From owner-proteins@net.bio.net Tue Dec 01 22:00:00 1998 Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!sunqbc.risq.qc.ca!news.uow.edu.au!news.nsw.CSIRO.AU!tastiger.dbe.csiro.au!user From: lloyd.graham@REMOVE-MEmolsci.csiro.au (Lloyd Graham) Subject: FPLC - Superdex 200HR vs. Superose 12HR columns X-Nntp-Posting-Host: tastiger.dbe.csiro.au Message-ID: Sender: news@news.nsw.CSIRO.AU Organization: CSIRO Date: Thu, 3 Dec 1998 06:03:52 GMT Lines: 13 Xref: biosci bionet.molbio.methds-reagnts:72645 bionet.molbio.proteins:13640 Can anyone comment on relative advantages (for protein size estimation and purification) of Pharmacia's new(ish) Superdex-200HR 10/30 column, as compared to their long-standing Superose-12HR 10/30 column? They both have similar nominal separation ranges (which are suitable to our needs) and both appear to have comparable theoretical plates & and flowrates despite having different matrix compositions. Feedback gratefully received. Thanks, Lloyd. -- Delete REMOVE-ME from email address to reply From owner-proteins@net.bio.net Tue Dec 01 22:00:00 1998 Path: biosci!ed.ac.uk!richard.adams From: richard.adams@ed.ac.uk (Richard Adams) Newsgroups: bionet.molbio.proteins Subject: sucrose gradients Date: 2 Dec 1998 11:32:42 -0800 Organization: University of Edinburgh Lines: 14 Sender: daemon@net.bio.net Distribution: world Message-ID: <36659593.7EF5@ed.ac.uk> NNTP-Posting-Host: net.bio.net Dear All, I'm purifying a large protein complex (12S) from Xenopus egg extracts. I have no functional assay for the complex and so have to test whether a particular treatment (e.g.,salt) disrupts the complex by running sucrose gradients containing that salt. I am aware that addition of salt will affect the migration of the protein and so am relying that my sedimentation markers (BSA, catalase, thyroglobulin) are equally affected. Is this a valid assumption? I.e., are all proteins equally affected by buffer changes to sucrose gradients? Thank You Richard Adams From owner-proteins@net.bio.net Wed Dec 02 22:00:00 1998 From: "Frank Nygaard" Newsgroups: bionet.molbio.proteins,bionet.software Subject: Calculation of Mg-ions in solution Lines: 32 X-Newsreader: Microsoft Outlook Express 4.72.2106.4 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.2106.4 Message-ID: Date: Thu, 3 Dec 1998 23:22:02 +0100 NNTP-Posting-Host: 194.234.169.226 X-Complaints-To: news-abuse@image.dk X-Trace: news010.image.dk 912724040 194.234.169.226 (Thu, 03 Dec 1998 23:27:20 MET DST) NNTP-Posting-Date: Thu, 03 Dec 1998 23:27:20 MET DST Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!news.gtei.net!newsfeed.berkeley.edu!howland.erols.net!newsfeed.cwix.com!193.162.146.37!miri.tele.dk!news4.image.dk!news010.image.dk.POSTED!not-for-mail Xref: biosci bionet.molbio.proteins:13643 bionet.software:22428 Hi, During a kinetic study of an enzyme requiring magnesium both as free ions as well as MgATP, I need to calculate the concentration of each component in my assay condition. A typical assay condition could be: 50 mM Tris-HCl, 50 mM Pi, 2 mM EGTA, 2 mM ATP and 5 mM MgCl2 at pH 8,2 - 37degree. My main concern binding of magnesium to Pi and Tris. I have tried two programs: "cabuffer" by Jochen Kleinschmit and a program by Karl-Josef Foehr and Wojciech Warchol which I think is called "calcium". The programs give similar results in the absence of Pi and Tris. The binding of Mg++ to Pi however are estimated differently by the two programs where cabuffer gives a relatively high concentration of MgPi compared to MgATP whereas "calcium" have very low binding of Mg++ to Pi. I've found a stability constant for MgHPO4 of 500 M-1 corresponding to a Kd of 2 mM which would obvious contribute to binding of Mg++. None of the programs calculate the binding of Mg++ to Tris. I've not been able to find any stability/dissociation constant for this complex but the dissociation constant for Mn-Tris complex have been reported to be 250 mM. Does anybody have experience using these programs, or know of similar programs for calculating the destribution af Mg-ions in solution? Thanks in advance Frank Nygaard Centre for Crystallographic Studies University of Copenhagen DK-2100 DENMARK frank@xray.ki.ku.dk From owner-proteins@net.bio.net Wed Dec 02 22:00:00 1998 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!news.gtei.net!news.pbi.net!151.142.220.3!WCG!backpost.satin.net!newsfeed.kabelfoon.nl!not-for-mail From: Bassie Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: FPLC - Superdex 200HR vs. Superose 12HR columns Date: Thu, 03 Dec 1998 10:26:37 +0100 Organization: Ikke Lines: 25 Message-ID: <3666594D.5CB8575D@kabelfoon.nl> References: NNTP-Posting-Host: k1nw025.dial.kabelfoon.nl Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: news.kabelfoon.nl 912676895 25978 195.193.20.25 (3 Dec 1998 09:21:35 GMT) X-Complaints-To: abuse@kabelfoon.nl NNTP-Posting-Date: 3 Dec 1998 09:21:35 GMT X-Mailer: Mozilla 4.06 [en] (Win95; I) Xref: biosci bionet.molbio.methds-reagnts:72648 bionet.molbio.proteins:13641 Lloyd Graham wrote: > Can anyone comment on relative advantages (for protein size estimation and > purification) of Pharmacia's new(ish) Superdex-200HR 10/30 column, as > compared to their long-standing Superose-12HR 10/30 column? They both have > similar nominal separation ranges (which are suitable to our needs) and > both appear to have comparable theoretical plates & and flowrates despite > having different matrix compositions. Feedback gratefully received. > > Thanks, > > Lloyd. > Lloyd, In my experience the Superose 12 behaves better and with sharper peaks. I use this one for analytical purposes. The Superdex 200 has a somewhat less separation behaviour and thus I use this one for small scale preparative purposes. Regards ---=== Bassie ===--- From owner-proteins@net.bio.net Wed Dec 02 22:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!newsfeed.nyu.edu!newsfeed.sgi.net!nntp.itis.com!news.doit.wisc.edu!Home From: klenchin@REMOVE_TO_REPLY.facstaff.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: FPLC - Superdex 200HR vs. Superose 12HR columns Date: Thu, 03 Dec 1998 15:42:19 GMT Organization: UW Lines: 14 Message-ID: <746bgu$1886$1@news.doit.wisc.edu> References: NNTP-Posting-Host: t-43-183-143.dialup.wisc.edu X-Newsreader: News Xpress 2.01 X-No-Archive: Yes Xref: biosci bionet.molbio.methds-reagnts:72659 bionet.molbio.proteins:13642 In article , lloyd.graham@REMOVE-MEmolsci.csiro.au (Lloyd Graham) wrote: >Can anyone comment on relative advantages (for protein size estimation and >purification) of Pharmacia's new(ish) Superdex-200HR 10/30 column, as >compared to their long-standing Superose-12HR 10/30 column? They both have >similar nominal separation ranges (which are suitable to our needs) and >both appear to have comparable theoretical plates & and flowrates despite >having different matrix compositions. Feedback gratefully received. > They are indeed very similar. In my experience limited to only several runs on both, Superdex has a bit wider separation range and yields of activities were higher. - Dima From owner-proteins@net.bio.net Thu Dec 03 22:00:00 1998 Path: biosci!news.stanford.edu!logbridge.uoregon.edu!hammer.uoregon.edu!leto.ou.edu!news.ecn.ou.edu!news1.optus.net.au!optus!news.usyd.edu.au!sbe From: sbe@biochem.usyd.edu.au (Simon Easterbrook-Smith) Newsgroups: bionet.molbio.proteins Subject: Re: km determination Date: Sat, 05 Dec 1998 17:41:11 +1100 Organization: Department of Biochemistry, University of Sydney Lines: 50 Message-ID: References: <36687378.1A19ED8B@sciborg.uwaterloo.ca> NNTP-Posting-Host: bio225.biochem.usyd.edu.au Mime-Version: 1.0 Content-Type: text/plain; charset=ISO-8859-1 Content-Transfer-Encoding: 8bit X-Trace: metro.ucc.usyd.edu.au 912839947 16112 129.78.130.225 (5 Dec 1998 06:39:07 GMT) X-Complaints-To: usenet@news.usyd.edu.au NNTP-Posting-Date: 5 Dec 1998 06:39:07 GMT X-Newsreader: Yet Another NewsWatcher 2.3.4 Dear Mike It is probably most reliable to fit the M-M equation (v = Vm*S/(Km + S) to the primary data (pairs of (v, S)) by non-linear regression analysis. The problem with the various linear plots (Lineweaver-Burke et al.) is that experimental errors can lead to distortion in the values of the parameter estimates. Eg, in a Lineweaver-Burke plot large values of 1/v and 1/S have the biggest effect on the parameter estimates but these data points (corresponding to small values of v and S) are usually the least accurate. Cheers Simon || Hello || I am currently working to determine the km's of our two different || enzymes of interest (isoforms), and am by no means a biochemist. I am || wondering which is the best re-arrangement to determine the actual km. || i have tried some initial data using Lineweaver-Burk plots, Hofstee || plats, Woolf plots and Scatchard plots. Only the Scatchard and || Lineweaver-Burk plot agreed. || || Is there a reference re which is the best plot depending on the data? || Should they all agree within a reasonable error? || || Thanks in advance || || Mike Allen || M.Sc. Student || University of Waterloo || Ontario, Canada || m3allen@sciborg.uwaterloo.ca -- _______________________________________________________________ Dr Simon B Easterbrook-Smith Voice: (+) 612 9351 3905 Department of Biochemistry FAX: (+) 612 9351 4726 University of Sydney Email: sbe@biochem.usyd.edu.au Sydney NSW 2006 AUSTRALIA _______________________________________________________________ Under U.S. Code, Title 47, Chapter 5, Subchapter II, ß227, all unsolicited commercial E-mail sent to my address from a U.S. account is subject to a fee of $500.00 U.S. By E-mailing me you accept these terms. cf. _______________________________________________________________ From owner-proteins@net.bio.net Thu Dec 03 22:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!sunqbc.risq.qc.ca!torn!kwon!watserv3.uwaterloo.ca!not-for-mail From: Michael Allen Newsgroups: bionet.molbio.proteins Subject: km determination Date: Fri, 04 Dec 1998 18:42:48 -0500 Organization: University of Waterloo Lines: 18 Message-ID: <36687378.1A19ED8B@sciborg.uwaterloo.ca> NNTP-Posting-Host: gelrecdr.uwaterloo.ca Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.05 [en] (Win95; I) Hello I am currently working to determine the km's of our two different enzymes of interest (isoforms), and am by no means a biochemist. I am wondering which is the best re-arrangement to determine the actual km. i have tried some initial data using Lineweaver-Burk plots, Hofstee plats, Woolf plots and Scatchard plots. Only the Scatchard and Lineweaver-Burk plot agreed. Is there a reference re which is the best plot depending on the data? Should they all agree within a reasonable error? Thanks in advance Mike Allen M.Sc. Student University of Waterloo Ontario, Canada m3allen@sciborg.uwaterloo.ca From owner-proteins@net.bio.net Thu Dec 03 22:00:00 1998 Message-ID: <36682BF7.3699C5BC@ibm.net> Date: Fri, 04 Dec 1998 14:37:43 -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 - Principal Scientist, Protein Chemistry Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit NNTP-Posting-Host: 166.72.219.25 X-Trace: 4 Dec 1998 19:19:06 GMT, 166.72.219.25 Organization: IBM.NET Lines: 27 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!166.72.219.25 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 Principal Scientist, Protein Chemistry. Duties: focus on the design of multi-dimensional (multi-modal) chromatographic separations of peptides; and create unique separation strategies for complex mixtures of naturally processed biomolecules. Experience: microbore and microcapillary LC separations of biomolecules. Expertise in microcapillary peptide and protein HPLC separations. Ph.D. in Analytical Chemistry or Biochemistry or Chemical Engineering. Very competitive compensation. ALL FEES AND EXPENSES PAID BY THE COMPANY. Leonard Kalvert President Kalvert Personnel Service, Inc. [est. 1966] P.O. Box 1579 Stony Brook, NY 11790 Phone (516) 331-3388 Fax: (516) 331-1886 e-mail: kalvert@ibm.net From owner-proteins@net.bio.net Thu Dec 03 22:00:00 1998 Message-ID: <36681908.A8D091A0@ibm.net> Date: Fri, 04 Dec 1998 13:16:57 -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 - Principal Scientist, Protein Chemistry Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit NNTP-Posting-Host: 32.100.112.252 X-Trace: 4 Dec 1998 17:58:15 GMT, 32.100.112.252 Organization: IBM.NET Lines: 37 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!newsfeed.cwix.com!165.87.194.242!newsm2.ibm.net!ibm.net!news1.ibm.net!32.100.112.252 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 Principal Scientist, Protein Chemistry. Duties: focus on the design of multi-dimensional (multi-modal) chromatographic separations of peptides; and create unique separation strategies for complex mixtures of naturally processed biomolecules. Experience: microbore and microcapillary LC separations of biomolecules. Expertise in microcapillary peptide and protein HPLC separations. Ph.D. in Analyical Chemistry or Biochemistry or Chemical Engineering. Very competitive compensation. ALL FEES AND EXPENSES PAID BY THE COMPANY. Leonard Kalvert President Kalvert Personnel Service, Inc. [est. 1966] P.O. Box 1579 Stony Brook, NY 11790 Phone (516) 331-3388 Fax: (516) 331-1886 e-mail: kalvert@ibm.net From owner-proteins@net.bio.net Thu Dec 03 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!news-peer.gip.net!news-lond.gip.net!news.gsl.net!gip.