nov 1998 08:23:14 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 3 Sender: daemon@net.bio.net Distribution: world Message-ID: <3641D0BC.D96A4BFF@tc3net.com> NNTP-Posting-Host: net.bio.net Hi, I've cut out a band from a 12-20% Tricine gel and would like to run this through an HPLC. How would I prepare the sample? Thanks Nicolas From owner-proteins@net.bio.net Wed Nov 04 22:00:00 1998 Path: biosci!news.stanford.edu!logbridge.uoregon.edu!news.idt.net!newsfeed.cwix.com!158.43.192.17!rill.news.pipex.net!pipex!sun4nl!194.178.9.131.MISMATCH!newsfeed.kabelfoon.nl!not-for-mail From: Bassie Newsgroups: bionet.molbio.proteins,bionet.molbio.methds-reagents Subject: Re: Protein purification - getting rid of DNA in lysate Date: Thu, 05 Nov 1998 15:13:09 +0100 Organization: Kabelfoon B.V. Lines: 33 Message-ID: <3641B275.E8C1AB8C@kabelfoon.nl> References: <36416FFD.60D5FF31@mrc-lmb.cam.ac.uk> NNTP-Posting-Host: k1nw067.dial.kabelfoon.nl Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: news.kabelfoon.nl 910274937 13187 195.193.20.67 (5 Nov 1998 14:08:56 GMT) X-Complaints-To: abuse@kabelfoon.nl NNTP-Posting-Date: 5 Nov 1998 14:08:56 GMT X-Mailer: Mozilla 4.06 [en] (Win95; I) Yu Wai Chen wrote: > Dear all, > > As a first step in purifying my protein, I sonicated my culture and got > a very thick lysate. This material was too thick to be filtered (e.g. > only < 5 ml can go through each 0.45um syringe filter). I tried loading > onto a column without filtering and it clogged the column. > > What is the best way of clearing the DNA in the lysate? I came across > reports that one can use a reagent called "PEI", does anybody know what > this is? Is it good? Some other people use DNAse. Can somebody share > with me their experiences of which method is the best and cleanest? > Thanks. > The first thing we usually try is addition of a Calcium salt and sometimes combined with Phosphate. At elevated Ca (20-50 mM) concs DNA binds and precipitates, addition of extra phosphate generates high density solids that can be removed easily. When your next step is Ionexchange chromatography this can be a problem as you will have to remove the added salts. PEI and other so-called Flocculants can be used as well but you will never be sure every thing is removed afterwards. They are highly cationic and can bind to almost anything. Next to that they won't function very well in high salt (>200 mM ) conditions (you need much more) Succes ---=== Bassie ===--- From owner-proteins@net.bio.net Wed Nov 04 22:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!btnet-peer!btnet!nntp.news.xara.net!xara.net!server6.netnews.ja.net!news.york.ac.uk!not-for-mail From: David Scott Newsgroups: bionet.molbio.proteins Subject: Re: inclusion bodies Date: Thu, 05 Nov 1998 13:47:49 +0000 Organization: University of York Lines: 60 Sender: djs17@york.ac.uk Message-ID: <3641AC85.261A4857@york.ac.uk> References: <363E3E45.A01D50ED@bmb.leeds.ac.uk> NNTP-Posting-Host: biolpc289.york.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: pump1.york.ac.uk 910273630 1280 144.32.84.112 (5 Nov 1998 13:47:10 GMT) X-Complaints-To: abuse@york.ac.uk NNTP-Posting-Date: 5 Nov 1998 13:47:10 GMT X-Mailer: Mozilla 4.05 [en] (Win95; I) Hia, We regularly solubilse proteins expressed into inclusion bodies in cells grown in minimal media. Assuming its E.coli your talking about.... Lyse cells and pellet debris. Then resuspend in buffer with 1M salt (takes a loonnnnngggg time), the spin, resuspend in 1% triton, spin, resuspend and spin 3 times to wash away detergent, and then resuspend in buffer + 8M Urea. N.B. in last step keep the concentration low < 1 mg/ml protein. This is so to avoid problems of aggreagation in following step. Dialyse O/N against the buffer of your choice. Purify as per your protocol.... Hope this helps Dave. jeremy murray wrote: > hi y'all > just a quick survey into > methods for dealing with inclusion bodies... > > has anybody had any experience good, bad or indifferent, > when growing cells and over-expressing proteins > prone to inclusion body formation in minimal media? > > TIA > j > > -- > . .-. .-. .-. .-. .-. .-. .-. .-. .-. .-. .-. .-. .-. .- > |\ /|||X|||\ /|||X|||\ /|||X|||\ /|||X|||\ /|||X|||\ /|||X|||\ /|||X|| > ||X|||/ \|||X|||/ \|||X|||/ \|||X|||/ \|||X|||/ \|||X|||/ \|||X|||/ \| > -' `-' `-' `-' `-' `-' `-' `-' `-' `-' `-' `-' `-' `-' ` > Jeremy Murray MAIL: bmbjmm@bmb.nospam.leeds.ac.uk > Biochemistry & Molecular Biology TEL: +44 113 233 2591 > Leeds University, LEEDS LS2 9JT FAX: +44 113 233 3167 > http://www.smb.leeds.ac.uk/wwwsb/jez.html > . .-. .-. .-. .-. .-. .-. .-. .-. .-. .-. .-. .-. .-. .- > |\ /|||X|||\ /|||X|||\ /|||X|||\ /|||X|||\ /|||X|||\ /|||X|||\ /|||X|| > ||X|||/ \|||X|||/ \|||X|||/ \|||X|||/ \|||X|||/ \|||X|||/ \|||X|||/ \| > -' `-' `-' `-' `-' `-' `-' `-' `-' `-' `-' `-' `-' `-' ` -- ********************************* * Dr. David Scott * * Dept. Biology * * University of York * * Heslington * * YORK * * UK * * Y01 5DD * * Phone: +44 1904 432868 * * Fax: +44 1904 432860 * * Email:djs17@york.ac.uk * ********************************* From owner-proteins@net.bio.net Wed Nov 04 22:00:00 1998 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!news.gtei.net!cpk-news-hub1.bbnplanet.com!news.news.gtei.net!howland.erols.net!woodstock.news.demon.net!demon!news.demon.co.uk!demon!cammol1.demon.co.uk!see_sig From: see_sig@cmtech.co.delete.uk (Richard P. Grant) Newsgroups: bionet.molbio.proteins,bionet.molbio.methds-reagents Subject: Re: Protein purification - getting rid of DNA in lysate Date: Thu, 05 Nov 1998 10:39:15 +0000 Organization: CMT Message-ID: References: <36416FFD.60D5FF31@mrc-lmb.cam.ac.uk> Reply-To: rgrant@netscape.net NNTP-Posting-Host: cammol1.demon.co.uk X-NNTP-Posting-Host: cammol1.demon.co.uk:212.228.79.177 X-Trace: news.demon.co.uk 910262438 nnrp-09:3293 NO-IDENT cammol1.demon.co.uk:212.228.79.177 X-Complaints-To: abuse@demon.net X-Newsreader: MT-NewsWatcher 2.4.4 X-Face: E%wu[EoEhNnjFG!0[ 7cH@VuxC//iy$!n-+bh9Q`+SHW@&}E.&Ly(y'Tvrd5amG/o35>c3zF6QvXX, Yu Wai Chen wrote: > Dear all, > > As a first step in purifying my protein, I sonicated my culture and got > a very thick lysate. This material was too thick to be filtered (e.g. > only < 5 ml can go through each 0.45um syringe filter). I tried loading > onto a column without filtering and it clogged the column. You have to watch your lysis conditions here. Routinely I used to take 500 ml culture, spin down, resus in ~ 6 ml PBS, drop onto liquid N2 until frozen (you can break here and store at -80), stick on ice to 'crack', then sonicate ON ICE for either 3 x 30 s or 4 x 20 s with 30 s intervals, using a probe sonicator at (IIRC) 3 - 5 um amplitude. You don't want to over sonicate. Then I'd do a 2 um or 0.8 um filter followed by 0.2 um. Yes, it took some oomph to get the stuff through, but > What is the best way of clearing the DNA in the lysate? we never did this. The pre-filter helped. > I came across > reports that one can use a reagent called "PEI", does anybody know what > this is? Is it good? Some other people use DNAse. Can somebody share > with me their experiences of which method is the best and cleanest? > Thanks. If you must clear DNA, I'd recommend DNase. It's cheap! :-)) R -- Richard P. Grant MA DPhil | rgrant at cmtech.co.uk Senior R&D Scientist | work: www.cmtech.co.uk Cambridge Molecular | home: www.avnet.co.uk/adastra -- +44 1223 508345 - the Number of the Yeast -- From owner-proteins@net.bio.net Wed Nov 04 22:00:00 1998 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!news.gtei.net!newsfeed.direct.ca!peerfeed.ncal.verio.net!newshub1.home.com!news.home.com!news.rdc1.bc.wave.home.com.POSTED!not-for-mail From: "Achim Recktenwald" Newsgroups: bionet.molbio.proteins Subject: JOB: Victoria,BC,Canada: Protein Purification/Process Development/Manufacturing Lines: 44 X-Newsreader: Microsoft Outlook Express 4.72.3155.0 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3155.0 Message-ID: Date: Thu, 05 Nov 1998 19:27:35 GMT NNTP-Posting-Host: 24.64.220.105 NNTP-Posting-Date: Thu, 05 Nov 1998 11:27:35 PDT Organization: @Home Network Canada StressGen Biotechnologies, Corp. 4243 Glanford Avenue Victoria, BC, V8Z 4B9 Canada StressGen Biotechnologies is a growing biopharmaceutical company developing medical applications of the cellular stress response. StressGen’s R&D programs focus on utilizing stress proteins as immunomodulatory agents in therapeutic applications. Research Associate / Senior Research Associate +++++++++++++++++++++++++++++++++++++++ StressGen Biotechnologies, Corp. has an immediate opening for an experienced technically qualified scientist to join our Protein Chemistry Group. The candidate should have a M.Sc. or B.Sc. with 2+ years industrial experience in the purification of biopharmaceutical proteins, preferentially up to pilot-scale. A broad knowledge of column chromatographic methods and equipment (ÄKTA/ FPLC) is essential. In addition, familiarity with standard analytical and electrophoretic methods (PAGE, Western blotting), as well as exposure to fermentation and cell disintegration procedures, is a plus. Please send C.V. c/o: Dr. Achim Recktenwald StressGen Biotechnologies, Corp. 4243 Glanford Avenue Victoria, BC, V8Z 4B9 Canada Fax: (250) 744-2877 Email: ARecktenwald@StressGen.Com We thank all candidates for applying. Only those under consideration will be contacted. Please – no phone calls or drops ins. From owner-proteins@net.bio.net Thu Nov 05 22:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!news-feed.inet.tele.dk!bofh.vszbr.cz!news.daimi.au.dk!not-for-mail