From owner-proteins@net.bio.net Fri Jan 01 22:00:00 1999 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!news.gtei.net!newsfeed.wli.net!news-feed.inet.tele.dk!bofh.vszbr.cz!newsfeed.tli.de!unlisys!news.snafu.de!cs.tu-berlin.de!news.uni-hamburg.de!not-for-mail From: behrends@plexus.uke.uni-hamburg.de (Soenke Behrends) Newsgroups: bionet.molbio.proteins Subject: KLH vs MAP peptide antibodies Date: Sat, 02 Jan 1999 23:34:25 GMT Organization: University of Hamburg -- Germany Lines: 16 Message-ID: <368eab4f.2898912@news.uni-hamburg.de> NNTP-Posting-Host: virchow.uke.uni-hamburg.de X-Newsreader: Forte Free Agent 1.11/32.235 Dear Antibody-specialists, I desperately need a very good antibody against a human cytosolic protein for Immunofluorescence, IP and Western. Has anyone tried using the multiple antigenic peptide approach? Did that seem better than coupling the peptide to KLH? Any hints or ideas are appreciated. Thanks a lot for your time Soenke From owner-proteins@net.bio.net Fri Jan 01 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!news-peer1.sprintlink.net!news-in-east1.sprintlink.net!news.sprintlink.net!itssrv1.ucsf.edu!macmac-2.ucsf.edu!user From: bpmurray*STUFFER*@socrates.ucsf.edu (Bernard P. Murray, PhD) Newsgroups: bionet.molbio.proteins Subject: Re: KLH vs MAP peptide antibodies Date: Sat, 02 Jan 1999 17:59:49 -0700 Organization: University of California, San Francisco Lines: 55 Message-ID: References: <368eab4f.2898912@news.uni-hamburg.de> NNTP-Posting-Host: macmac-2.ucsf.edu In article <368eab4f.2898912@news.uni-hamburg.de>, behrends@plexus.uke.uni-hamburg.de (Soenke Behrends) wrote: > Dear Antibody-specialists, > I desperately need a very good antibody against > a human cytosolic protein for Immunofluorescence, > IP and Western. > Has anyone tried using the multiple antigenic > peptide approach? Did that seem better than > coupling the peptide to KLH? > Any hints or ideas are appreciated. > Thanks a lot for your time > Soenke The only time we did a direct comparison between MAP and KLH-MBS-cysteine-peptide we saw no good antibody production with the MAP. We synthesised the MAP ourselves and had it fully characterised and the anti-KLH-peptide antibody bound strongly to the MAP. The only explanation we could come up with is that we were using shorter (7mer) peptides than most people (although I have seen 7mer MAPs used with good results). We later immunised the MAP rabbit with the KLH conjugate and saw a very good response (actually better than the rabbit with KLH-peptide alone). The KLH conjugate antibodies work well in westerns (unpurified) in ELISA (purified) and immunohistochemistry (purified) and I believe they have also been used for immunoprecipitation (probably purified). Purified = IgG by ammonium sulphate and then depleted of anti-KLH-MBS-cysteine by affinity chromatography. Purification using the peptide doesn't work. I realise that many people use the MAP with very good results but I thought it would be best to warn you that it may not always work whereas we always saw *something* with a KLH-peptide (even if it didn't cross-react well with the target antigen). I believe it is now routine practice to include some sort of T-cell response modulator during immunisation so this may increase the chances of a MAP working. Try both if you have the cash and you are short on time. If you only have one shot at it then I would go for the KLH in the first instance - and then after you have the grant money rolling in because of your beautiful blots you can get the MAP synthesised :-) Good luck, Bernard -- Bernard P. Murray, PhD Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA From owner-proteins@net.bio.net Sat Jan 02 22:00:00 1999 Path: biosci!news.stanford.edu!logbridge.uoregon.edu!news.maxwell.syr.edu!newsfeed.cwix.com!204.71.76.137!news.campus.mci.net!uky.edu!news.cuny.edu!NewsWatcher!user From: laurette@msvax.mssm.edu (laurette) Newsgroups: bionet.molbio.proteins Subject: Re: Dnatools, sequencing software Date: 3 Jan 1999 22:20:39 GMT Lines: 18 Distribution: world Message-ID: References: <36880B5C.FE8908F4@crc.dk> NNTP-Posting-Host: 146.203.107.132 