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.methds-reagnts,bionet.molbio.proteins Subject: Re: FPLC - Superdex 200HR vs. Superose 12HR columns Date: Fri, 04 Dec 1998 09:31:20 +0100 Organization: noone Lines: 21 Message-ID: References: <746bgu$1886$1@news.doit.wisc.edu> NNTP-Posting-Host: med180.bham.ac.uk Xref: biosci bionet.molbio.methds-reagnts:72682 bionet.molbio.proteins:13644 > >Can anyone comment on relative advantages (for protein size estimation and > >purification) of Pharmacia's new(ish) Superdex-200HR 10/30 column, as > >compared to their long-standing Superose-12HR 10/30 column? They both have > >similar nominal separation ranges (which are suitable to our needs) and > >both appear to have comparable theoretical plates & and flowrates despite > >having different matrix compositions. Feedback gratefully received. > > > > They are indeed very similar. In my experience limited to only several > runs on both, Superdex has a bit wider separation range and yields of > activities were higher. > > - Dima I also believe that the Superdex 200 has a wider separation range. I used it successfully for a few high molecular weight proteins of approx. 300 kDa. IMHO Superose 12 is still the best choice though when it comes to proteins up to, say, 50-80 kDa. Peter From owner-proteins@net.bio.net Thu Dec 03 22:00:00 1998 Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Path: biosci!news.stanford.edu!logbridge.uoregon.edu!sunqbc.risq.qc.ca!news.uow.edu.au!news.nsw.CSIRO.AU!tastiger.dbe.csiro.au!user From: lloyd.graham@REMOVE-MEmolsci.csiro.au (Lloyd Graham) Subject: Re: FPLC - Superdex 200HR vs. Superose 12HR columns X-Nntp-Posting-Host: tastiger.dbe.csiro.au Message-ID: Sender: news@news.nsw.CSIRO.AU Organization: CSIRO References: <746bgu$1886$1@news.doit.wisc.edu> Date: Sat, 5 Dec 1998 02:18:01 GMT Lines: 37 Xref: biosci bionet.molbio.methds-reagnts:72700 bionet.molbio.proteins:13648 In article , noone@cancer.bham.ac.uk (noone) wrote: > > >Can anyone comment on relative advantages (for protein size estimation and > > >purification) of Pharmacia's new(ish) Superdex-200HR 10/30 column, as > > >compared to their long-standing Superose-12HR 10/30 column? They both have > > >similar nominal separation ranges (which are suitable to our needs) and > > >both appear to have comparable theoretical plates & and flowrates despite > > >having different matrix compositions. Feedback gratefully received. > > > > > > > They are indeed very similar. In my experience limited to only several > > runs on both, Superdex has a bit wider separation range and yields of > > activities were higher. > > > > - Dima > > I also believe that the Superdex 200 has a wider separation range. I used > it successfully for a few high molecular weight proteins of approx. 300 > kDa. IMHO Superose 12 is still the best choice though when it comes to > proteins up to, say, 50-80 kDa. > > Peter Dima, Peter - When you say the Superdex has a wider separation range, this suggests to me that a 40 kDa protein and its 80 kDa dimer will be closer together (less well resolved) on the Superdex column. Is this actually what you mean? Many thanks for the replies, Lloyd. -- Delete REMOVE-ME from email address to reply From owner-proteins@net.bio.net Fri Dec 04 22:00:00 1998 Path: biosci!agate!eve!keith From: keith@eve.cchem.berkeley.edu (Keith Rickert) Newsgroups: bionet.molbio.proteins Subject: Re: km determination Date: 6 Dec 1998 04:02:53 GMT Organization: University of California, Berkeley Lines: 29 Message-ID: <74cvld$dmh$1@agate.berkeley.edu> References: <36687378.1A19ED8B@sciborg.uwaterloo.ca> <7opiGDf98QgB-pn2-dy9Z1ZHxmmZ4@rnaworld.bio.ukans.edu> NNTP-Posting-Host: eve.cchem.berkeley.edu X-Newsreader: NN version 6.5.0 #10 (NOV) In <7opiGDf98QgB-pn2-dy9Z1ZHxmmZ4@rnaworld.bio.ukans.edu> PGegen@UKans.nolospamare.edu (Dr. Peter Gegenheimer) writes: >Yes -- it IS _always_ most accurate to fit the untransformed data to the equation >which describes the relationship between X and Y; you are guaranteed to get the best >estimate for the kinetic constants. Not only for the reasons Simon mentions, but also >because when you transform the variables, you also transform their experimental error >so that it no longer has a ~Gaussian distribution. Further, the transformations in >which one axis contains both V and S violate the assumtions of regression analysis, >in that the X and Y axis variables are no longer separate (you no longer have an >independent and a dependent variable), and (so certain transforms) the experimental >error is no longer exclusively in the Y axis. While I generally agree with all of these points, I have to say that I still think there's a place for the linear transform plots. If your data has some kind of systematic deviation from Michaelis-Menten kinetics, it's often much easier to recognize that on a linear transform than on the non-linear plot. So I generally do both plots, but only derive constants from the nonlinear fit. BTW, it would be much easier to read your posts if you could keep to standard Usenet convention of <75-78 characters per line. Keith -- Keith Rickert | "Imprisoned for a crime I didn't commit. keith@eve.cchem.berkeley.edu | Attempted murder. Now honestly, what is that? rickertk@netcom.com | Do they give a Nobel Prize for attempted keith@imppig.caltech.edu | chemistry?" Sideshow Bob, The Simpsons From owner-proteins@net.bio.net Fri Dec 04 22:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!newsfeed.cwix.com!198.82.160.249!solaris.cc.vt.edu!news.vt.edu!news.cc.ukans.edu!not-for-mail From: PGegen@UKans.nolospamare.edu (Dr. Peter Gegenheimer) Newsgroups: bionet.molbio.proteins Subject: Re: km determination Date: 6 Dec 1998 03:47:59 GMT Organization: Univ. Kansas (BCMB) Lines: 47 Message-ID: <7opiGDf98QgB-pn2-dy9Z1ZHxmmZ4@rnaworld.bio.ukans.edu> References: <36687378.1A19ED8B@sciborg.uwaterloo.ca> Reply-To: PGegen@UKans.nolospamare.edu NNTP-Posting-Host: rnaworld.bio.ukans.edu Mime-Version: 1.0 Content-Type: text/plain; charset="iso-8859-1" Content-Transfer-Encoding: quoted-printable X-Newsreader: ProNews/2 Version 1.00 Yes -- it IS _always_ most accurate to fit the untransformed data to the equation which describes the relationship between X and Y; you are guaranteed to get the best estimate for the kinetic constants. Not only for the reasons Simon mentions, but also because when you transform the variables, you also transform their experimental error so that it no longer has a ~Gaussian distribution. Further, the transformations in which one axis contains both V and S violate the assumtions of regression analysis, in that the X and Y axis variables are no longer separate (you no longer have an independent and a dependent variable), and (so certain transforms) the experimental error is no longer exclusively in the Y axis. For more info, you should look at the materials at the GraphPad "Prism" web site, http://www.graphpad.com/ . On Sat, 5 Dec 1998 06:41:11, sbe@biochem.usyd.edu.au (Simon Easterbrook-Smith) wrote: > > Dear Mike > > It is probably most reliable to fit the M-M equation (v =3D Vm*S/(Km + S) to > the primary data (pairs of (v, S)) by non-linear regression analysis. > > The problem with the various linear plots (Lineweaver-Burke et al.) is that > experimental errors can lead to distortion in the values of the parameter > estimates. Eg, in a Lineweaver-Burke plot large values of 1/v and 1/S have > the biggest effect on the parameter estimates but these data points > (corresponding to small values of v and S) are usually the least accurate. > || Hello > || I am currently working to determine the km's of our two different > || enzymes of interest (isoforms), and am by no means a biochemist. I am > || wondering which is the best re-arrangement to determine the actual km. > || i have tried some initial data using Lineweaver-Burk plots, Hofstee > || plats, Woolf plots and Scatchard plots. Only the Scatchard and > || Lineweaver-Burk plot agreed. > || > || Is there a reference re which is the best plot depending on the data? > || Should they all agree within a reasonable error? o----------------------------------------------------------------------o | Dr. Peter Gegenheimer | Vox: 785-864-3939 FAX: 785-864-5321 | | Department of | PGegen@UKans.nospam.edu | | Molecular Biosciences | http://rnaworld.bio.ukans.edu/ | | & Dept. Evol Biology | | | University of Kansas |"When you have excluded the impossible, | | 2045 Haworth Hall | whatever remains, however improbable, | | Lawrence KS 66045-2106 | must be the truth." S. Holmes | o_____________________________|________________________________________o From owner-proteins@net.bio.net Sat Dec 05 22:00:00 1998 Path: biosci!news.stanford.edu!logbridge.uoregon.edu!news-feed.inet.tele.dk!bofh.vszbr.cz!news.daimi.au.dk!not-for-mail From: Rasmus Wendelbo Nielsen Newsgroups: bionet.molbio.proteins Subject: ANS-probe conditions Date: Sun, 06 Dec 1998 16:34:09 +0100 Organization: University of Aarhus, Department of Computer Science (DAIMI) Lines: 14 Message-ID: <366AA3F0.9BE54DAA@diku.dk> NNTP-Posting-Host: imsb-212.imsb.au.dk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: xinwen.daimi.au.dk 912958272 7977 255.255.255.255 (6 Dec 1998 15:31:12 GMT) X-Complaints-To: news@daimi.au.dk NNTP-Posting-Date: 6 Dec 1998 15:31:12 GMT X-Mailer: Mozilla 4.07 [en] (Win95; I) Have anyone any experience in using 8-anilino-1-naphtalene sulfonate or other isomers of ANS? I'm especially interested in comments on what buffer-type(s) you have used, ANS concentration and the temperature dependence on ANS fluorescence intensity. Right now I'm planning to use a 100 mM Tris-HCl, pH 8.1 buffer. Not to waste too much of my protein, I want to use as low concentrations as possible i.e. a ANS concentration of below 5 uM and a fivefold excess of ANS (1uM protein) as standard conditions. It would obviously be easiest to do it at room temperature. Many thanks for any suggestions, Rasmus W. Nielsen From owner-proteins@net.bio.net Sat Dec 05 22:00:00 1998 From: "S.W. Rasmussen" Newsgroups: bionet.molbio.proteins Subject: Dnatools, sequence software Date: Mon, 07 Dec 1998 00:27:06 +0100 Organization: Carlsberg Laboratory Lines: 17 Message-ID: <366B12CA.B79E6CA7@crc.dk> Reply-To: swr@crc.dk NNTP-Posting-Host: 130.226.184.128 Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Trace: news.net.uni-c.dk 912987014 41838 (None) 130.226.184.128 X-Complaints-To: usenet@news.net.uni-c.dk X-Mailer: Mozilla 4.04 [en] (Win95; I) Path: biosci!news.ic.sunysb.edu!news-nysernet-16.sprintlink.net!news-east1.sprintlink.net!news-peer1.sprintlink.net!news.sprintlink.net!news.maxwell.syr.edu!news-peer.gip.net!news-lond.gip.net!news.gsl.net!gip.net!newsfeed.ecrc.net!news.net.uni-c.dk!not-for-mail DNATools is a program package for handling and analysis of DNA and protein sequences. It includes a number of general facilities for sequence editing, restriction enzyme mapping, translation into protein etc. but also several more complex functions for creation of codon tables, primer design and ordering, SAGE analysis, etc. Visit our home page at: http://www.crc.dk/phys/A01B04_dnatools.htm Dr. Søren W. Rasmussen Carlsberg Laboratory Dept. of Physiology Copenhagen, Denmark http://www.crc.dk/phys/index.htm From owner-proteins@net.bio.net Sat Dec 05 22:00:00 1998 Path: biosci!pravda.ucr.edu!ihnp4.ucsd.edu!newsfeed.berkeley.edu!news.maxwell.syr.edu!news-was.dfn.de!news-fra1.dfn.