From: "Max Søgaard" Newsgroups: bionet.molbio.proteins Subject: Spacer-arm between thrombin site and GST tag? Date: Fri, 06 Nov 1998 11:46:32 +0100 Organization: Inst. of Mol and Struct. Biol. at the university of Aarhus Lines: 43 Message-ID: <3642D388.79CB0E80@mbio.aau.dk> Reply-To: tmms@mbio.aau.dk NNTP-Posting-Host: mbio-30.mbio.aau.dk Mime-Version: 1.0 Content-Type: multipart/mixed; boundary="------------75B4250B151582FAF33D3189" X-Trace: xinwen.daimi.au.dk 910349068 4815 255.255.255.255 (6 Nov 1998 10:44:28 GMT) X-Complaints-To: news@daimi.au.dk NNTP-Posting-Date: 6 Nov 1998 10:44:28 GMT X-Mailer: Mozilla 4.05 [en] (Win95; I) This is a multi-part message in MIME format. --------------75B4250B151582FAF33D3189 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit Dear netters. I am sitting here making plans for the construction of an expressionplasmid based on the pGEX vectors. The GST tag shuld be cut off by thrombin during the purification. I know these things depend strongly on the protein of interest but have anyone has experience with putting in a space arm between the GST-tag and the thrombin site so as to minimize the risk of steric hinderance of the clevage-site. What would be a good primary seq. of a spacer for this purpose? Any input appreciated greatly! Yours T. Max M. Soegaard Inst of mol. and struct. biol. University of Aarhus, Denmark. --------------75B4250B151582FAF33D3189 Content-Type: text/x-vcard; charset=us-ascii; name="vcard.vcf" Content-Transfer-Encoding: 7bit Content-Description: Card for Max Soegaard Content-Disposition: attachment; filename="vcard.vcf" begin: vcard fn: Max Soegaard n: Soegaard;Max org: University of Aarhus, Inst of Mol. and Struct. Biol. adr;dom: C.F. Moellers Alle;;;;;; email;internet: tmms@mbio.aau.dk title: Stud. Scient x-mozilla-cpt: ;0 x-mozilla-html: TRUE version: 2.1 end: vcard --------------75B4250B151582FAF33D3189-- From owner-proteins@net.bio.net Thu Nov 05 22:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!newshub.northeast.verio.net!news-pen-3.sprintlink.net!news-in-east1.sprintlink.net!news.sprintlink.net!wesley.videotron.net!Pollux.Teleglobe.net!orion.cst.tpsa.pl!news.task.gda.pl!news.man.poznan.pl!news.icm.edu.pl!news.nask.pl!news.icmp.lviv.ua!prima.meduniv.lviv.ua!not-for-mail From: drobot@biochem.lviv.ua.for_pete Newsgroups: bionet.molbio.proteins Subject: for russian student Date: 5 Nov 1998 17:30:20 GMT Organization: Your Organization Lines: 5 Message-ID: <71snbc$3em$3@prima.meduniv.lviv.ua> NNTP-Posting-Host: 194.44.178.81 Mime-Version: 1.0 Content-Type: Text/Plain; charset=US-ASCII X-Newsreader: WinVN 0.99.9 (Released Version) (x86 32bit) Who can give my the new article about PI3'-kinase or about others proteins, including in cell signalisation. Thank you From owner-proteins@net.bio.net Thu Nov 05 22:00:00 1998 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!news.gtei.net!cpk-news-hub1.bbnplanet.com!news.news.gtei.net!newspeer.monmouth.com!newsfeed.axxsys.net!newsfeed.nyu.edu!rockyd.rockefeller.edu!not-for-mail Message-ID: <36435B76.2781@rockvax.rockefeller.edu> From: Satish Nair X-Mailer: Mozilla 2.02 (X11; I; IRIX 5.3 IP22) MIME-Version: 1.0 Newsgroups: bionet.molbio.proteins,bionet.molbio.methds-reagents Subject: Re: Protein purification - getting rid of DNA in lysate References: <36416FFD.60D5FF31@mrc-lmb.cam.ac.uk> Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit Lines: 23 Date: Fri, 06 Nov 1998 15:26:30 -0500 NNTP-Posting-Host: 129.85.33.121 X-Trace: rockyd.rockefeller.edu 910384503 129.85.33.121 (Fri, 06 Nov 1998 15:35:03 EDT) NNTP-Posting-Date: Fri, 06 Nov 1998 15:35:03 EDT Organization: The Rockefeller University Yu Wai Chen wrote: > > Dear all, > > > What is the best way of clearing the DNA in the lysate? I came across > reports that one can use a reagent called "PEI", does anybody know > what this is? Is it good? Some other people use DNAse. Can > somebody share with me their experiences of which method is the > best and cleanest? Thanks. > PEI is poly(ethylene)amine which will precipitate DNA (and your protein if it is (a) acidic or (b) binds DNA). It's a nuisance to use as it's average MW is close to 50,000 Da (polymeric) and as it tends to bind to cation exchange columns, you have to find some way to get rid of it. We usually treat lysate with DNase and then add neutralize PEI (use a 1% solution to 1:10 dilution and add very slowly) to precipitate DNA. Following PEI addition, I crash my protein with ammonium sulfate (PEI remains in the supernatant) and resuspend in buffer. From owner-proteins@net.bio.net Thu Nov 05 22:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.concentric.net!newsfeed.direct.ca!btnet-peer!btnet!dispose.news.demon.net!demon!news.demon.co.uk!demon!cammol1.demon.co.uk!see_sig From: see_sig@cmtech.co.delete.uk (Richard P. Grant) Newsgroups: bionet.molbio.proteins,bionet.molbio.methds-reagents Subject: Re: Protein purification - getting rid of DNA in lysate Date: Fri, 06 Nov 1998 08:09:43 +0000 Organization: CMT Approved: My approval does not come from men Message-ID: References: <36416FFD.60D5FF31@mrc-lmb.cam.ac.uk> Reply-To: rgrant@netscape.net NNTP-Posting-Host: cammol1.demon.co.uk X-NNTP-Posting-Host: cammol1.demon.co.uk:212.228.79.177 X-Trace: news.demon.co.uk 910339806 nnrp-03:18524 NO-IDENT cammol1.demon.co.uk:212.228.79.177 X-Complaints-To: abuse@demon.net X-Newsreader: MT-NewsWatcher 2.4.4 X-Face: E%wu[EoEhNnjFG!0[ 7cH@VuxC//iy$!n-+bh9Q`+SHW@&}E.&Ly(y'Tvrd5amG/o35>c3zF6QvXX, rgrant@netscape.net wrote: > In article <36416FFD.60D5FF31@mrc-lmb.cam.ac.uk>, Yu Wai Chen > wrote: > > > Dear all, > > > > As a first step in purifying my protein, I sonicated my culture and got > > a very thick lysate. This material was too thick to be filtered (e.g. > > only < 5 ml can go through each 0.45um syringe filter). I tried loading > > onto a column without filtering and it clogged the column. > > You have to watch your lysis conditions here. Routinely I used to take > 500 ml culture, spin down, resus in ~ 6 ml PBS, drop onto liquid N2 until > frozen (you can break here and store at -80), stick on ice to 'crack', > then sonicate ON ICE for either 3 x 30 s or 4 x 20 s with 30 s intervals, > using a probe sonicator at (IIRC) 3 - 5 um amplitude. You don't want to > over sonicate. Ooops. You need to spin here, quite hard. Use the s/n in following steps. That's probably where the DNA goes - with the membranes and other gunk in the pellet. > Then I'd do a 2 um or 0.8 um filter followed by 0.2 um. Yes, it took some > oomph to get the stuff through, but > -- Richard P. Grant MA DPhil | rgrant at cmtech.co.uk Senior R&D Scientist | work: www.cmtech.co.uk Cambridge Molecular | home: www.avnet.co.uk/adastra -- 'Practising biochemistry without a licence' - E. Chargaff -- From owner-proteins@net.bio.net Fri Nov 06 22:00:00 1998 Path: biosci!263.NET!weijunli From: weijunli@263.NET (weijunli) Newsgroups: bionet.molbio.proteins Subject: MPEG-Protein analysis Date: 7 Nov 1998 05:19:43 -0800 Organization: the State Key Lab. of Biochemical Engineering, CAS,P.R.China Lines: 35 Sender: daemon@net.bio.net Distribution: world Message-ID: <36483CC6.27E317BA@263.net> Reply-To: weijunli@263.net NNTP-Posting-Host: net.bio.net This is a multi-part message in MIME format. --------------083B606795B577EE19E4C350 Content-Type: text/plain; charset=gb2312 Content-Transfer-Encoding: 7bit I want to use MPEG modify the lysines of some protein(such as:hemoglobin,SOD,albumin,Rnase A etc.),and I want to use CE(capillary electrophoresis) analysising the modification drgee and modifiation sites.Could anybody give me some papers about this analysis method? also could someone tell me how to modify lysine through some reagents to get fluescene light to identify the modification degree?I can't find the better reagents. thank for your help! weijun li --------------083B606795B577EE19E4C350 Content-Type: text/x-vcard; charset=gb2312; name="vcard.vcf" Content-Transfer-Encoding: 7bit Content-Description: Card for Weijun Li Content-Disposition: attachment; filename="vcard.vcf" begin: vcard fn: Weijun Li n: Li;Weijun adr;dom: P.O.box 2749-88-14, Beijing ,P.R.China ,100080;;;;;; email;internet: weijunli@263.net x-mozilla-cpt: ;0 x-mozilla-html: FALSE version: 2.1 end: vcard --------------083B606795B577EE19E4C350-- From owner-proteins@net.bio.net Fri Nov 06 22:00:00 1998 Path: biosci!NIC.BMI.AC.CN!wangqm From: wangqm@NIC.BMI.AC.CN (wangqm) Newsgroups: bionet.molbio.proteins Subject: Subject: Spacer-arm between thrombin site and GST tag? Date: 7 Nov 1998 18:14:59 -0800 Organization: amms Lines: 19 Sender: daemon@net.bio.net Distribution: world Message-ID: <3644FDD4.4CFC8465@nic.bmi.ac.cn> NNTP-Posting-Host: net.bio.net I had expressed TPO using pGEX vector.When making the construction, I use aaaaa as a linker and thrombin can cut correctly. But my target is also been cut by thrombin, so I can not purify it. Unluckly, the chimeric protein is so large that it is hard to purify it. Would you please give me some advice. -- Zhang Bo Department of Experimental Hematology Institute of Radiation Medicine 27# Taiping Road Beijing 100850 P.R.China wangqm@nic.bmi.ac.cn From owner-proteins@net.bio.net Fri Nov 06 22:00:00 1998 Path: biosci!FI.UDC.ES!buu98 From: buu98@FI.UDC.ES (Photo Transfer Specialties) Newsgroups: bionet.molbio.proteins Subject: Photo Mousepads..... Great Gift Idea! Date: 7 Nov 1998 06:11:44 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 154 Sender: daemon@net.bio.net Distribution: world Message-ID: <199811032350MAA33181@Photomousepadgift.de> NNTP-Posting-Host: net.bio.net GREAT CHRISTMAS GIFT IDEA ! PHOTO TRANSFER SPECIALTIES MOUSEPADS WITH YOUR FAVORITE PHOTOS! Now you can have your favorite photos printed onto mousepads! Photo mousepads are our most popular gift year after year! In the past we have provided our HIGH QUALITY mousepads to thousands of customers. Most are so pleased they reorder each year! Photo mousepads make a PERFECT GIFT for ANYONE! Replace boring, dirty, frayed mousepads! Instead of a plain, generic or boring corporate logo you can have PERSONALIZED photo mousepads brighten your desktop! PHOTO Mousepads are DAILY REMINDERS of your favorite: CHILDREN, PETS, EVENTS....... WEDDING PHOTOS, FAMILY, GRADUATION..... LOVED ONES,HOLIDAY PHOTOS,GRANDCHILDREN..... Anything you choose! 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SA/ From owner-proteins@net.bio.net Fri Nov 06 22:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!news.msfc.nasa.gov!bcm.tmc.edu!news From: Simon Hoffenberg Newsgroups: bionet.molbio.proteins Subject: Silent exons Date: Sat, 07 Nov 1998 21:59:18 -0600 Organization: Baylor College of Medicine, Houston, Tx Lines: 8 Message-ID: <36451715.873F3524@bcm.tmc.edu> NNTP-Posting-Host: 128.249.70.57 Mime-Version: 1.0 Content-Type: text/plain; charset=koi8-r Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.5 [en] (Win95; I) X-Accept-Language: ru Dear netters, Is there such thing as silent exons, i.e. present in cDNA but not expressed? If the answer is yes, how can one identify them? Simon Hoffenberg Baylor College of Medicine From owner-proteins@net.bio.net Sat Nov 07 22:00:00 1998 Path: biosci!AOL.COM!Cutegirl21 From: Cutegirl21@AOL.COM Newsgroups: bionet.molbio.proteins Subject: WANT A NEW CELL PHONE? YOU'RE APPROVED! 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CLICK HERE NOW --part0_910533626_boundary-- From owner-proteins@net.bio.net Sun Nov 08 22:00:00 1998 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!news.gtei.net!cpk-news-hub1.bbnplanet.com!news.news.gtei.net!rill.news.pipex.net!pipex!server1.netnews.ja.net!bham!med180.bham.ac.uk!user From: noone@cancer.bham.ac.uk (noone) Newsgroups: bionet.molbio.proteins,bionet.molbio.methds-reagents Subject: Re: Protein purification - getting rid of DNA in lysate Date: Thu, 05 Nov 1998 15:38:37 +0100 Organization: noone Lines: 19 Message-ID: References: <36416FFD.60D5FF31@mrc-lmb.cam.ac.uk> NNTP-Posting-Host: med180.bham.ac.uk In article , rgrant@netscape.net wrote: > In article <36416FFD.60D5FF31@mrc-lmb.cam.ac.uk>, Yu Wai Chen > wrote: > > > Dear all, > > > > As a first step in purifying my protein, I sonicated my culture and got > > a very thick lysate. This material was too thick to be filtered (e.g. > > only < 5 ml can go through each 0.45um syringe filter). I tried loading > > onto a column without filtering and it clogged the column. You could try spinning your sample in an ultracentrifuge at 100.000g for about 30 min. This usually gets rid of microsomes, DNA etc. when the density of the buffer is not too high. With DNAse you better make shure that it's protease-free. Hope this helps, Peter From owner-proteins@net.bio.net Sun Nov 08 22:00:00 1998 Newsgroups: bionet.molbio.proteins,bionet.molbio.methds-reagents Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!news.gtei.net!newsfeed.corridex.com!news.maxwell.syr.edu!news.new-york.net!newsfeed.wirehub.nl!baron.netcom.net.uk!netcom.net.uk!server3.netnews.ja.net!ucl.ac.uk!bcc.ac.uk!chen From: chen@bsm.bioc.ucl.ac.uk (Chen Ho An) Subject: Re: Protein purification - getting rid of DNA in lysate Message-ID: <1998Nov9.194707.77162@ucl.ac.uk> Date: Mon, 9 Nov 1998 19:47:07 GMT References: <36416FFD.60D5FF31@mrc-lmb.cam.ac.uk> <36435B76.2781@rockvax.rockefeller.edu> Organization: University College London X-Newsreader: TIN [version 1.2 PL2] Followup-To: bionet.molbio.proteins,bionet.molbio.methds-reagents Lines: 21 Satish Nair (nairs@rockvax.rockefeller.edu) wrote: : Yu Wai Chen wrote: : > : > What is the best way of clearing the DNA in the lysate? I came across : > reports that one can use a reagent called "PEI", does anybody know : > what this is? Is it good? Some other people use DNAse. Can : > somebody share with me their experiences of which method is the : > best and cleanest? Thanks. : PEI is poly(ethylene)amine which will precipitate DNA (and your : protein if it is (a) acidic or (b) binds DNA). It's a nuisance to : use as it's average MW is close to 50,000 Da (polymeric) and as it : tends to bind to cation exchange columns, you have to find some way : to get rid of it. Other methods of removing DNA are using streptomycin (ref:Jorin et al, JBC, 244,2996)) and protamine sulphate (a DNA binding protein). Protamine sulphate is dissolved in neutral pH buffer (10 mg/ml), and added dropwise to cell lysate. Nucleic acid can then be removed by centrifugation. See the Scopes book on protein purification for more details. From owner-proteins@net.bio.net Sun Nov 08 22:00:00 1998 Path: biosci!AUG.UKL.UNI-FREIBURG.DE!kulmburg From: kulmburg@AUG.UKL.UNI-FREIBURG.DE (Peter Kulmburg) Newsgroups: bionet.molbio.proteins Subject: Protein domain that helps importing large proteins into cytoplasm Date: 9 Nov 1998 07:09:20 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 24 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Dear collegues, I would like to introduce GFP (the whole protein and not just a short peptide) into the cytoplasm of murine or human cells. I can direct the protein to a specific receptor on the cell surface by fusing an scFv to GFP but then, the protein is degraded in the endosomes. Are there peptides or protein domains known to help endosomal escape when fused to another protein? There is a lot of information about translocation domains in immunotoxins, however, fusion proteins containing those fragments are difficult to produce, highly immunogenic (although that is not so important, since GFP is immunogenic, too). Do you know about a peptide that just helps escaping a fusion protein from the endosome degradation? Human proteins preferred :-) Please send any answers to my private e-mail address since I do not adhere to your list. Thank you for your help in advance! Sincerely yours Peter Kulmburg Peter KULMBURG at home Tel./Fax:+33 3 89.68.28.83 at work: Tel.: +49 761 270/7208 or 7207 Fax: /7217 From owner-proteins@net.bio.net Mon Nov 09 22:00:00 1998 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!news.gtei.net!newsfeed.wli.net!howland.erols.net!news-peer.gip.net!news-lond.gip.net!news.gsl.net!gip.net!nntp.news.xara.net!xara.net!server5.netnews.ja.net!daresbury!not-for-mail From: Luc CAMOIN Newsgroups: bionet.molbio.proteins Subject: Blocked N-terminal residues Date: 10 Nov 1998 15:49:20 -0000 Organization: Daresbury Laboratory, Warrington, U.K. Lines: 48 Message-ID: <729na0$apq$1@mserv2.dl.ac.uk> NNTP-Posting-Host: mserv2.dl.ac.uk Original-To: proteins@dl.ac.uk content-length: 1089 Return-Path: X-MIME-Autoconverted: from 8bit to quoted-printable by mserv1.dl.ac.uk id PAA11879 Many natives proteins have blocked N-terminal residues, commonly through acetylation, formylation, or cyclization of N-terminal glutamyl residues. The presence of these groups blocked the Edman degradation. I wonder, if in the case of a recombinant protein with a modified N-terminal by adding an His tag this kind of blockage is always possible. This construct was already used for different recombinant proteins in our laboratory and never such blocage was identified.=20 He think, It is very unlikely to have a blocage of the N-terminal for a recombinant protein. Do you get already a blocked recombinant protein? Has anybody identified already a blocked residues for a N-terminal tagged protein? Thanks in advance. Luc CAMOIN Institut Cochin de G=E9n=E9tique Mol=E9culaire / Groupe Chimie des Prot=E9= ines Laboratoire d'Immuno-Pharmacologie Mol=E9culaire / CNRS UPR 415 =20 22 rue Mechain 75014 Paris France Luc CAMOIN Tel:+33 1 40 51 64 98 Fax:+33 1 40 51 72 10 =09 Internet: http://www.cochin.inserm.fr/upr415/UPR415E2.htm Intranet: http://193.52.76.98 From owner-proteins@net.bio.net Mon Nov 09 22:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!news.maxwell.syr.edu!news-ge.switch.ch!news-zh.switch.ch!news.belwue.de!news.uni-freiburg.de!sun2.ruf.uni-freiburg.de!roth From: roth@sun2.ruf.uni-freiburg.de (Sabine Roth) Newsgroups: bionet.molbio.proteins Subject: Isolating peptides Date: 10 Nov 1998 23:14:20 GMT Organization: Rechenzentrum der Universitaet Freiburg, Germany Lines: 16 Message-ID: <72ahcc$5uk$1@n.ruf.uni-freiburg.de> NNTP-Posting-Host: sun2.ruf.uni-freiburg.de X-Newsreader: TIN [version 1.2 PL2] -- _______________________________________________________________________________ Sabine Roth Clinical Research Unit for Rheumatology University Freiburg, Medical Center Breisacher Str. 64 79106 Freiburg Germany From owner-proteins@net.bio.net Mon Nov 09 22:00:00 1998 Message-ID: <36488496.8A87760D@ibm.net> Date: Tue, 10 Nov 1998 14:23:18 -0400 From: lenkalvert@ibm.net Reply-To: lenkalvert@ibm.net X-Mailer: Mozilla 4.04 [en] (Win95; I) MIME-Version: 1.0 Newsgroups: bionet.molbio.proteins Subject: Position Available - Group Leader Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit NNTP-Posting-Host: 32.100.250.73 X-Trace: 10 Nov 1998 19:06:47 GMT, 32.100.250.73 Organization: IBM.NET Lines: 28 X-Notice: Items posted that violate the IBM.NET Acceptable Use Policy X-Notice: should be reported to postmaster@ibm.net X-Complaints-To: postmaster@ibm.net Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!news-stock.gip.net!news-peer.gip.net!news.gsl.net!gip.net!newsm2.ibm.net!ibm.net!news1.ibm.net!32.100.250.73 Kalvert Personnel Service, Inc. is a professional recruitment firm specializing in the Biotechnology, Pharmaceutical and Diagnostics industries. Our extensive contacts, knowledge and understanding of these industries offer candidates a wide spectrum of career opportunities. We represent a biotechnology company seeking a Group Leader - Expression Systems. Research: Gene Therapy, recombinant expression in eukaryotic systems, bacterial genetics. Ph.D in Molecular Biology or Bacterial Genetics. Recognized expertise and publications in expression systems is essential. The company is discovering and developing drugs which modulate the immune system. Very copetitive compensation. ALL FEES AND EXPENSES ARE PAID BY THE COMPANY. Leonard Kalvert President Kalvert Personnel Service, Inc. (est. 1966) P.O. Box 1579 Stony Brook, NY 11790 (516) 331-3388 (tel) (516) 331-1886 (fax) kalvert@ibm.net (e-mail) From owner-proteins@net.bio.net Tue Nov 10 22:00:00 1998 Path: biosci!AOL.COM!NIEMAN60 From: NIEMAN60@AOL.COM Newsgroups: bionet.molbio.proteins Subject: Photo Mousepads...........Great Gifts ! Date: 11 Nov 1998 00:03:29 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 160 Sender: daemon@net.bio.net Distribution: world Message-ID: <1f67be5f.36492e78@aol.com> NNTP-Posting-Host: net.bio.net GREAT CHRISTMAS GIFT IDEA! PHOTO TRANSFER SPECIALTIES! MOUSEPADS WITH YOUR FAVORITE PHOTOS! Now you can have your favorite photos printed onto mousepads! Photo mousepads are our most popular gift year after year! In the past we have provided our HIGH QUALITY mousepads to thousands of customers. Most are so pleased they reorder each year! Photo mousepads make a PERFECT GIFT for ANYONE! Replace boring, dirty, frayed mousepads! Instead of plain, generic, boring corporate logos you can have PERSONALIZED photo mousepads brighten your desktop! PHOTO Mousepads are DAILY REMINDERS of your favorite: CHILDREN, PETS, EVENTS....... WEDDING PHOTOS, FAMILY, GRADUATION..... LOVED ONES,HOLIDAY PHOTOS,GRANDCHILDREN..... Anything you choose! Use your imagination! 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From owner-proteins@net.bio.net Tue Nov 10 22:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!howland.erols.net!rill.news.pipex.net!pipex!wuff.mayn.de!news-nue1.dfn.de!news-lei1.dfn.de!news-ber1.dfn.de!news-ham1.dfn.de!news.mu-luebeck.de!not-for-mail From: "Lars Komorowski" Newsgroups: bionet.molbio.proteins Subject: Native gel with starch Date: Wed, 11 Nov 1998 14:05:32 +0100 Organization: Med. Universitaet zu Luebeck Lines: 7 Message-ID: <72c2c2$8hr$1@gwsun.medinf.mu-luebeck.de> NNTP-Posting-Host: 141.83.167.171 X-Newsreader: Microsoft Outlook Express 4.72.3110.5 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3110.3 Who can give me advise and a good recipe for a native PAA-gel for proteins. In addition I would like to add starch to the gel to test for alpha-amylase-activity (maybe 2D). Can give someone recommendations about the amount of starch necessary for iodine-coloring ? Lars From owner-proteins@net.bio.net Tue Nov 10 22:00:00 1998 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.gtei.net!howland.erols.net!rill.news.pipex.net!pipex!server1.netnews.ja.net!server5.netnews.ja.net!daresbury!not-for-mail From: NIEMAN60@aol.com Newsgroups: bionet.molbio.proteins Subject: Photo Mousepads...........Great Gifts ! Date: 11 Nov 1998 07:58:55 -0000 Organization: Daresbury Laboratory, Warrington, U.K. Lines: 160 Message-ID: <72bg3v$g84$1@mserv2.dl.ac.uk> NNTP-Posting-Host: mserv2.dl.ac.uk content-length: 3698 Return-Path: Apparently-To: GREAT CHRISTMAS GIFT IDEA! PHOTO TRANSFER SPECIALTIES! MOUSEPADS WITH YOUR FAVORITE PHOTOS! Now you can have your favorite photos printed onto mousepads! Photo mousepads are our most popular gift year after year! In the past we have provided our HIGH QUALITY mousepads to thousands of customers. Most are so pleased they reorder each year! 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From owner-proteins@net.bio.net Tue Nov 10 22:00:00 1998 Path: biosci!AOL.COM!BEEASYE From: BEEASYE@AOL.COM Newsgroups: bionet.molbio.proteins Subject: HEAVY DUTY SEWING MACHINE SALE !! Date: 11 Nov 1998 13:18:36 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 101 Sender: daemon@net.bio.net Distribution: world Message-ID: <9b4ae815.3649f7d0@aol.com> NNTP-Posting-Host: net.bio.net This is a multi-part message in MIME format. --part0_910817234_boundary Content-ID: <0_910817234@inet_out.mail.aol.com.1> Content-type: text/plain; charset=US-ASCII --part0_910817234_boundary Content-ID: <0_910817234@inet_out.mail.aol.com.2> Content-type: message/rfc822 Content-transfer-encoding: 7bit Content-disposition: inline From: BEEASYE@aol.com Return-path: To: BEEASYE@aol.com Subject: f Date: Wed, 11 Nov 1998 15:38:41 EST Mime-Version: 1.0 Content-type: text/plain; charset=US-ASCII Content-transfer-encoding: 7bit This is a 1 Time Mailing ** PUBLIC SALE !! ** UNSOLD ORDERS OF SEW AND SERGE SEWING MACHINES _________________________________________________________________ *** To view machine click below! *** or visit: http://209.149.255.49 The Necchi Company produced a large quantity of their 1998 Sew and Serge Sewing Machines, anticipating increased sales. But due to the current market conditions, these orders remain unsold. THESE MACHINES MUST BE SOLD! These heavy duty machines are made of metal, with metal gears. They have been prepared for sale to schools and cottage industries nationally. The manufacturer warrants these machines for 5 years against parts failure. WHY PAY THOUSANDS OF $ FOR A SERGER AND SEWING MACHINE WHEN A SEW AND SERGE SEWING MACHINE DOES IT ALL! _________________________________________________________________ Today Necchi offers a machine that performs all of your sewing tasks! ** 1st: As a sophisticated sewing machine, it performs: Buttonhole (any size), Stretch, Invisible Blind hems, Monograms, Quilting, Elastic Stitch, Top Stitching Applique, and Decorative sewing. Turn the dial and sew magic! ** 2nd : The Built-In Professional Serging stitch: allows you to sew super strong seams and over edge the material in one step...Our optional cutter trims as you sew. ** 3rd: No more complicated threading and you don't have to tie off seams like you do with a serger! They handle all fabrics with ease. They even sew on leather and vinyl. _________________________________________________________________ ALL MACHINES ARE CABINET READY IN FACTORY SEALED CARTONS. Your price when mentioning this ad. ** $198 ** _________________________________________________________________ LIMITED STOCK! CALL NOW WHILE SUPPLIES LAST! ** 1-800-992-4(SEW) 739 ** Phone orders accepted with major credit cards To view machine and or place your order click below! or visit: http://209.149.255.49 --part0_910817234_boundary-- From owner-proteins@net.bio.net Tue Nov 10 22:00:00 1998 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!news.gtei.net!newsfeed.corridex.com!newsfeed.cwix.com!194.162.162.196!newsfeed.nacamar.de!nntp.news.xara.net!xara.net!server6.netnews.ja.net!daresbury!not-for-mail From: BEEASYE@aol.com Newsgroups: bionet.molbio.proteins Subject: HEAVY DUTY SEWING MACHINE SALE !! Date: 11 Nov 1998 21:14:31 -0000 Organization: Daresbury Laboratory, Warrington, U.K. Lines: 101 Message-ID: <72cunn$na3$1@mserv2.dl.ac.uk> NNTP-Posting-Host: mserv2.dl.ac.uk content-length: 2638 Return-Path: Apparently-To: This is a multi-part message in MIME format. --part0_910817234_boundary Content-ID: <0_910817234@inet_out.mail.aol.com.1> Content-type: text/plain; charset=US-ASCII --part0_910817234_boundary Content-ID: <0_910817234@inet_out.mail.aol.com.2> Content-type: message/rfc822 Content-transfer-encoding: 7bit Content-disposition: inline From: BEEASYE@aol.com Return-path: To: BEEASYE@aol.com Subject: f Date: Wed, 11 Nov 1998 15:38:41 EST Mime-Version: 1.0 Content-type: text/plain; charset=US-ASCII Content-transfer-encoding: 7bit This is a 1 Time Mailing ** PUBLIC SALE !! ** UNSOLD ORDERS OF SEW AND SERGE SEWING MACHINES _________________________________________________________________ *** To view machine click below! *** or visit: http://209.149.255.49 The Necchi Company produced a large quantity of their 1998 Sew and Serge Sewing Machines, anticipating increased sales. But due to the current market conditions, these orders remain unsold. THESE MACHINES MUST BE SOLD! These heavy duty machines are made of metal, with metal gears. They have been prepared for sale to schools and cottage industries nationally. The manufacturer warrants these machines for 5 years against parts failure. WHY PAY THOUSANDS OF $ FOR A SERGER AND SEWING MACHINE WHEN A SEW AND SERGE SEWING MACHINE DOES IT ALL! _________________________________________________________________ Today Necchi offers a machine that performs all of your sewing tasks! ** 1st: As a sophisticated sewing machine, it performs: Buttonhole (any size), Stretch, Invisible Blind hems, Monograms, Quilting, Elastic Stitch, Top Stitching Applique, and Decorative sewing. Turn the dial and sew magic! ** 2nd : The Built-In Professional Serging stitch: allows you to sew super strong seams and over edge the material in one step...Our optional cutter trims as you sew. ** 3rd: No more complicated threading and you don't have to tie off seams like you do with a serger! They handle all fabrics with ease. They even sew on leather and vinyl. _________________________________________________________________ ALL MACHINES ARE CABINET READY IN FACTORY SEALED CARTONS. Your price when mentioning this ad. ** $198 ** _________________________________________________________________ LIMITED STOCK! CALL NOW WHILE SUPPLIES LAST! ** 1-800-992-4(SEW) 739 ** Phone orders accepted with major credit cards To view machine and or place your order click below! or visit: http://209.149.255.49 --part0_910817234_boundary-- From owner-proteins@net.bio.net Tue Nov 10 22:00:00 1998 Path: biosci!butler.edu!rkarn From: rkarn@butler.edu ("Robert Karn") Newsgroups: bionet.molbio.proteins Subject: Re: Native gel with starch Date: 11 Nov 1998 06:32:20 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 38 Sender: daemon@net.bio.net Distribution: world Message-ID: References: <72c2c2$8hr$1@gwsun.medinf.mu-luebeck.de> NNTP-Posting-Host: net.bio.net We published several recipes in: Taggart et al., 1978. Vertical thin-layer slab polyacrylamide gel electrophoresis of selected human polymorphic proteins. Electrophoresis '78 (Catsimpoolas, N., ed.) pp. 231-242, Elsevier North Holland, Inc. In applying polyacrylamide electrophoresis to analyzing amylase, we soaked the gel, following electrophoresis, in a starch solution and then stained with iodine. See: Karn et al., 1973. Evidence for posttranscriptional modification of human salivary amylase (Amy1) isozymes. Biochem. Genet. 