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: Cleavage of glycosidic moiety Date: Sun, 6 Dec 1998 18:54:07 +0100 Organization: Med. Universitaet zu Luebeck Lines: 6 Message-ID: <74egng$5ff$1@gwsun.medinf.mu-luebeck.de> NNTP-Posting-Host: 141.83.167.171 X-Newsreader: Microsoft Outlook Express 4.72.3110.5 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3110.3 Who can tell my an efficient and inexpensive way to quantitatively remove the glycosidic moiety of a glycosidated protein to determine the mass of the unglycosilated protein ? Lars Komorowski From owner-proteins@net.bio.net Sat Dec 05 22:00:00 1998 Path: biosci!AOL.COM!DOCTORHIM From: DOCTORHIM@AOL.COM Newsgroups: bionet.molbio.proteins Subject: Re: Cleavage of glycosidic moiety Date: 6 Dec 1998 21:33:20 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 3 Sender: daemon@net.bio.net Distribution: world Message-ID: <2efc1c6f.366b678a@aol.com> NNTP-Posting-Host: net.bio.net Try enzymatic deglycosylation. You can read a kit booklet from http://www.prozyme.com/glycoenzymes/welcome.html to see if it fits your requirments. From owner-proteins@net.bio.net Sun Dec 06 22:00:00 1998 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!news.gtei.net!logbridge.uoregon.edu!news.u.washington.edu!not-for-mail From: Maca Newsgroups: bionet.molbio.proteins Subject: Re: 2D Electrophoresis sample prep Date: Mon, 07 Dec 1998 19:28:32 -0800 Organization: University of Washington Lines: 66 Message-ID: <366C9CE0.406991E1@hotmail.com> References: <73kken$aat$1@newsreader3.core.theplanet.net> NNTP-Posting-Host: cs111-4.u.washington.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: nntp4.u.washington.edu 913087743 37326 (None) 140.142.17.35 X-Complaints-To: help@cac.washington.edu X-Mailer: Mozilla 4.06 [en] (Win98; I) Andrew and Glen, The presence of a small amount of SDS in your sample preparations is fine and will not result in perturbation of your proteins' apparent pIs from your gel. In fact, as Andrew has found, including it can improve your final results. Boiling of samples in SDS is sometimes the only way to stop proteolysis in your sample. Proteolysis is highly undesirable in 2D gels as it leads to artefacts. The trick is to keep the SDS at a concentration that is 4 times lower than the concentration of the non-ionic detergent (I assume that you're using CHAPS). At these types of concentrations, CHAPS will sequester the SDS; forming micelles and taking the charge from the solubilised protein. You can work out these things empirically if you look at the Critical Micellular Concentrations (CMCs) of the detergents that you're working with and the temperature that you do the experiments. Some references for you: (The Rabilloud paper is a "bible" for 2D gels. Look at the first paragraph on p825 for the SDS problem). Rabilloud, T. Electrophoresis 17 1996 Solubilization of proteins for electrophoretic analyses pp 813-829. Ames G.F.L., Nikaido K. Biochemistry 15 1976 pp616-623. Weber, K, Kuter D.J., J. Biol. Chem 246 1971 pp4504-4509. Remy R, Ambard-Bretteville, F., Methods Enzymol 148 1987 pp623-632. Hope this helps - post back to the group with your thoughts! Maca. Glen Tamura wrote: > Andrew: > > I agree with you. There is very little question in my mind that this will > result in alterations in the apparent pI. On the other hand, if the amount > of SDS which binds is constant, you might still get nice separation. I > certainly would use any pI information from such gels with a very large > grain of salt (or is it a small grain?). > > Glen Tamura > > On Thu, 26 Nov 1998, Andrew Pridmore wrote: > > > I have achieved excellent two-dimensional separations of bacterial proteins > > using a sample prep described in a Japanese paper, which includes SDS as > > well as nonionic detergent in the first dimension sample buffer. > > > > Why does this work? > > > > Surely the negative charge imparted to the proteins by the SDS would disrupt > > the isoelectric focussing? Or does the nonionic detergent displace the > > negative charge? > > > > Does anyone have a reference which demonstrates what is happening? > > > > Thanks for reading this! > > > > Andrew Pridmore > > > > > > > > From owner-proteins@net.bio.net Sun Dec 06 22:00:00 1998 Path: biosci!agate!not-for-mail From: lhom@OCF.Berkeley.EDU (Louis Hom) Newsgroups: bionet.molbio.proteins Subject: Re: km determination Date: 8 Dec 1998 02:09:36 GMT Organization: Univ. of California Berkeley Open Computing Facility Lines: 13 Message-ID: <74i1p0$su7$1@agate.berkeley.edu> References: <36687378.1A19ED8B@sciborg.uwaterloo.ca> <7opiGDf98QgB-pn2-dy9Z1ZHxmmZ4@rnaworld.bio.ukans.edu> <74cvld$dmh$1@agate.berkeley.edu> NNTP-Posting-Host: conquest.ocf.berkeley.edu In article <74cvld$dmh$1@agate.berkeley.edu>, Keith Rickert wrote: > >While I generally agree with all of these points, I have to say >that I still think there's a place for the linear transform plots. e.g., when computers haven't yet been invented? (sorry, couldn't help myself) -- ______________________________________________________________________________ Lou Hom >K'93 lhom@ocf.berkeley.edu http://www.ocf.berkeley.edu/~lhom/ From owner-proteins@net.bio.net Sun Dec 06 22:00:00 1998 Path: biosci!daresbury!not-for-mail From: Cutie85523@aol.com Newsgroups: bionet.molbio.proteins Subject: Photo Mousepads ....... Great Christmas Gifts ! Date: 7 Dec 1998 10:28:05 -0000 Organization: Daresbury Laboratory, Warrington, U.K. Lines: 160 Message-ID: <74gajl$2om$1@mserv2.dl.ac.uk> NNTP-Posting-Host: mserv2.dl.ac.uk content-length: 3699 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 Sun Dec 06 22:00:00 1998 Path: biosci!news.stanford.edu!nntp.cs.ubc.ca!cyclone.bc.net!news.maxwell.syr.edu!nntp2.dejanews.com!nnrp1.dejanews.com!not-for-mail From: kll@crc.dk Newsgroups: bionet.molbio.proteins Subject: Cupper staining of gels Date: Mon, 07 Dec 1998 10:21:32 GMT Organization: Deja News - The Leader in Internet Discussion Lines: 18 Message-ID: <74ga79$6kn$1@nnrp1.dejanews.com> NNTP-Posting-Host: 130.226.1.36 X-Article-Creation-Date: Mon Dec 07 10:21:32 1998 GMT X-Http-User-Agent: Mozilla/4.5 [en] (Win95; I) X-Http-Proxy: 1.0 www2.crc.dk:3128 (Squid/1.1.20), 1.0 www-cache.net.uni-c.dk:3128 (Squid/1.1.20), 1.0 x4.dejanews.com:80 (Squid/1.1.22) for client 130.226.182.217, 130.226.183.6, 130.226.1.36 Could anybody tell me how to do a (*rapid*) copper staining of a SDS-PA gel. Will any quality of CuCl_2 do? Can the CuCl_2 sol. be reused (how many times)? The protocol I have simply says that I should rinse the gel in H_2O and then soak it in 0.3 M CuCl_2 for appox. 5 min. Any other tricks which will improve efficiency? Thanks Kresten -- Kresten Lindorff Larsen Dept. Yeast Genetics Carlsberg Laboratory Denmark -----------== Posted via Deja News, The Discussion Network ==---------- http://www.dejanews.com/ Search, Read, Discuss, or Start Your Own From owner-proteins@net.bio.net Sun Dec 06 22:00:00 1998 Path: biosci!AOL.COM!Cutie85523 From: Cutie85523@AOL.COM Newsgroups: bionet.molbio.proteins Subject: Photo Mousepads ....... Great Christmas Gifts ! Date: 7 Dec 1998 02:22:35 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 160 Sender: daemon@net.bio.net Distribution: world Message-ID: <4bf470fe.366ba40d@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! 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From owner-proteins@net.bio.net Sun Dec 06 22:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!howland.erols.net!frankfurt.de.uu.net!news.uni-stuttgart.de!news.belwue.de!newsserv.zdv.uni-tuebingen.de!not-for-mail From: cbiei01@uni-tuebingen.de (H.V.Taylor) Newsgroups: bionet.molbio.proteins Subject: Re: Cleavage of glycosidic moiety Date: Mon, 07 Dec 1998 17:15:45 GMT Organization: ZDV Uni-Tuebingen Lines: 44 Message-ID: <366c0c56.30400348@newsserv.zdv.uni-tuebingen.de> References: <74egng$5ff$1@gwsun.medinf.mu-luebeck.de> NNTP-Posting-Host: eis4.pci.chemie.uni-tuebingen.de Mime-Version: 1.0 Content-Type: text/plain; charset=ISO-8859-1 Content-Transfer-Encoding: 8bit X-Newsreader: Forte Agent 1.5/16.451 "Lars Komorowski" wrote: >Who can tell my an efficient and inexpensive way to quantitatively remove >the glycosidic moiety of a glycosidated protein to determine the mass of the >unglycosilated protein ? Dear Lars, although I've no experience in such things, here's a message which was posted in this group a while ago. Hope it helps, Harold V. Taylor ********************** Trifluoromethanesulfonic acid TMFSA (also called triflic acid. There is a paper by J. Horvath 1989 J. Neurosci. Res. which gives a simple method to follow. There are a few different methods to follow, the main difference between them is the completeness of removal of CHO, some will not remove the last GlcNAc/GalNAc residues. But for antibody production this is not usually crucial, this process gives undenatured proteins which you can use for Ab production. If you've got any more questions try the bionet.glycosci newsgroup or drop me an e-mail. Ian McFarlane In article , Anders Steenfeldt wrote: > Does anyone know of a non-enzymatic way to deglycosylate a protein without > rendering it denatured? > Please mail me if You do. > Anders Steenfeldt > Email torin@imsb.au.dk Harold V. Taylor Physiol. Chem. Institut Hoppe-Seyler-Str. 4 D-72076 Tübingen phone: 07071-297-3353 fax: 07071-29-4182 email: cbiei01@uni-tuebingen.de From owner-proteins@net.bio.net Sun Dec 06 22:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!howland.erols.net!newsfeed.nacamar.de!rill.news.pipex.net!pipex!server1.netnews.ja.net!pegasus.csx.cam.ac.uk!not-for-mail From: Marko Hyvonen Newsgroups: bionet.molbio.proteins Subject: Re: Cupper staining of gels Date: 07 Dec 1998 14:43:51 +0000 Organization: University of Cambridge, England Lines: 32 Message-ID: References: <74ga79$6kn$1@nnrp1.dejanews.com> NNTP-Posting-Host: hirta.bioc.cam.ac.uk X-Newsreader: Gnus v5.5/Emacs 20.2 You might wish try a variation of the original copper staining which uses imidazole soak before the copper (or even better, zinc) solution. All done in 10 minutes and fully reversible and very sensitive I use it all the time. In brief: - soak the gel in 0.2 M imidazole for 5-10 minutes - transfer to 0.3 M ZnCl (or other zinc salt) for ca. 30 seconds. Follow development of white background bye eye against dark background. When the gel doesn't get any more white, rinse with water. - View ona dark/black surface and store in water. - For destaining for subsequent steps like immunoblotting soak in 0.3% citric acid until transparent again References: C. Fernandez-Patron at al. (1992) Biotechniques 12:564- C. Fernandez-Patron at al. (1995) Analytical Biochemistry 224:203- Have fun, Marko -- <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> Marko Hyvonen tel: +44-(0)1223-766 030 / 766 027 fax: +44-(0)1223-766 082 Department of Biochemistry email: marko@cryst.bioc.cam.ac.uk University of Cambridge http://www-cryst.bioc.cam.ac.uk/~marko <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> From owner-proteins@net.bio.net Sun Dec 06 22:00:00 1998 From: Cornelius Krasel Subject: Re: Cupper staining of gels Newsgroups: bionet.molbio.proteins References: <74ga79$6kn$1@nnrp1.dejanews.com> User-Agent: tin/pre-1.4-980514 (UNIX) (Linux/2.0.34 (i486)) Date: Mon, 7 Dec 1998 12:54:34 +0100 Message-ID: NNTP-Posting-Host: wpxx02.toxi.uni-wuerzburg.de Lines: 15 Path: biosci!agate!newsfeed.berkeley.edu!howland.erols.net!EU.net!blackbush.xlink.net!rz.uni-karlsruhe.de!news.uni-stuttgart.de!uni-erlangen.de!uni-wuerzburg.de!not-for-mail kll@crc.dk wrote: > The protocol I have simply says that I should rinse the gel in H_2O and then > soak it in 0.3 M CuCl_2 for appox. 5 min. Worked for me (yes, it's really that simple!). I think reusing the CuCl_2 solution may be not a good idea because it becomes enriched in SDS. I am not sure whether CuSO_4 will do the trick as well. I used some old CuCl_2 from Sigma that I found on the shelves. --Cornelius. -- /* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */ /* D-97078 Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de SP4 */ /* "Science is the game we play with God to find out what His rules are." */ From owner-proteins@net.bio.net Sun Dec 06 22:00:00 1998 Path: biosci!news.stanford.edu!logbridge.uoregon.edu!arclight.uoregon.edu!news.cc.ukans.edu!not-for-mail From: PGegen@UKans.nolospamare.edu (Dr. Peter Gegenheimer) Newsgroups: bionet.molbio.proteins Subject: Re: km determination Date: 8 Dec 1998 03:45:52 GMT Organization: Univ. Kansas (BCMB) Lines: 43 Message-ID: <7opiGDf98QgB-pn2-d6WVPc9od3Us@rnaworld.bio.ukans.edu> References: <36687378.1A19ED8B@sciborg.uwaterloo.ca> <7opiGDf98QgB-pn2-dy9Z1ZHxmmZ4@rnaworld.bio.ukans.edu> <74cvld$dmh$1@agate.berkeley.edu> Reply-To: PGegen@UKans.nolospamare.edu NNTP-Posting-Host: rnaworld.bio.ukans.edu Mime-Version: 1.0 Content-Type: text/plain; charset="iso-8859-1" Content-Transfer-Encoding: quoted-printable X-Newsreader: ProNews/2 Version 1.00 On Sun, 6 Dec 1998 04:02:53, keith@eve.cchem.berkeley.edu (Keith Rickert) wrote: > In <7opiGDf98QgB-pn2-dy9Z1ZHxmmZ4@rnaworld.bio.ukans.edu> PGegen@UKans.nolospamare.edu (Dr. Peter Gegenheimer) writes: > > >Yes -- it IS _always_ most accurate to fit the untransformed data to the equation > >which describes the relationship between X and Y; you are guaranteed to get the best > >estimate for the kinetic constants. Not only for the reasons Simon mentions, but also > >because when you transform the variables, you also transform their experimental error > >so that it no longer has a ~Gaussian distribution. Further, the transformations in > >which one axis contains both V and S violate the assumtions of regression analysis, > >in that the X and Y axis variables are no longer separate (you no longer have an > >independent and a dependent variable), and (so certain transforms) the experimental > >error is no longer exclusively in the Y axis. > > While I generally agree with all of these points, I have to say > that I still think there's a place for the linear transform plots. > If your data has some kind of systematic deviation from > Michaelis-Menten kinetics, it's often much easier to recognize > that on a linear transform than on the non-linear plot. > So I generally do both plots, but only derive constants from > the nonlinear fit. I forgot to discuss this point: in any nonlinear curvefitting, you have to examine the plot of residuals. Indeed, the residual plot should be published along with the fitted curve. The residual plot is exactly what you'd get if you pulled the curve like a string to make it straight, and all the data points moved along with it -- in other words, it's a linear plot of the deviation of the data points from the fitted curve. For exactly the reason Keith Rickert gives, it is much easier to tell the goodness of fit from the residual plot. (I should acknowledge that the various linear plots are convenient ways to visualize various types of enzyme inhibition mechanisms. I've never used them.) o----------------------------------------------------------------------o | Dr. Peter Gegenheimer | Vox: 785-864-3939 FAX: 785-864-5321 | | Department of | PGegen@UKans.nospam.edu | | Molecular Biosciences | http://rnaworld.bio.ukans.edu/ | | & Dept. Evol Biology | | | University of Kansas |"When you have excluded the impossible, | | 2045 Haworth Hall | whatever remains, however improbable, | | Lawrence KS 66045-2106 | must be the truth." S. Holmes | o_____________________________|________________________________________o From owner-proteins@net.bio.net Mon Dec 07 22:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!easynet-fr!oleane!pasteur.fr!not-for-mail From: Pierre Rodrigues Newsgroups: bionet.molbio.proteins Subject: Imidazol induced oligomerization? Date: Tue, 08 Dec 1998 13:23:23 +0100 Organization: Institut Pasteur, Paris Lines: 15 Message-ID: <366D1A38.9FF502B9@pasteur.fr> NNTP-Posting-Host: terre.rtg.pasteur.fr Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii; x-mac-type="54455854"; x-mac-creator="4D4F5353" Content-Transfer-Encoding: 7bit X-Trace: desdemone.pasteur.fr 913119668 16079 157.99.2.24 (8 Dec 1998 12:21:08 GMT) X-Complaints-To: usenet@pasteur.fr NNTP-Posting-Date: 8 Dec 1998 12:21:08 GMT X-Mailer: Mozilla 4.5 (Macintosh; I; PPC) X-Accept-Language: en Hello, Does anybody knows if imidazol (used as eluant of his tag purification) can induce or at least strongly stabilize protein oligomerization? This because when I purify a receptor by histag technique I always obtain it as if it was an oligomer (detected by western-blot), even resistant to SDS-PAGE. When I look at it directly by western-blot on crude extracts I have it mainly as a monomer with some higher bands. So, could imidazol have stabilize the interaction? thanks From owner-proteins@net.bio.net Mon Dec 07 22:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!newshub.northeast.verio.net!newsserver.jvnc.net!dsinc!nntp.upenn.edu!obrien From: obrien@pharm.med.upenn.edu (Peter O'Brien) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: FPLC - Superdex 200HR vs. Superose 12HR columns Date: 8 Dec 1998 19:54:07 GMT Organization: University of Pennsylvania Lines: 2 Message-ID: References: <746bgu$1886$1@news.doit.wisc.edu> NNTP-Posting-Host: zolot02.med.upenn.edu X-Newsreader: MT-NewsWatcher 2.4.4 Xref: biosci bionet.molbio.methds-reagnts:72759 bionet.molbio.proteins:13670 I don't recall which, but one of the two (I think it's superdex) has somewhat greater tailing of peaks due to hydrophobic interactions. From owner-proteins@net.bio.net Mon Dec 07 22:00:00 1998 Path: biosci!NERVM.NERDC.UFL.EDU!pcs From: pcs@NERVM.NERDC.UFL.EDU (Paul Sehnke) Newsgroups: bionet.molbio.proteins Subject: Non reducible protein crosslinking of recombinant proteins question Date: 8 Dec 1998 08:42:59 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 19 Sender: daemon@net.bio.net Distribution: world Message-ID: <366D563B.38427C45@nervm.nerdc.ufl.edu> NNTP-Posting-Host: net.bio.net Hello, I had a question about a topic that I had heard discussed in a seminar some years ago. This has to do with the problem associated with purified proteins ( recombinant in this case) spontaneously crosslinking upon storage. By crosslinking I mean mulitmerization of the protein monomer which is not reducible in an Laemmli SDS -PAGE system. This also ocurs in various tissues. I believe it had something to do with amide bond formation between acidic proteins subunit. We are have a problem with certain plant tissues generating high percentages of these non-reducible multimers ( added 100 mM DTT/1% SDS- no effect) for several acidic proteins that we are studying. Any ideas regarding potential inhibitors to prevent this during tissue prep? We can switch to different buffers easliy if necessary. We usually use PBST or TBST 7.6. Any and all help would be most appreciated. Paul Sehnke UF From owner-proteins@net.bio.net Mon Dec 07 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!server4.netnews.ja.net!news.icnet!NewsWatcher!user From: i.mcfarlane@icrf.icnet.uk (Ian McFarlane) Newsgroups: bionet.molbio.proteins Subject: Re: Cleavage of glycosidic moiety Date: 8 Dec 1998 16:03:04 GMT Organization: Imperial Cancer Research Fund Lines: 54 Message-ID: References: <74egng$5ff$1@gwsun.medinf.mu-luebeck.de> <366c0c56.30400348@newsserv.zdv.uni-tuebingen.de> NNTP-Posting-Host: 143.65.17.54 However, There is another way. There is a method involving running several different concentrations of SDS-PAGE from which you can derive a plot which will give you a 'protein-only' M.Wt. More details are in the Gel Electrophoresis book of the Practical Series. Sorry I can't give you any more details. Ian Mc > "Lars Komorowski" wrote: > > >Who can tell my an efficient and inexpensive way to quantitatively remove > >the glycosidic moiety of a glycosidated protein to determine the mass of the > >unglycosilated protein ? > > Dear Lars, > > although I've no experience in such things, here's a message which was > posted in this group a while ago. Hope it helps, > Harold V. Taylor > > ********************** > Trifluoromethanesulfonic acid TMFSA (also called triflic acid. There > is a paper by J. Horvath 1989 J. Neurosci. Res. which gives a simple > method to follow. There are a few different methods to follow, the > main difference between them is the completeness of removal of CHO, > some will not remove the last GlcNAc/GalNAc residues. But for antibody > production this is not usually crucial, this process gives undenatured > proteins which you can use for Ab production. > > If you've got any more questions try the bionet.glycosci newsgroup or > drop me an e-mail. > > Ian McFarlane > > In article > , > Anders Steenfeldt wrote: > > > Does anyone know of a non-enzymatic way to deglycosylate a protein without > > rendering it denatured? > > Please mail me if You do. > > Anders Steenfeldt > > Email torin@imsb.au.dk > > Harold V. Taylor > Physiol. Chem. Institut > Hoppe-Seyler-Str. 4 > D-72076 Tübingen > > phone: 07071-297-3353 > fax: 07071-29-4182 > email: cbiei01@uni-tuebingen.de From owner-proteins@net.bio.net Mon Dec 07 22:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!newsfeed.nyu.edu!newsfeed.sgi.net!nntp.itis.com!news.doit.wisc.edu!Home From: klenchin@REMOVE_TO_REPLY.facstaff.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins Subject: Re: Imidazol induced oligomerization? Date: Tue, 08 Dec 1998 14:16:02 GMT Organization: UW Lines: 14 Message-ID: <74jcb7$ko8$1@news.doit.wisc.edu> References: <366D1A38.9FF502B9@pasteur.fr> NNTP-Posting-Host: t-35-182-197.dialup.wisc.edu X-Newsreader: News Xpress 2.01 X-No-Archive: Yes In article <366D1A38.9FF502B9@pasteur.fr>, Pierre Rodrigues wrote: >Hello, > >Does anybody knows if imidazol (used as eluant of his tag purification) >can induce or at least strongly stabilize protein oligomerization? > >This because when I purify a receptor by histag technique I always >obtain it as if it was an oligomer (detected by western-blot), even >resistant to SDS-PAGE. When I look at it directly by western-blot on >crude extracts I have it mainly as a monomer with some higher bands. So, >could imidazol have stabilize the interaction? NO. From owner-proteins@net.bio.net Mon Dec 07 22:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!news.maxwell.syr.edu!newsfeed.cwix.com!203.97.37.7!newsfeed.clear.net.nz!news.wlg.netlink.net.nz!not-for-mail From: Thomas Buckley Newsgroups: bionet.molbio.proteins Subject: ATPase secondary structure Date: Wed, 09 Dec 1998 16:09:40 +1300 Organization: VUW Lines: 9 Message-ID: <366DE9F4.93B624FC@vuw.ac.nz> Reply-To: Thomas.Buckley@vuw.ac.nz NNTP-Posting-Host: totara.its.vuw.ac.nz Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.04 [en] (Win95; I) Cache-Post-Path: totara.its.vuw.ac.nz!unknown@dhcp0717.sbs.vuw.ac.nz X-Cache: nntpcache 2.3.2.1 (see http://www.nntpcache.org/) Can someone please give me the most recent reference for the secondary structure of the ATPase protein. I am particularly interested in mitochondrial subunits 6 and 8. Please email me at: Thomas.Buckley@vuw.ac.nz Thanks, Thomas From owner-proteins@net.bio.net Mon Dec 07 22:00:00 1998 Path: biosci!cnn.nas.nasa.gov!newsfeed.berkeley.edu!news.maxwell.syr.edu!cpk-news-hub1.bbnplanet.com!news.gtei.net!rill.news.pipex.net!pipex!uunet!in2.uu.net!netnews.mc.net!not-for-mail From: "Blake Frantz" Newsgroups: bionet.molbio.proteins Subject: is this safe? -- correct reply address Date: Tue, 8 Dec 1998 20:33:23 -0600 Organization: McHenryCom Company, Chicago IL, USA Lines: 16 Message-ID: <74kn9k$re8$1@netnews.mc.net> NNTP-Posting-Host: woodstock1modem56.mc.net X-Newsreader: Microsoft Outlook Express 4.72.3110.1 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3110.3 In 1975 an Artical was pulished in the "Journal of Biological Chemistry." called "The Extraction & Purification of the Kidney Bean" written by Dr. Marshall and Dr. Lauda. The artical describes a process that produces a protein which produces a starch enzym. This enzym when taken prohibits the bodies ability to obsorb carbohydrates. Does any one know of this process, or know where I can obtain more info on it? Has anyone performed this and what were the results? And most importantly is it safe? Any help whould be most appreciated. Please reply by e-mail FFrantz666@aol.com From owner-proteins@net.bio.net Mon Dec 07 22:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!nntp2.dejanews.com!nnrp1.dejanews.com!not-for-mail From: jean-christophe.ravarini@genebio.com Newsgroups: bionet.molbio.proteins Subject: Retreive a MEDLINE entry ? Date: Tue, 08 Dec 1998 12:44:59 GMT Organization: Deja News - The Leader in Internet Discussion Lines: 16 Message-ID: <74j70b$mkm$1@nnrp1.dejanews.com> NNTP-Posting-Host: 194.6.173.103 X-Article-Creation-Date: Tue Dec 08 12:44:59 1998 GMT X-Http-User-Agent: Mozilla/4.0 (compatible; MSIE 4.01; Windows NT) X-Http-Proxy: 1.1 x5.dejanews.com:80 (Squid/1.1.22) for client 194.6.173.103 Hi, I need to be able to automatically retreive