10:341-350. If reviews on the subject of amylase would be useful, see: Merritt and Karn, 1977. The human alpha-amylases. In: Advances in Human Genetics (Harris and Hirschhorn, eds.) vol. 8, pp. 135-234. and: Karn and Malacinski, 1978. The comparative biochemistry, physiology and genetics of animal alpha-amylases. In: Adv. Comp. Biochem. Physiol. 7:1-103. Cheers, Bob Karn On Wed, 11 Nov 1998, Lars Komorowski wrote: > Who can give me advise and a good recipe for a native PAA-gel for proteins. > In addition I would like to add starch to the gel to test for > alpha-amylase-activity (maybe 2D). Can give someone recommendations about > the amount of starch necessary for iodine-coloring ? > Lars > > > > From owner-proteins@net.bio.net Tue Nov 10 22:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!netnews.com!newspeer.monmouth.com!newsin.agis.net!agis!news.inter.net.il!not-for-mail From: "Barak Akabayov" Newsgroups: bionet.molbio.proteins Subject: TATA Modulatoy Factor Date: Wed, 11 Nov 1998 14:10:03 +0200 Organization: Internet Gold Lines: 11 Message-ID: <72d16n$6si$1@news.inter.net.il> NNTP-Posting-Host: r-h-186-116.access.net.il X-Newsreader: Microsoft Outlook Express 4.72.3110.37 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3110.37 Hi My name is Barak, a 3rd year student to biochemistry in Bar-Ilan University in Israel. As a part of my project in the advence laboratory I need to know sequential motifs information about TMF. Any data about this protein and its sequence will be helpful. Thank you Barak From owner-proteins@net.bio.net Tue Nov 10 22:00:00 1998 Path: biosci!UMBI.UMD.EDU!collins From: collins@UMBI.UMD.EDU (john collins) Newsgroups: bionet.molbio.proteins Subject: Position available Date: 11 Nov 1998 15:30:16 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 21 Sender: daemon@net.bio.net Distribution: world Message-ID: <3.0.3.32.19981111182552.007a95b0@umbi.umd.edu> NNTP-Posting-Host: net.bio.net Senior Research Associate, non-tenure track. Requires (1) Ph.D. in biological science, (2) at least ten years of postdoctoral research, including three or more years at the level of Research Associate, in biochemical studies on the molecular structure and interactions of muscle proteins, (3) fifteen or more publications in the field, and (4) extensive expertise in protein sequencing, chemical modification, HPLC, site-directed mutagenesis and fluorescence spectroscopy. The successful applicant will carry out independent research or collaborate in group research at the advanced level, and train others (such as technical staff, graduate students, postdocs and other Research Associates) within the laboratory environment. Salary: $36,000-$39,000 per year. Position available upon completion of search. Send letter of application and curriculum vitae by e-mail to collins@umbi.umd.edu or to John H. Collins, Ph.D., Medical Biotechnology Center, University of Maryland Biotechnology Institute, 725 W. Lombard Street, Baltimore, MD 21201, USA, no later than December 15, 1998. E-mail applications are preferred. The University of Maryland Biotechnology Institute is an Equal Opportunity/Affirmative Action Employer. Women, minorities, veterans, and candidates with disabilities are encouraged to apply. From owner-proteins@net.bio.net Wed Nov 11 22:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!btnet-peer!btnet!nntp.news.xara.net!xara.net!server6.netnews.ja.net!daresbury!not-for-mail From: Cormac Shaw Newsgroups: bionet.molbio.proteins Subject: Re: Native gel with starch Date: 12 Nov 1998 17:48:59 -0000 Organization: University College Dublin Lines: 32 Message-ID: <72f72b$fem$1@mserv2.dl.ac.uk> Reply-To: cormac.shaw@ucd.ie NNTP-Posting-Host: mserv2.dl.ac.uk Original-To: Lars Komorowski , PROTEINS@DL.AC.UK content-length: 1600 Return-Path: On 11 Nov 98 at 14:05, Lars Komorowski wrote: >Who can give me advise and a good recipe for a native PAA-gel for proteins. >In addition I would like to add starch to the gel to test for >alpha-amylase-activity (maybe 2D). Can give someone recommendations about >the amount of starch necessary for iodine-coloring ? >Lars Hello Lars, I ran native PAGE gels (a la Weber & Osburn) containing 0.4% (w/v) soluble starch with very satisfactory results. The gels were somewhat firmer to the touch but proteins ran to the same distance in more or less the same time as gels with no starch. After electrophoresis, the gels were immersed in acetate buffer, pH 5 and popped in an incubator for half an hour. I think the key to getting as discrete a clear band as possible is to minimise the time of incubation after electrophoresis so that diffusssion does not occur. If I come across the paper that gave me the 0.4% figure I'll pass it on. Good Luck. Cormac. ________________________________________________________________________ Cormac Shaw, BSc, | Dept. of Industrial Microbiology, | mailto:Cormac.Shaw@ucd.ie University College Dublin, | phone: +353 (1) 706 1307 Dublin 4, Ireland | fax: +353 (1) 706 1183 ------------------------------------------------------------------------ "Under the most rigorously controlled conditions of pressure, temperature, volume, humidity, nutrients and other variables, the organism will do as it pleases." ________________________________________________________________________ From owner-proteins@net.bio.net Wed Nov 11 22:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!dca1-hub1.news.digex.net!digex!news-feed.fnsi.net!hammer.uoregon.edu!leto.ou.edu!news.ecn.ou.edu!news1.optus.net.au!optus!news.uwa.edu.au!mbi-mac-wendy.ichr.uwa.edu.au!user From: wendy@ichr.uwa.edu.au (Wendy-Anne Smith) Newsgroups: bionet.molbio.proteins Subject: His tag expressed proteins aggregates Date: Fri, 13 Nov 1998 12:52:08 +0800 Organization: TVW Telethon Institute for Child Health Lines: 41 Message-ID: Reply-To: wendy@ichr.uwa.edu.au NNTP-Posting-Host: mbi-mac-wendy.ichr.uwa.edu.au Hi, We have expressed a house dust mite trypsin protein using the pET-11d vector in BL21 (DE3)pLysS. We purify the 2mg/ml recombinant protein using the Qiagen Ni-NTA denaturing protocol getting nice yields of protein using buffer E which contains urea at a conc. of 8M. This is where we start to have problems. If we try to dialyse the protein into PBS, it appears that all the protein sticks to the dialysis tubing (it doesn't degrade.). So we thought about desalting the protein and improving the purity by passing it over a Sephadex 25 column but with filtration (Millipore filter) prior to loading on the column. The first time we did this we tried to filter 2-3 mg and lost it all. By adding 0.01% Tween 20 to the protein prior to filtration we were able to retain all our sample suggesting that we had broken down all the aggregates allowing filtration to occur. So it would appear that our recombinant protein is very sticky but we want to use the material in tissue culture and so don't want to use a protein prep with Tween20 in it. Now my boss says that it is sticky only because it has not been refolded properly and that if we refolded it prior to eluting off the column we would solve all our problems. I have seen briefly a paper which talked about refolding the protein while it was bound to the column. My boss is keen for me to try that but unfortunately we have misplaced the paper. So my questions are: 1) Has anyone read a paper where they described refolding a His tag protein whilst bound to a Nickel column & 2) What methods have people used to refold His tag proteins isolated from insoluble inclusion bodies which has given them a protein similar to, in activity, to it's native counterpart? Thanks -- Dr. Wendy-Anne Smith TVW Telethon Institute for Child Health Research (Company Limited by Guarantee) Western Australia Fax 61 9 388 3414 From owner-proteins@net.bio.net Wed Nov 11 22:00:00 1998 Path: biosci!ihnp4.ucsd.edu!not-for-mail From: "Antonin Tutter" Newsgroups: bionet.molbio.proteins Subject: Re: His tag expressed proteins aggregates Date: Thu, 12 Nov 1998 22:27:39 -0800 Organization: University of California, San Deigo Lines: 52 Message-ID: <910938440.958630@news1.ucsd.edu> References: NNTP-Posting-Host: news1.ucsd.edu Mime-Version: 1.0 Content-Type: text/plain; charset="US-ASCII" Content-Transfer-Encoding: 