a MEDLINE entry, using http://www.ncbi.nlm.nih.gov/PubMed or using FTP. Does anyone knows if flat-format MEDLINE entrys could be retreived that way ? Any help appreciated: thanks in advance. Regards, -- Jean Christophe Ravarini, Software Developper mailto:jean-christophe.ravarini@genebio.com -----------== Posted via Deja News, The Discussion Network ==---------- http://www.dejanews.com/ Search, Read, Discuss, or Start Your Own From owner-proteins@net.bio.net Tue Dec 08 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!rill.news.pipex.net!pipex!nntp.news.xara.net!xara.net!server6.netnews.ja.net!daresbury!not-for-mail From: Henning Pluecken Newsgroups: bionet.molbio.proteins Subject: Iodo acetamide blocking Date: 9 Dec 1998 17:09:52 -0000 Organization: Daresbury Laboratory, Warrington, U.K. Lines: 19 Message-ID: <74mat0$1el$1@mserv2.dl.ac.uk> NNTP-Posting-Host: mserv2.dl.ac.uk Original-To: proteins@dl.ac.uk content-length: 393 Return-Path: X-Sender: pluecken@mail.rz.uni-duesseldorf.de (Unverified) Hello everyone, does anyone know a recipe for blocking reduced SH-groups of proteins by using iodoacetamide in protein samples prior to loading them on an SDS-PAGE? Thanks for your help, Henning Pluecken Institut fuer Entwicklungs- und Molekularbiologie der Pflanzen Heinrich-Heine-Universitaet Duesseldorf Universitaetsstrasse 1 40225 Duesseldorf pluecken@mail.rz.uni-duesseldorf.de From owner-proteins@net.bio.net Wed Dec 09 22:00:00 1998 Path: biosci!news.stanford.edu!logbridge.uoregon.edu!nntp2.dejanews.com!nnrp1.dejanews.com!not-for-mail From: vinsin@my-dejanews.com Newsgroups: bionet.molbio.proteins Subject: Probing for lipase gene probe Date: Thu, 10 Dec 1998 13:37:30 GMT Organization: Deja News - The Leader in Internet Discussion Lines: 18 Message-ID: <74oiqq$8tv$1@nnrp1.dejanews.com> NNTP-Posting-Host: 202.54.56.36 X-Article-Creation-Date: Thu Dec 10 13:37:30 1998 GMT X-Http-User-Agent: Mozilla/4.06 [en] (Win95; I) X-Http-Proxy: 1.0 charak.sgpgi.ac.in:8080 (Squid/1.1.21), 1.0 x5.dejanews.com:80 (Squid/1.1.22) for client 192.100.10.251, 202.54.56.36 Dear friends, WE are trying to clone lipase gene from indegenous fungal and bacterial strains. Can anyone of you help us out by giving us information from where we can have universal probe for the purpose OR kindly gift us the probe it self. We will duely acknoledge the gift in all of our correspondaces. Thanks in advance. Anjna Dixit Division of Biochemistry Central Drug Research Institute, LUCKNOW, INDIA e-mail: root@cscdri.rec.nic.in -----------== 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 Dec 09 22:00:00 1998 Path: biosci!MANI.CBS.UMN.EDU!np From: np@MANI.CBS.UMN.EDU (Nora Plesofsky) Newsgroups: bionet.molbio.proteins Subject: antibody against ribosomal protein Date: 10 Dec 1998 09:43:05 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 8 Sender: daemon@net.bio.net Distribution: world Message-ID: <9812101736.AA01471@mani.cbs.umn.edu> Reply-To: nora@biosci.cbs.umn.edu NNTP-Posting-Host: net.bio.net We are interested in finding antibody against a protein in the large ribosomal subunit, L10, of eukaryotes. If anyone can tell me a personal or commercial source for this, I would appreciate it. Thanks alot. Nora Plesofsky nora@biosci.cbs.umn.edu From owner-proteins@net.bio.net Wed Dec 09 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!204.238.120.130!news-feeds.jump.net!nntp2.dejanews.com!nnrp1.dejanews.com!not-for-mail From: vinsin@my-dejanews.com Newsgroups: bionet.molbio.proteins Subject: Probing for lipase gene probe Date: Thu, 10 Dec 1998 13:36:21 GMT Organization: Deja News - The Leader in Internet Discussion Lines: 18 Message-ID: <74oiol$8td$1@nnrp1.dejanews.com> NNTP-Posting-Host: 202.54.56.36 X-Article-Creation-Date: Thu Dec 10 13:36:21 1998 GMT X-Http-User-Agent: Mozilla/4.06 [en] (Win95; I) X-Http-Proxy: 1.0 charak.sgpgi.ac.in:8080 (Squid/1.1.21), 1.0 x4.dejanews.com:80 (Squid/1.1.22) for client 192.100.10.251, 202.54.56.36 Dear friends, WE are trying to clone lipase gene from indegenous fungal and bacterial strains. Can anyone of you help us out by giving us information from where we can have universal probe for the purpose OR kindly gift us the probe it self. We will duely acknoledge the gift in all of our correspondaces. Thanks in advance. Anjna Dixit Division of Biochemistry Central Drug Research Institute, LUCKNOW, INDIA e-mail: root@cscdri.rec.nic.in -----------== 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 Dec 09 22:00:00 1998 Path: biosci!SLIP.NET!grizzly From: grizzly@SLIP.NET (Michael Sherrell) Newsgroups: bionet.molbio.proteins Subject: ACT 396 for sale Date: 10 Dec 1998 20:20:47 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 14 Sender: daemon@net.bio.net Distribution: world Message-ID: <01BE247A.0D409960@oak-pm2-1-193.dialup.slip.net> NNTP-Posting-Host: net.bio.net For sale: 1998 Advanced Chemtech Model 396 Multiple Biomolecular Synthesizer, unused, ~2/3 of new price. Also available: Perkin Elmer Sciex API III+ LC/MS/MS, electrospray, APCI, $79,000 Hewlett Packard 5989B LC/MS, electrospray, extend mass range, 1995 model, $45,000 Plus many ABI and other sequencers and synthesizers (and service arrangements for synthesizers), flow cytometers, NMRs and SEMs listed on my website. Michael Sherrell Grizzly Analytical 707 887 2919/fax 707 887 9834 www.grizzlyanalytical.com From owner-proteins@net.bio.net Wed Dec 09 22:00:00 1998 Path: biosci!news.stanford.edu!logbridge.uoregon.edu!cyclone.bc.net!crc-news.crc.ca!news.drenet.dnd.ca!newshost.dres.dnd.ca!cwbinc.dres.dnd.ca!user From: tdlaing@nospam.dres.dnd.ca (T.D. Laing) Newsgroups: bionet.molbio.proteins Subject: Re: Retreive a MEDLINE entry ? Date: 10 Dec 1998 17:37:30 GMT Organization: Canada West Biosciences Lines: 24 Message-ID: References: <74j70b$mkm$1@nnrp1.dejanews.com> NNTP-Posting-Host: cwbinc.dres.dnd.ca In article <74j70b$mkm$1@nnrp1.dejanews.com>, jean-christophe.ravarini@genebio.com wrote: > Hi, > > I need to be able to automatically retreive a MEDLINE entry, > using http://www.ncbi.nlm.nih.gov/PubMed or using FTP. > Does anyone knows if flat-format MEDLINE entrys > could be retreived that way ? > Any help appreciated: thanks in advance. Can't you just select the desired abstracts, scroll down to the bottom of the abstracts page and use the save feature to download them? You can download in PC, Mac or Unix format. T. -- T.D. Laing tdlaing@dres.dnd.ca Remove "nospam" from my e-mail address in the header to reply. All the standard disclaimers apply. From owner-proteins@net.bio.net Thu Dec 10 22:00:00 1998 Path: biosci!news.stanford.edu!logbridge.uoregon.edu!nntp2.dejanews.com!nnrp1.dejanews.com!not-for-mail From: tgururaja@rigelinc.com Newsgroups: bionet.molbio.proteins Subject: 2D-gel Database Creation Date: Fri, 11 Dec 1998 18:21:52 GMT Organization: Deja News - The Leader in Internet Discussion Lines: 13 Message-ID: <74rnrt$1lj$1@nnrp1.dejanews.com> NNTP-Posting-Host: 38.170.6.2 To: tgururaja@rigel.com X-Article-Creation-Date: Fri Dec 11 18:21:52 1998 GMT X-Http-User-Agent: Mozilla/4.01aC-DIAL (Macintosh; U; PPC) X-Http-Proxy: 1.0 x11.dejanews.com:80 (Squid/1.1.22) for client 38.170.6.2 Hi I am planning to setup a 2D-gel Database which can be latter accessed via Web browser. Can someone tell me, is there any software available to do so? I really appreciate your help in this regard. Thanking you. Guru -----------== Posted via Deja News, The Discussion Network ==---------- http://www.dejanews.com/ Search, Read, Discuss, or Start Your Own From owner-proteins@net.bio.net Thu Dec 10 22:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!news-peer1.sprintlink.net!news-backup-west.sprintlink.net!news.sprintlink.net!itssrv1.ucsf.edu!mac-daddy.ucsf.edu!user From: bpmurray*STUFFER*@socrates.ucsf.edu (Bernard P. Murray, PhD) Newsgroups: bionet.molbio.proteins Subject: Re: 2D-gel Database Creation Date: Fri, 11 Dec 1998 13:57:34 -0800 Organization: University of California, San Francisco Lines: 20 Message-ID: References: <74rnrt$1lj$1@nnrp1.dejanews.com> NNTP-Posting-Host: mac-daddy.ucsf.edu In article <74rnrt$1lj$1@nnrp1.dejanews.com>, tgururaja@rigelinc.com wrote: > Hi > > I am planning to setup a 2D-gel Database which can be latter > accessed via Web browser. Can someone tell me, is there any > software available to do so? I really appreciate your help > in this regard. > Thanking you. > Guru You may want to have a look at the ExPASy web site as they have a variety of 2D gel databases - nicely linked to SwissProt etc. Check them out at: http://expasy.hcuge.ch/ch2d/ Bernard -- Bernard P. Murray, PhD Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA From owner-proteins@net.bio.net Thu Dec 10 22:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!isdnet!pasteur.fr!not-for-mail From: Pierre Rodrigues Newsgroups: bionet.molbio.proteins Subject: actin blotting Date: Fri, 11 Dec 1998 10:11:42 +0100 Organization: Institut Pasteur, Paris Lines: 14 Message-ID: <3670E1CA.D348A71E@pasteur.fr> NNTP-Posting-Host: terre.rtg.pasteur.fr Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii; x-mac-type="54455854"; x-mac-creator="4D4F5353" Content-Transfer-Encoding: 7bit X-Trace: desdemone.pasteur.fr 913367356 24630 157.99.2.24 (11 Dec 1998 09:09:16 GMT) X-Complaints-To: usenet@pasteur.fr NNTP-Posting-Date: 11 Dec 1998 09:09:16 GMT X-Mailer: Mozilla 4.5 (Macintosh; I; PPC) X-Accept-Language: en Hi! I am trying to detect actin in western-blot after several treatments. I am using sigma AC-40 mAb. I can see in the blots a few additional bands at higher MW, which appear to be multiples of actin monomer. Can anybody tell me if these bands could be specific, or could be due to cross-reaction of the Ab with some parasitic band (when using the AC-40 Ab)? Is there any way to detect actin small order oligomers by gentle SDS-PAGE/WB of cell extracts? thanks Pierre Rodrigues From owner-proteins@net.bio.net Thu Dec 10 22:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!howland.erols.net!newscore.univie.ac.at!aconews.univie.ac.at!news.univie.ac.at!not-for-mail From: Christian Radauer Newsgroups: bionet.molbio.proteins Subject: Re: Imidazol induced oligomerization? Date: Fri, 11 Dec 1998 19:38:06 +0100 Organization: Dept. of General and Experimental Pathology, University of Vienna Lines: 25 Message-ID: <3671668E.605AAABF@emb1.bcc.univie.ac.at> References: <366D1A38.9FF502B9@pasteur.fr> <74jcb7$ko8$1@news.doit.wisc.edu> NNTP-Posting-Host: lemon.expatho.akh-wien.ac.at Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: www.univie.ac.at 913397912 95810 149.148.108.43 (11 Dec 1998 17:38:32 GMT) X-Complaints-To: news-adm@news.univie.ac.at NNTP-Posting-Date: 11 Dec 1998 17:38:32 GMT X-Mailer: Mozilla 4.5 [en] (Win95; I) X-Accept-Language: de,en,fr Dima Klenchin wrote: > > In article <366D1A38.9FF502B9@pasteur.fr>, Pierre Rodrigues wrote: > >Hello, > > > >Does anybody knows if imidazol (used as eluant of his tag purification) > >can induce or at least strongly stabilize protein oligomerization? > > > >This because when I purify a receptor by histag technique I always > >obtain it as if it was an oligomer (detected by western-blot), even > >resistant to SDS-PAGE. When I look at it directly by western-blot on > >crude extracts I have it mainly as a monomer with some higher bands. So, > >could imidazol have stabilize the interaction? > > NO. Hi! Indeed it is not the imidazol that causes oligomerization, but the His-tag, which is crosslinked by heavy metal ions. I have not tried it yet, but maybee adding EDTA to the PAGE sample buffer would help. Christian Radauer Institute of General and Experimental Pathology University of Vienna, Austria From owner-proteins@net.bio.net Thu Dec 10 22:00:00 1998 Path: biosci!news.stanford.edu!logbridge.uoregon.edu!newsfeed.direct.ca!torn!kwon!watserv3.uwaterloo.ca!not-for-mail From: Michael Allen Newsgroups: bionet.molbio.proteins Subject: Adenosine Kinase and ATP Date: Fri, 11 Dec 1998 10:32:35 -0500 Organization: University of Waterloo Lines: 29 Message-ID: <36713B13.E4ADDCB4@sciborg.uwaterloo.ca> NNTP-Posting-Host: gelrecdr.uwaterloo.ca Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.05 [en] (Win95; I) I am a Master's student working on an adenosine kinase assay. To cut the story short, I made a new batch of 0.1 M ATP (free acid from ICN) to use in the assay (in Hepes pH 7.2). The powder was dissolved in 85mM Hepes buffer, pH 7.5. The initial pH of the soln was 6.9, and I adjusted this to 7. I tested this against a previous batch of ATP which was made up with water (probably a bad idea, in retrospect) which had an initial pH of 3 (or so) which I adjusted quickly to 7. The interesting (read: frustrating) part of all this is that the ATP made with the water gives a higher count incorporation than the ATP made with Hepes. I had thought as a result of assay results that the ATP made with water was degraded, and that was why I made the new ATP. Is there any reason why having more ADP or AMP could increase the conversion of adenosine to AMP by Adenosine Kinase? I am at a loss as to what to do. How are stock solutions of ATP usually made, and in what buffer? Thanks in advance Mike Allen University Of Waterloo Ontario, Canada m3allen@sciborg.uwaterloo.ca From owner-proteins@net.bio.net Thu Dec 10 22:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!ihnp4.ucsd.edu!news.scripps.edu!not-for-mail From: Don Bashford Newsgroups: bionet.molbio.proteins Subject: Re: Nernst equation problems/information anyone?? Date: 11 Dec 1998 10:31:46 -0800 Organization: The Scripps Research Institute, La Jolla, CA, USA Lines: 4 Message-ID: References: NNTP-Posting-Host: gage.scripps.