7bit X-Trace: ihnp4.ucsd.edu 910938519 23357 132.239.1.221 (13 Nov 1998 06:28:39 GMT) X-Complaints-To: Abuse@UCSD.Edu NNTP-Posting-Date: 13 Nov 1998 06:28:39 GMT >We have expressed a house dust mite trypsin protein using the pET-11d >vector in BL21 (DE3)pLysS. We purify the 2mg/ml recombinant protein using >the Qiagen Ni-NTA denaturing protocol getting nice yields of protein using >buffer E which contains urea at a conc. of 8M. This is where we start to >have problems. If we try to dialyse the protein into PBS, it appears that >all the protein sticks to the dialysis tubing (it doesn't degrade.). So >we thought about desalting the protein and improving the purity by passing >it over a Sephadex 25 column but with filtration (Millipore filter) prior >to loading on the column. The first time we did this we tried to filter >2-3 mg and lost it all. By adding 0.01% Tween 20 to the protein prior to >filtration we were able to retain all our sample suggesting that we had >broken down all the aggregates allowing filtration to occur. So it would >appear that our recombinant protein is very sticky but we want to use the >material in tissue culture and so don't want to use a protein prep with >Tween20 in it. Now my boss says that it is sticky only because it has not >been refolded properly and that if we refolded it prior to eluting off the >column we would solve all our problems. I have seen briefly a paper which >talked about refolding the protein while it was bound to the column. My >boss is keen for me to try that but unfortunately we have misplaced the >paper. > >So my questions are: >1) Has anyone read a paper where they described refolding a His tag >protein whilst bound to a Nickel column & > >2) What methods have people used to refold His tag proteins isolated from >insoluble inclusion bodies which has given them a protein similar to, in >activity, to it's native counterpart? The aggregation may not be due to some intrinsic quality of your protein, but rather due to the His tag itself. It is possible to refold the protein on the column or off, but you didn't mention whether you dialysed sequentially, or whether you are eluting by pH or by competition with imidazole. Basically you want the protein to refold gradually, so after the final elution dialyse against 6M urea, then 4M, 2M, 1M, 0.5M, and 0.0M. Include Tween during these, but for the final dialysis in 0M Urea, leave out the tween. Also starting with the 4M step, make sure to maintain ionic character, say 100mM NaCl, and pH7.9. Also keep EDTA in the buffers too...trace Ni++ can cause aggregation via the tags. Good luck, _______________________________________ Antonin Tutter Salk Institute for Biological Studies RBIO-J 10010 N. Torrey Pines Rd. La Jolla, CA 92037 email: atutter@aim.salk.edu web: http://www-biology.ucsd.edu/~atutter/ From owner-proteins@net.bio.net Wed Nov 11 22:00:00 1998 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.gtei.net!newshub.northeast.verio.net!btnet-peer!btnet!newscore.univie.ac.at!aconews.univie.ac.at!mail.boku.ac.at!news From: Steinkellner Herta Newsgroups: bionet.molbio.proteins Subject: His tag in plants Date: Thu, 12 Nov 1998 11:04:08 +0100 Organization: BOKU WIEN Lines: 4 Message-ID: <364AB298.1566@edv2.boku.ac.at> NNTP-Posting-Host: 141.244.38.118 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0 (Win95; I) Dear scientists has anybody experience about using the His tag in plants. Can I use both codons (UGC and UGU) for His expression in plants? From owner-proteins@net.bio.net Wed Nov 11 22:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!sunqbc.risq.qc.ca!wesley.videotron.net!Pollux.Teleglobe.net!server-b.cs.interbusiness.it!not-for-mail From: Dimasi Nazzareno Newsgroups: bionet.molbio.proteins Subject: Re: help(supersecondary prediction) Date: Thu, 12 Nov 1998 16:55:57 +0100 Organization: IRBM P. Angeletti S.p.A. Lines: 36 Message-ID: <364B050D.A0C359F@irbm.it> References: <364AF14B.8B8C5673@nic.bmi.ac.cn> NNTP-Posting-Host: 195.103.47.194 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.5 [en] (Win95; I) X-Accept-Language: en Try: http://www.ebi.ac.uk/queries/queries.html wangqm wrote: > Hi,everybody > Can you inform me a web site where I can predict the supersecondary > structure of my protein? > Thanks a lot. > > -- > Zhang Bo > Department of Experimental Hematology > Institute of Radiation Medicine > 27# Taiping Road > Beijing 100850 > P.R.China > wangqm@nic.bmi.ac.cn -- +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ NAZZARENO DIMASI, PhD Student Department of Protein Engineering and Biocrystallography IRBM "P. Angeletti" Via Pontina Km 30,600 00040 Pomezia (Rome) Italy ------------------------------------------------------------- Phone: (int+39)-06-91093415 Email: Dimasi@irbm.it Fax: (int+39)-06-91093225 URL: http://www.irbm.it ------------------------------------------------------------- Personal Home Page: http://www.geocities.com/TimesSquare/Stadium/1847/ +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ From owner-proteins@net.bio.net Wed Nov 11 22:00:00 1998 Path: biosci!NIC.BMI.AC.CN!wangqm From: wangqm@NIC.BMI.AC.CN (wangqm) Newsgroups: bionet.molbio.proteins Subject: help(supersecondary prediction) Date: 12 Nov 1998 06:35:03 -0800 Organization: amms Lines: 15 Sender: daemon@net.bio.net Distribution: world Message-ID: <364AF14B.8B8C5673@nic.bmi.ac.cn> NNTP-Posting-Host: net.bio.net Hi,everybody Can you inform me a web site where I can predict the supersecondary structure of my protein? Thanks a lot. -- Zhang Bo Department of Experimental Hematology Institute of Radiation Medicine 27# Taiping Road Beijing 100850 P.R.China wangqm@nic.bmi.ac.cn From owner-proteins@net.bio.net Wed Nov 11 22:00:00 1998 Path: biosci!MAILBOX.UQ.EDU.AU!cfarah From: cfarah@MAILBOX.UQ.EDU.AU (Camile S Farah) Newsgroups: bionet.molbio.proteins Subject: histidine marker for CATIONIC PAGE Date: 12 Nov 1998 19:46:02 -0800 Organization: Oral Biology and Pathology, School of Dentistry, UQ Lines: 11 Sender: daemon@net.bio.net Distribution: world Message-ID: <364BAA87.1FB5@mailbox.uq.edu.au> Reply-To: cfarah@mailbox.uq.edu.au NNTP-Posting-Host: net.bio.net Can u help i am looking for a marker that will run on cationic page gels using acrylamide and acetic acid/KOH. histidines are postively charged low mol. weight proteins (3-5kd) thanks C Farah Oral Biology and Pathology Email: c.farah@mailbox.uq.edu.au From owner-proteins@net.bio.net Wed Nov 11 22:00:00 1998 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!news.gtei.net!newsfeed.corridex.com!news.maxwell.syr.edu!bignews.mediaways.net!fu-berlin.de!news.uni-leipzig.de!news.uni-halle.de!not-for-mail From: Georg Wille Newsgroups: bionet.molbio.proteins,bionet.molbio.methds-reagnts Subject: FAD in culture medium? Date: Thu, 12 Nov 1998 21:17:55 +0100 Organization: University Halle, Germany Lines: 26 Message-ID: <364B4273.3E0F@mlucom6.urz.uni-halle.de> Reply-To: mocutoutqch@mlucom6.urz.uni-halle.de NNTP-Posting-Host: dial-25.urz.uni-halle.de Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.02 (OS/2; I) Xref: biosci bionet.molbio.proteins:13558 bionet.molbio.methds-reagnts:72168 Hi all, I'm about to express a recombinant protein in E.coli that requires FAD as a cofactor, otherwise it doesn't fold correctly - at least in vitro. I'm concerned that if protein expression (T7 promoter) is faster than FAD synthesis I'll end up with large amounts of denatured protein, inclusion bodies possibly - as has happened already in a similar experiment. Those are very, very difficult to refold in my case so I'd like to avoid them. Do you have any experience whether additional FAD in the culture medium or some cheaper precursor like FMN or riboflavin would be beneficial here? As far as I know E.coli can make FAD from "scratch", but can it utilize one of the mentioned precursors more efficiently? Thanks a lot! Georg ------------------------------------------- To reply, remove "cutout" from the name in the email-address, leaving "moqch". From owner-proteins@net.bio.net Thu Nov 12 22:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!btnet-peer!btnet!news-feed1.eu.concert.net!fu-berlin.de!molbio4.nawi.uni-wh.DE!not-for-mail From: Andreas Savelsbergh Newsgroups: bionet.molbio.proteins Subject: Re: His tag expressed proteins aggregates Date: Fri, 13 Nov 1998 12:24:51 +0100 Lines: 51 Message-ID: <364C1703.70F0C68D@gmx.de> References: NNTP-Posting-Host: molbio4.nawi.uni-wh.de (193.175.78.33) Mime-Version: 1.0 Content-Type: multipart/alternative; boundary="------------917399D62C96CBAC12082AF1" X-Access: 16 1597 1598 X-Trace: fu-berlin.de 910956339 28617 (none) 193.175.78.33 X-Mailer: Mozilla 4.03 [en] (Win95; I) --------------917399D62C96CBAC12082AF1 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit Hi Wendy, I have used the refolding on the column many times, and it worked very well. I followed more or less Qiagen's protocol from "The Expressionist" (manual for Ni-NTA). In my PhD-thesis I have described many more of such purifications, but I'm afraid this won't help: it's in german :-) The activities were (in cases where comparison was possible) the same as wild-type proteins prepared under non-denaturing conditions. For a reference: although not expicitely mentioned in materials and methods (characters were limited), we have published a paper with a protein purified by this method: Rodnina et al. (1997), Nature (385), 37-41. Good luck, Andreas --------------917399D62C96CBAC12082AF1 Content-Type: text/html; charset=us-ascii Content-Transfer-Encoding: 7bit Hi Wendy,