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Newsreader: Gnus v5.5/Emacs 20.2 Have a look at "Ionic Channels of Excitable Membranes" by Bertil Hille. Also, a good P-Chem or Stat Phys book might help clarify why logs of concentrations (or activities) are related to potentials. From owner-proteins@net.bio.net Fri Dec 11 22:00:00 1998 Message-ID: <3671F418.774@antibodyresource.com> Date: Fri, 11 Dec 1998 22:43:33 -0600 From: The Antibody Resource Page X-Mailer: Mozilla 3.01-C-MACOS8 (Macintosh; I; PPC) MIME-Version: 1.0 Newsgroups: bionet.molbio.proteins Subject: Looking for custom monoclonal/polyclonal antibodies? Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit NNTP-Posting-Host: master1-102.dialup.chi.idsonline.com Lines: 9 Path: biosci!agate!newsfeed.berkeley.edu!newsfeed.enteract.com!chicago-news-feed1.bbnplanet.com!news.gtei.net!news.idsonline.com!master1-102.dialup.chi.idsonline.com The Antibody Resource Page (http://www.antibodyresource.com/) now maintains a list of custom antibody suppliers. If you are interested in the production of custom monoclonal or polyclonal antibodies take a look at this invaluable guide at: http://www.antibodyresource.com/customantibody.html ps. The ARP was voted among the top 25 biotechnology webpages for 1997 by Genetic Engineering News! From owner-proteins@net.bio.net Fri Dec 11 22:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!newsfeed.wirehub.nl!news.belnet.be!news.vub.ac.be!not-for-mail From: laurent houzet Newsgroups: bionet.molbio.proteins Subject: protein secretion inhibitor... Date: Sat, 12 Dec 1998 22:29:25 +0100 Organization: Brussels Free Universities VUB/ULB Lines: 13 Message-ID: <3672E035.127EAF7E@alize.ulb.ac.be> NNTP-Posting-Host: 134.184.80.128 Mime-Version: 1.0 Content-Type: text/html; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.5 [en] (Win95; I) X-Accept-Language: en Hi everybody,
I'm working with transfected Hela cells producing a secreted protein; I would like to detect this protein in cells by immunoflurescence and in extract cells by western blott to caracterise it. I tried few times with different conditions but I never saw something. I think it's because of the small quantity present inside the cell, so I wondered if anybody knew a protein secretion inhibitor which would make it accumulating into the cell,

Thanks a lot,
Laurent From owner-proteins@net.bio.net Fri Dec 11 22:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!newsfeed.cwix.com!198.82.160.249!solaris.cc.vt.edu!news.vt.edu!not-for-mail From: "Ashley" Newsgroups: bionet.molbio.proteins Subject: Please help!! Date: Sat, 12 Dec 1998 11:31:56 -0500 Organization: Virginia Tech, Blacksburg, Virginia, USA Lines: 46 Message-ID: <74u5lk$jgt$1@solaris.cc.vt.edu> NNTP-Posting-Host: apuig.campus.vt.edu X-Trace: solaris.cc.vt.edu 913480180 19997 198.82.96.178 (12 Dec 1998 16:29:40 GMT) X-Complaints-To: abuse@vt.edu NNTP-Posting-Date: 12 Dec 1998 16:29:40 GMT X-Newsreader: Microsoft Outlook Express 4.72.3110.1 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3110.3 This is the third time I've tried to post this because it wouldn't let me post to multiple newsgroups!! To whom it may concern: I am a freshman at Virginia Tech, and I need help with an Honors Biology assignment. This is our final exam grade and is called a "creative experience." We are allowed to ask anyone for ideas, except people in our class. Here is the problem: We read a sci-fi story called "The Giving Plague" by David Brin. It was actually interesting and I highly recommend it to anyone! It is about a fictional disease called ALAS (Acquired Lavish Altruism Syndrome), which is a blood-to-blood transmitted virus which causes its victims to acquire a false altruism, beginning with the insatiable desire to donate blood. If you have ideas on any one or more of the following questions, please email me at apuig@vt.edu. Thanks in advance!! :) (By the way, this is not the entire set of questions- I am not asking anyone to do my homework for me! I just need more ideas) They are worded a little differently to avoid having to explain the entire story: 1. Design an experiment to test if another (deadly) virus evolved from ALAS. What data would you expect to get from your experimental design and how would you interpret these data? 2. The ALAS genome may incorporate itself into the host genome. How would you determine if it does? How would you separate it from the nucleotide sequences of the host organism? (I know that restriction enzymes are used to cut specific sequences- but are there any problems that might arise from this being a virus??) 3. You are on a team to design a way to prevent the spread of ALAS or inhibit it from propagating in the host. How could you achieve this goal specifically? Again, thanks to anyone who takes the time to answer any of the above questions. I really appreciate your assistance!! 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If you have received this message in error, reply with the word unsubscribe in the subject. * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * From owner-proteins@net.bio.net Sat Dec 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 Dec 1998 02:00:10 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 233 Sender: daemon@net.bio.net Distribution: world Message-ID: <199812131000.CAA08316@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 newsgroup. The discussions leaders and their e-mail addresses are listed in the BIOSCI Information Sheet which is available on the Web at http://www.bio.net/. If a newsgroup is being hit with too many junk postings, please contact the discussion leader for that group and see if there is interest in moderating the group. Please do not assume that by simply posting a complaint to the newsgroup itself, anyone on the BIOSCI staff will act on your complaint. With close to 100 newsgroups to run, the BIOSCI staff has to rely on the discussion leaders of each newsgroup to report problems directly to us at biosci-help@net.bio.net. We will moderate any of our newsgroups if the discussion leader tells us that the readership of the group wishes to do so and if a moderator is willing to do the work. For most BIOSCI/bionet groups, this entails only a few minutes of work each day. Moderating a newsgroup will resolve probably 95% of the junk postings on the USENET distribution. Unfortunately there are easy ways for determined spammers to override the moderation mechanism on USENET, but we can protect our e-mail subscribers from unwanted postings if the newsgroup is moderated. You can also access our newsgroups over the WWW at URL http://www.bio.net. While this Web interface will not stop spammers from trying to post to the groups, this will give you yet another way, besides using USENET news, to keep the junk out of your personal mail files. For those of you with local USENET news systems, the Web interface will also give you faster access to new newsgroups and recent postings. 3) Examples of subscribing and unsubscribing to the mailing lists. ------------------------------------------------------------------ PLEASE NOTE: The BIOSCI management does NOT act on subscription/unsubscription requests that are posted improperly to the newsgroups and mailing lists. People who do this only bother everyone on the lists to no avail. Please be sure to follow the proper procedures below. Gory details are in the BIOSCI Information sheets on the Web at http://www.bio.net. Below we give an example utilizing the METHODS-AND-REAGENTS list at both of our two BIOSCI sites: Users in the Americas and Pacific Rim countries who use the BIOSCI ------------------------------------------------------------------ node at computer net.bio.net: ---------------------------- A) Determine the "listname" which is the <=8 character mail address ^^^^^^^^^^^^^ for the group. These can be found in the BIOSCI Info. Sheet. For the METHODS-AND-REAGENTS group the mailing address is methods@net.bio.net. The listname is the portion of the address to the left of the @ sign, i.e., "methods". The listname is used with the "subscribe" and "unsubscribe" commands illustrated below. B) Mail all commands in the body of a mail message addressed to biosci-server@net.bio.net. Do NOT send commands to the newsgroup posting addresses! Leave the Subject: line blank, any text on it will be ignored. C) In the body of your message put one or more of the following commands with an "end" command on the last line, e.g., subscribe methods unsubscribe methods end Do NOT put your e-mail address or other text on these lines. The server only allows you to cancel your subscription if the address on your mail header matches the address on our mailing list. Please ask for help at biosci-help@net.bio.net if your address has changed, e.g., if you know you are on the list but the server tells you that you are not a member. Users in Europe, Africa, and Central Asia who use the BIOSCI node at -------------------------------------------------------------------- computer daresbury.ac.uk (also known as dl.ac.uk): ------------------------------------------------- To subscribe and unsubscribe to/from the BIOSCI lists, you need to specify the full USENET newsgroup name with "bionet-news." prepended. The USENET newsgroup names are listed in the BIOSCI Information sheet on the Web at http://www.bio.net/. For the METHODS-AND-REAGENTS list the USENET newsgroup name is bionet.molbio.methds-reagnts, thus the appropriate commands are sub bionet-news.bionet.molbio.methds-reagnts unsub bionet-news.bionet.molbio.methds-reagnts These commands are included in a message addressed to mxt@dl.ac.uk, NOT to the newsgroup mailing addresses. As usual, include the text in the body of the message as text on the Subject: line is ignored. To unsubscribe from all the lists at the UK node, use unsub bionet-news Please note that if the address in the list is different than the one in your mail message header, you will not be able to unsubscribe by this method. If you have problems, please mail biosci@daresbury.ac.uk. 4) The BIOSCI user address and research interest directory. ----------------------------------------------------------- Please take this opportunity to add your name, address, and research interest information to the BIOSCI User Address Database if you have not already done so. You can fill out the address form directly through our Web page at URL http://www.bio.net/adrform.html. The address database is reindexed nightly for WWW access (the URL is http://www.bio.net/). If you are not directly on the Internet but can reach it by e-mail, please use our waismail server to access the user directory. waismail use is described above. You can also request a user address form by e-mail from biosci-help@net.bio.net. Please check your database entry from time-to-time to see if your address information is still up-to-date. Because of our limited personnel resources, we ask that you resubmit a *complete* form to revise your entry; we only replace complete entries and do not have resources to edit old forms. From owner-proteins@net.bio.net Sat Dec 12 22:00:00 1998 Path: biosci!bcm.tmc.edu!ah690549 From: ah690549@bcm.tmc.edu (Annette C. Hollmann) Newsgroups: bionet.molbio.proteins Subject: Re: actin blotting Date: 13 Dec 1998 19:25:04 GMT Organization: Baylor College of Medicine, Houston, Tx Lines: 32 Message-ID: <7514ag$h9a@ns2.bcm.tmc.edu> References: <3670E1CA.D348A71E@pasteur.fr> NNTP-Posting-Host: condor.mbcr.bcm.tmc.edu In article <3670E1CA.D348A71E@pasteur.fr> Pierre Rodrigues writes: >Hi! > >I am trying to detect actin in western-blot after several treatments. I >am using sigma AC-40 mAb. I can see in the blots a few additional bands >at higher MW, which appear to be multiples of actin monomer. Can anybody >tell me if these bands could be specific, or could be due to >cross-reaction of the Ab with some parasitic band (when using the AC-40 >Ab)? >Is there any way to detect actin small order oligomers by gentle >SDS-PAGE/WB of cell extracts? I have tried to use this same antibody for Western blot, and never did get it to work. AC-40 works marvellously for immunofluorescence, but appears to be useless for Western blots. Annette From owner-proteins@net.bio.net Sat Dec 12 22:00:00 1998 Path: biosci!news.stanford.edu!logbridge.uoregon.edu!news6.mr.net!solomon.io.com!news.tamu.edu!not-for-mail From: Ernie Maynard Newsgroups: bionet.molbio.proteins Subject: enzyme kinetics newsgroup Date: Sun, 13 Dec 1998 18:24:12 -0600 Organization: Texas A&M University Lines: 13 Message-ID: <36745AAC.30BE@mail.chem.tamu.edu> NNTP-Posting-Host: burgess7.chem.tamu.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit NNTP-Posting-Date: 14 Dec 1998 00:22:05 GMT X-Mailer: Mozilla 3.04Gold (Win95; I) Is there a good enzyme kinetics news group out there??? I am interseted in allosteric interactions between proteins and the kinetics describing such systems. -- Ernie Maynard Graduate Student Department of Chemistry Texas A&M University Phone: 409-845-4724 Fax: 409-845-4719 From owner-proteins@net.bio.net Sat Dec 12 22:00:00 1998 Path: biosci!news.stanford.edu!logbridge.uoregon.edu!news.maxwell.syr.edu!newsfeed.cwix.com!198.82.160.249!solaris.cc.vt.edu!news.vt.edu!not-for-mail From: "Ashley" Newsgroups: bionet.molbio.proteins Subject: Thank you from "Please help" Date: Mon, 14 Dec 1998 02:15:53 -0500 Organization: Virginia Tech, Blacksburg, Virginia, USA Lines: 13 Message-ID: <752dt3$sjg$1@solaris.cc.vt.edu> NNTP-Posting-Host: apuig.campus.vt.edu X-Trace: solaris.cc.vt.edu 913619683 29296 198.82.96.178 (14 Dec 1998 07:14:43 GMT) X-Complaints-To: abuse@vt.edu NNTP-Posting-Date: 14 Dec 1998 07:14:43 GMT X-Newsreader: Microsoft Outlook Express 4.72.3110.1 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3110.3 To everybody-- A big thank you to those who made an effort to respond to my post about the ALAS virus. Once again, I really appreciated your help. To those who had read my message and thought about sending a reply, I believe I already have some good info to go with, so I will not be needing any more replies. Many thanks if you were thinking about doing so, and especially to those who already did! God bless!! Ashley :) From owner-proteins@net.bio.net Sat Dec 12 22:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!news-peer.gip.net!news-lond.gip.net!news.gsl.net!gip.net!newsfeed.uk.ibm.net!news.ibm.net.il!ibm.net!news.biu.ac.il!news.huji.ac.il!mangal.cs.huji.ac.il!jill From: Bejerano Gill Newsgroups: bionet.molbio.proteins Subject: the concept of a protein family Date: Sun, 13 Dec 1998 16:33:37 +0200 Organization: The hebrew University of Jerusalem Lines: 40 Message-ID: NNTP-Posting-Host: centauri.cs.huji.ac.il Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII Greetings All, Being a newcomer to (computational) molecular biology I would like to try and raise a couple of related points regarding proteins and protein classification. I find these issues more on the CONCEPTUAL level, and have thus opted for this newsgroup. Feel free to direct me elsewhere (I have refrained from cross-posting). Is the concept "Protein family" really so undefined as some of the lit. claims it to be? 1) does the term refer to a group of homologous proteins? if so - how far back should their common ancestor be? is that measured in time, in sequence similarity or perhaps in function similarity? 2) or does it refer to functionally analogous proteins? how different is this concept from the previous? Is convergent evolution the only reason to blame? how common is it? 3) Or is it simply that every database proprietor (eg PROSITE, Pfam etc.) simply devises, directly or indirectly, his own "taxonomy" into groups? 4) Is it therefore for one to deduce that no real benchmark or criterion for comparing any two such taxonomies exists, except one's personal taste? 5) Or perhaps all these methods should be compared only in terms of the use researchers find in them - say, when a new protein sequence is searched against them? 6) What is exactly the picture there? It seems the wealth of partial tools as well as DBs is rather overwhelming... Well, hope I haven't tired you all :-) Answers, opinions and refs. would be most welcomed! - Gill p.s Even if you choose to post please cc me DIRECTLY as our news gateway seem to be losing items from time to time. my e-mail (as above): jill@cs.huji.ac.il From owner-proteins@net.bio.net Sat Dec 12 22:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!news-peer1.sprintlink.net!news-in-east1.sprintlink.net!news.sprintlink.net!newsfeed00.btx.dtag.de!news.btx.dtag.de!not-for-mail From: Gym_Warstein@t-online.de (Gymnasium Warstein) Newsgroups: bionet.molbio.proteins Subject: proteins from draff extraction fluid Date: 11 Dec 1998 14:20:50 GMT Lines: 23 Message-ID: <74r9o2$41o$1@news00.btx.dtag.de> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Sender: 700800000644-0001@t-online.de X-Mailer: Mozilla 4.05 [de]C-DT (Win95; I) Hello, we are a group of studends at the age of 16 who are concerned with the research of beer-draff. A liquid is pressed out when the draff goes through a screw conveyor. The dry matter of this liquid, called "treberpresssaft" ("draff extraction fluid"), consists to 50% of proteins. We want to have a closer look at this liquid. We have some questions: - Does anybody know which proteins are ins this liquid or how we can find that out? - Are there other persons/groups that research draff? - Which methods can we use to find out which amino acids are in our substance? (hydrolysis by acid or enzymes, etc.) - Are any uses of draff or amino acids known? - Are allergies to special amino acids known? Our e-mail adress is: Gym_Warstein@t-online.de Thanks for answers wish the studens from the beer-town Warstein From owner-proteins@net.bio.net Sun Dec 13 22:00:00 1998 Path: biosci!SLIP.NET!grizzly From: grizzly@SLIP.NET (Michael Sherrell) Newsgroups: bionet.molbio.proteins Subject: ABI Procise 492 for sale Date: 14 Dec 1998 15:48:56 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 15 Sender: daemon@net.bio.net Distribution: world Message-ID: <01BE2778.B0D720C0@oak-pm1-58-186.dialup.slip.net> NNTP-Posting-Host: net.bio.net ABI 492 Procise protein sequencer, was $132,000 new, not used in 2 years, $59,000 For sale: 1998 Advanced Chemtech Model 396 Multiple Biomolecular Synthesizer, unused, ~2/3 of new price. Please call or email if you'd like more details. Also available: many ABI and other sequencers and synthesizers (and service arrangements for synthesizers), HPLC/MSs, flow cytometers, NMRs and SEMs listed on my website. Michael Sherrell Grizzly Analytical 707 887 2919/fax 707 887 9834 www.grizzlyanalytical.com From owner-proteins@net.bio.net Sun Dec 13 22:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!news.maxwell.syr.edu!cpk-news-hub1.bbnplanet.com!news.gtei.net!portc02.blue.aol.com!pitt.edu!not-for-mail From: pxpst2@unixs.cis.pitt.edu (Peter) Newsgroups: bionet.molbio.proteins Subject: Re: enzyme kinetics newsgroup Date: Mon, 14 Dec 1998 08:53:48 -0500 Organization: University Of Pittsburgh Lines: 9 Message-ID: References: <36745AAC.30BE@mail.chem.tamu.edu> NNTP-Posting-Host: pelli.pathology.pitt.edu X-Newsreader: MT-NewsWatcher 2.4.4 In article <36745AAC.30BE@mail.chem.tamu.edu>, Ernie Maynard wrote: > Is there a good enzyme kinetics news group out there??? I am interseted > in allosteric interactions between proteins and the kinetics describing > such systems. I have not seen one but it would be a realy cool group. Maybe we can ask Bionet to start one From owner-proteins@net.bio.net Sun Dec 13 22:00:00 1998 Message-ID: <367528FF.1BE93DCE@bioreason.com> Date: Mon, 14 Dec 1998 07:04:31 -0800 From: Andrew Dalke Organization: Bioreason, Inc. X-Mailer: Mozilla 4.05 [en] (X11; U; IRIX 6.2 IP22) MIME-Version: 1.0 Newsgroups: bionet.molbio.proteins To: Bejerano Gill Subject: Re: the concept of a protein family References: Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit NNTP-Posting-Host: dialup142.trail.com Lines: 72 Path: biosci!news.stanford.edu!logbridge.uoregon.edu!newspeer1.nac.net!newspeer.monmouth.com!news.lightlink.com!news4.his.com!dialup142.trail.com Bejerano Gill asked: > Is the concept "Protein family" really so undefined as some of the lit. > claims it to be? > 1) does the term refer to a group of homologous proteins? > if so - how far back should their common ancestor be? is that > measured in time, in sequence similarity or perhaps in function > similarity? Yes to the first one, but there's no fixed definition on this. For that matter, there are different metrics for homology, both for sequence similarity and structure similarity. Time might not be the best way to measure similarity. Some proteins are extremely well conserved through time while others are much more variable. Most phylogeny plots have time measured in arbitrary units, though there are some where they've made an estimate based on the likely mutation rate. > 2) or does it refer to functionally analogous proteins? how different > is this concept from the previous? Is convergent evolution the only > reason to blame? how common is it? No, it doesn't. There are proteins that are functionally similar but by sequence and structure very different. A standard example is the chymotrypsin and subtilisin. They have different sequences and different folds but the arrangement around the catalytic triad is the same and they have the same function. There are some classifications based on function, such as the Enzyme Classification (E.C.). > 3) Or is it simply that every database proprietor (eg PROSITE, Pfam > etc.) simply devises, directly or indirectly, his own "taxonomy" > into groups? When is someone "tall"? When did a reptile become a bird? Is there an ideal essense of a chair? Does a dog have Zen natur-- oops, sorry, got carried away there :) I think the answer is there is broad consensus, but the edges differ based on the person. > 4) Is it therefore for one to deduce that no real benchmark or > criterion for comparing any two such taxonomies exists, except > one's personal taste? See previous :) But it turns out the various definitions are useful; for example, they have some explanatory and predictive power. Sequences that are very similar almost always have similar function. The problem is extending that to weaker and weaker similarities. > 5) Or perhaps all these methods should be compared only in terms of > the use researchers find in them - say, when a new protein sequence > is searched against them? A pragmatic definition, but hopefully there is some agreement on when to use FASTA or BLAST or Smith-Watterman, or why certain values of pairwise similarity or gap penalty shouldn't be used. In other words, you can't try everything and there's reasons why it's okay to ignore some possibilities. As with many fields, you build up knowledge on what is useful and what is not based on your experience and that of others. > 6) What is exactly the picture there? It seems the wealth of > partial tools as well as DBs is rather overwhelming... That means there's a lot of data and we don't know all the answers yet. Andrew Dalke dalke@bioreason.com From owner-proteins@net.bio.net Sun Dec 13 22:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!news.maxwell.syr.edu!news-ge.switch.ch!in2p3.fr!univ-lyon1.fr!cri.ens-lyon.fr!news From: Joel Baguet Newsgroups: bionet.molbio.proteins Subject: DNA binding protein purification Date: Mon, 14 Dec 1998 16:37:15 +0000 Organization: Ecole Normale Superieure de Lyon Lines: 11 Message-ID: <36753EBB.1516@ens-lyon.fr> Reply-To: Joel.Baguet@ens-lyon.fr NNTP-Posting-Host: mac-lr5-118a-oncovirus.ens-lyon.fr Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0 (Macintosh; I; 68K) I need to purify