I have used the refolding on the column many times, and it worked very well. I followed more or less Qiagen's protocol from "The  Expressionist" (manual for Ni-NTA). In my PhD-thesis I have described many more of such purifications, but I'm afraid this won't help: it's in german :-) The activities were (in cases where comparison was possible) the same as wild-type proteins prepared under non-denaturing conditions.

For a reference: although not expicitely mentioned in materials and methods (characters were limited), we have published a paper with a protein purified by this method: Rodnina et al. (1997), Nature (385), 37-41.

Good luck,

Andreas
 
  --------------917399D62C96CBAC12082AF1-- From owner-proteins@net.bio.net Thu Nov 12 22:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!chippy.visi.com!news-out.visi.com!news-feed.inet.tele.dk!bofh.vszbr.cz!news.daimi.au.dk!not-for-mail From: Per Mygind Newsgroups: bionet.molbio.proteins Subject: Re: His tag expressed proteins aggregates Date: Fri, 13 Nov 1998 15:09:08 +0000 Organization: University of Aarhus, Department of Computer Science (DAIMI) Lines: 166 Message-ID: <364C4B94.6137EFE4@biobase.dk> References: NNTP-Posting-Host: majestetix.medmicro.aau.dk Mime-Version: 1.0 Content-Type: multipart/alternative; boundary="------------7371EB72C60667E2B64125AD" X-Trace: xinwen.daimi.au.dk 910965575 5794 255.255.255.255 (13 Nov 1998 13:59:35 GMT) X-Complaints-To: news@daimi.au.dk NNTP-Posting-Date: 13 Nov 1998 13:59:35 GMT X-Mailer: Mozilla 4.07 [en] (X11; I; SunOS 5.5.1 sun4m) --------------7371EB72C60667E2B64125AD Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit Wendy-Anne Smith wrote: > Hi, > > We have expressed a house dust mite trypsin protein using the pET-11d > vector in BL21 (DE3)pLysS. We purify the 2mg/ml recombinant protein using > the Qiagen Ni-NTA denaturing protocol getting nice yields of protein using > buffer E which contains urea at a conc. of 8M. This is where we start to > have problems. If we try to dialyse the protein into PBS, it appears that > all the protein sticks to the dialysis tubing (it doesn't degrade.). So > we thought about desalting the protein and improving the purity by passing > it over a Sephadex 25 column but with filtration (Millipore filter) prior > to loading on the column. The first time we did this we tried to filter > 2-3 mg and lost it all. By adding 0.01% Tween 20 to the protein prior to > filtration we were able to retain all our sample suggesting that we had > broken down all the aggregates allowing filtration to occur. So it would > appear that our recombinant protein is very sticky but we want to use the > material in tissue culture and so don't want to use a protein prep with > Tween20 in it. Now my boss says that it is sticky only because it has not > been refolded properly and that if we refolded it prior to eluting off the > column we would solve all our problems. I have seen briefly a paper which > talked about refolding the protein while it was bound to the column. My > boss is keen for me to try that but unfortunately we have misplaced the > paper. > > So my questions are: > 1) Has anyone read a paper where they described refolding a His tag > protein whilst bound to a Nickel column & > > 2) What methods have people used to refold His tag proteins isolated from > insoluble inclusion bodies which has given them a protein similar to, in > activity, to it's native counterpart? > > Thanks > > -- > Dr. Wendy-Anne Smith > > TVW Telethon Institute for Child Health Research > (Company Limited by Guarantee) > Western Australia > > Fax 61 9 388 3414 Try out this article: Refolding of E. coli produced membrane protein inclusion bodies immobilised by nickel chelating chromatography. Rogl et al, FEBS Letters 432(1998):21-26 I think it is availible on the internet. -- Regards Per Mygind ************************************************************************ If you are are not part of the solution, you are part of the precipitate ************************************************************************ Per Mygind, Cand.scient Department of Medical Microbiology and Immunology The Bartholin Building, University of Aarhus, Denmark phone : 89 42 17 47, fax : 86 19 61 28 ************************************************************************ It's hard work and great art to make life not so serious ************************************************************************ --------------7371EB72C60667E2B64125AD Content-Type: text/html; charset=us-ascii Content-Transfer-Encoding: 7bit Wendy-Anne Smith wrote:

Hi,

We have expressed a house dust mite trypsin protein using the pET-11d
vector in BL21 (DE3)pLysS.  We purify the 2mg/ml recombinant protein using
the Qiagen Ni-NTA denaturing protocol getting nice yields of protein using
buffer E which contains urea at a conc. of 8M.  This is where we start to
have problems.  If we try to dialyse the protein into PBS, it appears that
all the protein sticks to the dialysis tubing (it doesn't degrade.).  So
we thought about desalting the protein and improving the purity by passing
it over a Sephadex 25 column but with filtration (Millipore filter) prior
to loading on the column.  The first time we did this we tried to filter
2-3 mg and lost it all.  By adding 0.01% Tween 20 to the protein prior to
filtration we were able to retain all our sample suggesting that we had
broken down all the aggregates allowing filtration to occur.  So it would
appear that our recombinant  protein is very sticky but we want to use the
material in tissue culture and so don't want to use a protein prep with
Tween20 in it.  Now my boss says that it is sticky only because it has not
been refolded properly and that if we refolded it prior to eluting off the
column we would solve all our problems.  I have seen briefly a paper which
talked about refolding the protein while it was bound to the column.  My
boss is keen for me to try that but unfortunately we have misplaced the
paper.

So my questions are:
1)  Has anyone read a paper where they described refolding a His tag
protein whilst bound to a Nickel column &

2)  What methods have people used to refold His tag proteins isolated from
insoluble inclusion bodies which has given them a protein similar to, in
activity, to it's native counterpart?

Thanks

--
Dr. Wendy-Anne Smith

TVW Telethon Institute for Child Health Research
(Company Limited by Guarantee)
Western Australia

Fax 61 9 388 3414

Try out this article:
Refolding of E. coli produced membrane protein inclusion bodies immobilised by nickel chelating chromatography.
Rogl et al, FEBS Letters 432(1998):21-26
I think it is availible on the internet. 
-- 
Regards


Per Mygind

************************************************************************
If you are are not part of the solution, you are part of the precipitate
************************************************************************

Per Mygind, Cand.scient

Department of Medical Microbiology and Immunology
The Bartholin Building, University of Aarhus, Denmark
phone : 89 42 17 47, fax   : 86 19 61 28

************************************************************************
It's hard work and great art to make life not so serious
************************************************************************
  --------------7371EB72C60667E2B64125AD-- From owner-proteins@net.bio.net Thu Nov 12 22:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!newshub.northeast.verio.net!news.idt.net!news-feed.fnsi.net!hammer.uoregon.edu!csulb.edu!drivel.ics.uci.edu!news.service.uci.edu!rigel.oac.uci.edu!kkeikhan From: Kurosh K Newsgroups: bionet.molbio.proteins Subject: Nernst equation problems/information anyone?? Date: Fri, 13 Nov 1998 17:16:45 -0800 Organization: University of California, Irvine Lines: 22 Message-ID: NNTP-Posting-Host: rigel.oac.uci.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII Hi folks, I realize that this is a molec bio newsgroup rather than a cell bio newsgroup but i am hoping someone in here might be able to help me. I am looking for some information/problems (i'd appreciate problems with answers) regarding nernst equation. Unfortunately my book only talks about it in brief and has a few general (and seemingly easy questions) about it. If anyone out there can point me in the direction of more indept twisted and abstract questions (i,e not the basic problems) involving the nernst equation, i would truely appreciate it. I am currently taking cell bio class at my university and i have a feeling that my professor expects us to be able to think a little more than the basic stuff that our book explains. Here is how my book gives the equation as: V=RT/zF lnC1/C2.. where V=nernst potential, C1=concentration on the "outside" and C2= concentration on the "inside". thank you Kurosh From owner-proteins@net.bio.net Thu Nov 12 22:00:00 1998 Path: biosci!internet!biosci!not-for-mail From: biohelp (BIOSCI Administrator) Newsgroups: bionet.molbio.proteins Subject: BIOSCI/bionet miniFAQ & Fundraiser Date: 13 Nov 1998 02:00:18 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 233 Sender: daemon@net.bio.net Distribution: world Message-ID: <199811131000.CAA08366@net.bio.net> NNTP-Posting-Host: net.bio.net (LAST REVISION: 30-JUL-95) This BIOSCI "miniFAQ" is designed to answer the questions that come up the *most frequently*. The main BIOSCI FAQ (Frequently Asked Questions) is accessible on the World Wide Web at URL http://www.bio.net/. If you can not find an answer to your question in this or other documentation, the BIOSCI technical support staff answers e-mail queries sent to biosci-help@net.bio.net We can only answer questions about the use of the newsgroups and mailing lists. We unfortunately do not have the staff to do Internet information searches or answer scientific questions. Please post those to the appropriate BIOSCI/bionet newsgroups. Contents: -------- 0) BIOSCI NEEDS YOUR SUPPORT!! 1) Using the WWW to access the BIOSCI/bionet newsgroups. 2) What to do about "spams," i.e., junk mail, ads, etc. 3) Examples of subscribing and unsubscribing to the mailing lists. 4) The BIOSCI user address and research interest directory. 0) BIOSCI NEEDS YOUR SUPPORT!! ------------------------------ BIOSCI's government funding has been expended, and we are now operating solely from advertising revenue that we have raised from our Web site at http://www.bio.net/. We need just a few minutes of your time to help us serve you. You can do two important things which will take very little time for you individually and will immensely help us continue to help you. First, please use our WWW system at http://www.bio.net/ to access the archives. You can post or reply to messages via your Web browser as described in item #1 below. Your usage helps attract sponsors. If you contact any of our sponsors, please be sure to thank them for supporting BIOSCI. It is critical for them to get this feedback if they are to continue their sponsorship for the long term. Second, if you work for a company or organization that provides products or services of interest to the biology community, please pass this message on to your marketing or marketing communications department or other appropriate group. Please ask them to help support BIOSCI by sponsoring our Web site and explain the uses and benefits of the system to the biology community. If they are interested, they can then contact us for further information at our tech support address, biosci-help@net.bio.net. 1) Using the WWW to access the BIOSCI/bionet newsgroups. -------------------------------------------------------- As of 10 December 1995, all BIOSCI/bionet full newsgroups are accessible through the World Wide Web (WWW) at URL http://www.bio.net. One can read and reply publicly or privately to both recent postings and archived messages through one's Web browser if it is configured properly to send e-mail. Each newsgroup is equipped with its own WAIS index. The main BIOSCI home page also has access to the BIO-JOURNALS Table of Contents database WAIS index and the BIOSCI user address database described in another item further below. 2) What to do about "spams," i.e., junk mail, ads, etc. ------------------------------------------------------- BIOSCI is a set of parallel USENET newsgroups (the "bionet" groups), mailing lists, and a hypermail archive at URL http://www.bio.net/. The same postings are distributed on all media (except for a small number of mailing-list-only groups at net.bio.net). Unfortunately it is becoming a despicable practice on the Internet (by a few people out to make a fast buck) to do automated mass postings to thousands of newsgroups and mailing lists. These attempts to grab free advertising are refered to as "spams" in the usual, somewhat boneheaded, net terminology. USENET is more susceptible to this practice, and many spams originate on the USENET groups and then are passed on to the mailing lists. However, spammers also get lists of mailing addresses and hit these too, so neither medium is immune. What should you do personally if you get junk mail? --------------------------------------------------- Just delete it and move on without reading it further. Filing a protest is becoming increasingly useless because spammers are often disguising the addresses where the messages are sent from. Unless you really understand Internet mail systems, your attempt at protest by sending replies to the message will often end up being sent to the address of an innocent person that the spammer is victimizing. What can BIOSCI/bionet do to protect its newsgroups? ---------------------------------------------------- The only solution currently available is to moderate the newsgroup. If this newsgroup is already moderated, then you are in good shape. Moderation protects the USENET distribution from about 95% of the spams that are being sent to date and protects the mailing lists completely. Moderation means, however, that someone has to take the time to review each message before it goes out. We have set up software here that simply allows the moderator to forward to an address at net.bio.net messages that (s)he wishes to have distributed. This takes no more time than that needed to read the message and pass it on, say about 1 min. per message. Most newsgroups currently have a discussion leader who is responsible for their newsgro