some proteins which bind to DNA. I use DNA binding protein purification kit from Boerhinger with long concatamers of protein-binding oligonucleotide (obtainrd using self-primed PCR technique) ligated streptavidin magnetic particles. The protein-binding conditions are those for gel retardation assay. The results are very poor (about 1% with 35S in vitro synthetized proteins). Does anybody have some experience about that?? From owner-proteins@net.bio.net Sun Dec 13 22:00:00 1998 Path: biosci!news.stanford.edu!logbridge.uoregon.edu!enews.sgi.com!newshub1.home.com!news.home.com!news.rdc1.bc.wave.home.com.POSTED!not-for-mail From: "Achim Recktenwald" Newsgroups: bionet.molbio.proteins References: <36753EBB.1516@ens-lyon.fr> Subject: Re: DNA binding protein purification Lines: 21 X-Newsreader: Microsoft Outlook Express 4.72.3155.0 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3155.0 Message-ID: Date: Tue, 15 Dec 1998 02:54:07 GMT NNTP-Posting-Host: 24.64.220.105 X-Complaints-To: news@home.net X-Trace: news.rdc1.bc.wave.home.com 913690447 24.64.220.105 (Mon, 14 Dec 1998 18:54:07 PDT) NNTP-Posting-Date: Mon, 14 Dec 1998 18:54:07 PDT Organization: @Home Network Canada I have worked in the past with DNA-binding enzymes. Most of them have rather high pI-values and can usually be purified by cation-exchange chromatography. Achim Joel Baguet wrote in message <36753EBB.1516@ens-lyon.fr>... >I need to purify some proteins which bind to DNA. I use DNA binding >protein purification kit from Boerhinger with long concatamers of >protein-binding >oligonucleotide (obtainrd using self-primed PCR technique) ligated >streptavidin magnetic particles. The protein-binding conditions are >those for gel retardation assay. > >The results are very poor (about 1% with 35S in vitro synthetized >proteins). > >Does anybody have some experience about that?? From owner-proteins@net.bio.net Mon Dec 14 22:00:00 1998 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!sunqbc.risq.qc.ca!torn!kwon!watserv3.uwaterloo.ca!not-for-mail From: @sciborg.uwaterloo.ca Newsgroups: bionet.molbio.proteins Subject: sulfate group on protein Date: Tue, 15 Dec 1998 21:04:45 -0500 Organization: UW Lines: 9 Message-ID: <3677153D.7C2079F2@sciborg.uwaterloo.ca> Reply-To: @sciborg.uwaterloo.ca NNTP-Posting-Host: jetlab2.uwaterloo.ca Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.05 [en] (Win95; I) Hi there, Does anyone have any information or know of any references about how a protein can be sulfated? ie. solely on a carbohydrate group or possibly an amino acid? Thanks in advance Carol Froese, University of Waterloo, Waterloo, Ont. Canada enightshade@hotmail.com (please respond to me directly if possilbe) From owner-proteins@net.bio.net Mon Dec 14 22:00:00 1998 Path: biosci!news.stanford.edu!logbridge.uoregon.edu!feeder.qis.net!nntp.abs.net!news-xfer.siscom.net!streamer1.cleveland.iagnet.net!NewsNG.Chicago.Qual.Net!news.indiana.edu!news.iupui.edu!not-for-mail From: cmantel Newsgroups: bionet.molbio.proteins Subject: Re: enzyme kinetics newsgroup Date: Tue, 15 Dec 1998 16:02:46 -0600 Organization: Indiana University - Purdue Univeristy At Indianapols,IN Lines: 14 Message-ID: <3676DC85.D9B134EC@iupui.edu> References: <36745AAC.30BE@mail.chem.tamu.edu> NNTP-Posting-Host: 149.166.238.12 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.5 [en] (Win98; I) X-Accept-Language: en I second that motion! Peter wrote: > In article <36745AAC.30BE@mail.chem.tamu.edu>, Ernie Maynard > wrote: > > > Is there a good enzyme kinetics news group out there??? I am interseted > > in allosteric interactions between proteins and the kinetics describing > > such systems. > > I have not seen one but it would be a realy cool group. Maybe we can ask > Bionet to start one From owner-proteins@net.bio.net Tue Dec 15 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: kll@crc.dk Newsgroups: bionet.molbio.proteins Subject: Fusion protein cleavage Date: Wed, 16 Dec 1998 11:41:59 GMT Organization: Deja News - The Leader in Internet Discussion Lines: 22 Message-ID: <7586a7$74n$1@nnrp1.dejanews.com> NNTP-Posting-Host: 130.226.1.36 X-Article-Creation-Date: Wed Dec 16 11:41:59 1998 GMT X-Http-User-Agent: Mozilla/4.5 [en] (Win95; I) X-Http-Proxy: 1.0 www2.crc.dk:3128 (Squid/1.1.20), 1.0 www-cache.net.uni-c.dk:3128 (Squid/1.1.20), 1.0 x8.dejanews.com:80 (Squid/1.1.22) for client 130.226.182.217, 130.226.183.6, 130.226.1.36 I am going to make a fusion protein for purification purposes and I would like to know about good and bad experiences with different types of cleavage enzymes. As far as I know the most popular proteases for cleavage are: Thrombin Factor Xa Enterokinase Can anybody recommend any of these (or other) enzymes with regards to effectivity, specificity, price, removal of protease etc. Thanks Kresten -- Kresten Lindorff Larsen Dept. Yeast Genetics Carlsberg Laboratory Denmark -----------== Posted via Deja News, The Discussion Network ==---------- http://www.dejanews.com/ Search, Read, Discuss, or Start Your Own From owner-proteins@net.bio.net Tue Dec 15 22:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!howland.erols.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 Subject: Re: sulfate group on protein Date: Wed, 16 Dec 1998 09:45:09 +0100 Organization: noone Lines: 16 Message-ID: References: <3677153D.7C2079F2@sciborg.uwaterloo.ca> NNTP-Posting-Host: med180.bham.ac.uk In article <3677153D.7C2079F2@sciborg.uwaterloo.ca>, @sciborg.uwaterloo.ca wrote: > Hi there, > Does anyone have any information or know of any references about how a > protein can be sulfated? ie. solely on a carbohydrate group or possibly > an amino acid? > Thanks in advance > Carol Froese, University of Waterloo, Waterloo, Ont. Canada > enightshade@hotmail.com > (please respond to me directly if possilbe) If I'm not mistaken, the thrombin inhibitor hirudin contains a sulfate group on it's Tyrosine residue. Peter From owner-proteins@net.bio.net Tue Dec 15 22:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!howland.erols.net!portc02.blue.aol.com!pitt.edu!not-for-mail From: Rich Dudley Newsgroups: bionet.molbio.proteins Subject: Re: Fusion protein cleavage Date: Wed, 16 Dec 1998 08:14:21 -0500 Organization: University of Pittsburgh, Dept. of Cell Biology and Physiology Lines: 52 Message-ID: <3677B22C.74C50D74@pitt.edu> References: <7586a7$74n$1@nnrp1.dejanews.com> Reply-To: rdudley+@pitt.edu NNTP-Posting-Host: whill.cbp.pitt.edu 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 kll@crc.dk wrote: > I am going to make a fusion protein for purification purposes and I would like > to know about good and bad experiences with different types of cleavage > enzymes. As far as I know the most popular proteases for cleavage are: > > Thrombin > Factor Xa > Enterokinase > > Can anybody recommend any of these (or other) enzymes with regards to > effectivity, specificity, price, removal of protease etc. > > Thanks > Kresten > > -- > Kresten Lindorff Larsen > Dept. Yeast Genetics > Carlsberg Laboratory > Denmark > > -----------== Posted via Deja News, The Discussion Network ==---------- > http://www.dejanews.com/ Search, Read, Discuss, or Start Your Own Those three are pretty much it for specific cleavage of proteins. Which one you use will depend greatly on the composition of your protein and fusion partner. Factor Xa is expensive in large quantities from any supplier. Enterokinase (or enteropeptidase) is relatively common but may contain some contaminants. If you're studying a pancreatic proenzyme (e.g., trypsin), you would want to avoid enterokinase, since that also activates trypsin. Thrombin is cheap, but has a degenerate cleavage specificity. You should examine your protein's AA sequence to ensure there isn't a thrombin site in there. There are a couple of tools on the WWW for doing this, but if your fusion partner isn't too long, it's just as good to do it by eye. You can find a couple of references in Medline about thrombin's specificity. Good luck! rich -- --- --- --- -- -- -- --- --- --- Richard J. Dudley (rdudley+@pitt.edu) Research Specialist V Dept. of Cell Biology and Physiology University of Pittsburgh http://www.cbp.pitt.edu ---> search BIONET archives at http://www.bio.net <--- From owner-proteins@net.bio.net Tue Dec 15 22:00:00 1998 Path: biosci!agate!newsfeed.berkeley.edu!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: Fusion protein cleavage Date: Wed, 16 Dec 1998 11:16:36 -0500 Organization: Purdue University Lines: 55 Message-ID: <3677DCE4.762C1B60@biochem.purdue.edu> References: <7586a7$74n$1@nnrp1.dejanews.com> NNTP-Posting-Host: hermod323a.biochem.purdue.edu Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: quoted-printable X-Mailer: Mozilla 4.04 [en] (Win95; I) =A0=A0=A0=A0 Dear Kresten! =A0=A0=A0=A0 I worked with Factor Xa and thrombin. Adjustment of conditio= ns for cleavage of various fusion proteins took a lot of time. Eventually I am not very h= appy about results of cleavage. It appeared that even very specific enzyme, li= ke factor Xa, tends to either cut your protein in some non-specific sites (for long= er cleavage time), either incompletely cut your substrate protein (for short= time). As a result, further protein purification is desired after cleavage (and=A0= you lose your protein on that step, of course).=A0 Any way, I think=A0 it's better= to avoid the insertion of the cleavage site for protease in the fusion protein (if you= have huge amount of=A0 fusion protein than it makes a difference, and another purification step is not a problem). Short 6-His-tag is one of the choice= s. =A0=A0=A0=A0 Good luck! =A0=A0=A0=A0 Lena. =A0 kll@crc.dk wrote: > I am going to make a fusion protein for purification purposes and I wou= ld like > to know about good and bad experiences with different types of cleavage= > enzymes. As far as I know the most popular proteases for cleavage are: > > Thrombin > Factor Xa > Enterokinase > > Can anybody recommend any of these (or other) enzymes with regards to > effectivity, specificity, price, removal of protease etc. > > Thanks > Kresten > > -- > Kresten Lindorff Larsen > Dept. Yeast Genetics > Carlsberg Laboratory > Denmark > > -----------=3D=3D Posted via Deja News, The Discussion Network =3D=3D--= -------- > http://www.dejanews.com/=A0=A0=A0=A0=A0=A0 Search, Read, Discuss, or St= art Your Own =A0 From owner-proteins@net.bio.net Tue Dec 15 22:00:00 1998 Path: biosci!CSHL.ORG!leemor From: leemor@CSHL.ORG (Leemor Joshua-Tor) Newsgroups: bionet.molbio.proteins Subject: Re: Fusion protein cleavage Date: 16 Dec 1998 08:38:40 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 22 Sender: daemon@net.bio.net Distribution: world Message-ID: References: <3677DCE4.762C1B60@biochem.purdue.edu> NNTP-Posting-Host: net.bio.net Another very good restriction protease is TEV protease. It's a recombinant protease which eliminated problems of nonspecific protease contamination. Specificity and purity is good compared to the others. The specificity is - Glu-Asn-Leu-Tyr-Phe-Gln-|-Gly. The Gly is left on fusion protein. You can buy it from BRL life technologies. ******************************************************************* Leemor Joshua-Tor, Ph.D. Assistant Investigator Keck Structural Biology Cold Spring Harbor Laboratory Tel. (516) 367 8821 1 Bungtown Road Fax (516) 367 8873 Cold Spring Harbor, NY 11724 e-mail: leemor@cshl.org ******************************************************************* From owner-proteins@net.bio.net Tue Dec 15 22:00:00 1998 Path: biosci!news.stanford.edu!logbridge.uoregon.edu!howland.erols.net!newsfeed.metronet.de!RRZ.Uni-Koeln.DE!gmd.de!news.ruhr-uni-bochum.de!not-for-mail From: heiko.koch@ruhr-uni-bochum.de (Heiko Koch) Newsgroups: bionet.molbio.proteins Subject: Calculating secundary structure from CD-Spectra Date: Wed, 16 Dec 1998 16:41:38 GMT Organization: Ruhr-Universitaet Bochum, Rechenzentrum Lines: 12 Message-ID: <3677e1ad.34830612@news.ruhr-uni-bochum.de> NNTP-Posting-Host: r06-596.molgen.ruhr-uni-bochum.de X-Trace: sun579.rz.ruhr-uni-bochum.de 913826559 25965 134.147.135.206 (16 Dec 1998 16:42:39 GMT) NNTP-Posting-Date: 16 Dec 1998 16:42:39 GMT X-Newsreader: Forte Free Agent 1.11/32.235 Hello, I want to ask if someone knew a source to download free programms to analyse spectra from cirkular dichroism (CD). I wanted to calculate the secondary structure of a protein aut of these data thanks for answering Heiko Heiko.koch@ruhr-uni-bochum.de From owner-proteins@net.bio.ne