Do you have a Macintosh version of the software? In article <36880B5C.FE8908F4@crc.dk>, swr@crc.dk wrote: > Dear DNATools user, > > Look at the latest news at our homepage: > > http://www.crc.dk/phys/A01B04_dnatools.htm > > S. W. Rasmussen > > -- > Dr. scient. Søren W. Rasmussen > Carlsberg Laboratory, Department of Physiology > 10 Gl. Carlsbergvej, DK-2500, Copenhagen, Denmark > Phone 45 3327 5230 / 45 3616 2259, Fax 45 3327 4766 > E-mail swr@crc.dk, http://www.crc.dk/phys/index.htm From owner-proteins@net.bio.net Wed Jan 06 22:00:00 1999 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!su-news-hub1.bbnplanet.com!news.gtei.net!newsfeed.berkeley.edu!newsfeed.wirehub.nl!surfnet.nl!news.leidenuniv.nl!not-for-mail From: Flip Hoedemaeker Newsgroups: bionet.molbio.proteins Subject: Re: A simple question... Date: Wed, 06 Jan 1999 18:47:41 +0100 Organization: RUL Lines: 13 Message-ID: <3693A1BD.D4684A69@chem.leidenuniv.nl> References: <368FFF9D.4A33F46C@telerama.com> NNTP-Posting-Host: chema111.leidenuniv.nl Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: highway.leidenuniv.nl 915644857 16619 132.229.169.111 (6 Jan 1999 17:47:37 GMT) X-Complaints-To: usenet@highway.leidenuniv.nl NNTP-Posting-Date: 6 Jan 1999 17:47:37 GMT X-Mailer: Mozilla 4.5 [en] (X11; I; IRIX64 6.4 IP30) X-Accept-Language: en orfB probably means open reading frame B. So it's the second one, and they don't know what the protein is! Flip -- --------------------------------------------------------- P.J. Hoedemaeker Ph.D. Biofysische Struktuurchemie, Gorlaeus Laboratoria L010 Einsteineg 55/PO box 9502 2300 RA Leiden, The Netherlands Tel: +31-71-527-4211/4414 Fax: +31-71-5274349 http://chemg20.leidenuniv.nl/~flip/home.html --------------------------------------------------------- From owner-proteins@net.bio.net Wed Jan 06 22:00:00 1999 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!atl-news-feed1.bbnplanet.com!news.gtei.net!maze.dpo.uab.edu!usenet From: levy@uab.edu (David N. Levy) Newsgroups: bionet.molbio.proteins,bionet.cellbiol Subject: Weird protein behavior in cells - help. Date: 6 Jan 1999 20:24:23 GMT Organization: University of Alabama at Birmingham Lines: 38 Sender: -Not-Authenticated-[7889] Message-ID: <770gpn$iav@maze.dpo.uab.edu> NNTP-Posting-Host: dlevy.dom.uab.edu X-Posted-From: InterNews 1.1@dlevy.dom.uab.edu Xdisclaimer: No attempt was made to authenticate the sender's name. Xref: biosci bionet.molbio.proteins:13777 bionet.cellbiol:11082 I am studying a putative viral protein, whose behavior I have described once before in this group, and received some useful responses. I have a new behavior I would like to describe which is quite baffling to me, to see if anyone has a clue about it. I sure don't. I have expressed this protein as a fusion product with GFP. As I described in a previous post, this protein is then seen to be either in the cytoplasm or the nucleus, one or the other exclusively, in most cells. Now I have made a fusion protein of just the first 43 amino acids (of 78) of my putative protein to GFP. This protein is now found in the cytoplasm, in a perinuclear pattern early (14 hours) after transfection, then the interesting thing happens: The cells become bags of glowing balls. Each cell is now comprised of about 50 to 100 round glowing vessicles. In fact the whole cytoplasmic architecture is gone and only these round vessicles are left. I observed the same phenomenon by fusing the entire putative gene from a divergent strain of virus to GFP, but I only saw this phenomenon when I accidentally lysed the cells (unfixed) by lifting up the coverglass, thus causing shear forces to disturb the cells. I am posting a photo of this phenomenon on my computer at: ftp://138.26.49.17/Spelunking%20Homunculus/Public/Viral%20protein%20phot os/HeLa%20%26%20fusion%20protein.jpg You can use Netscape to see this photo or download it and view it in Photoshop or maybe other viewers (I don't know.) Thanks. David N. Levy University of Alabama at Birmingham Birmingham, AL 35294-0007 levy@uab.edu From owner-proteins@net.bio.net Wed Jan 06 22:00:00 1999 Path: biosci!news.stanford.edu!nntp.cs.ubc.ca!news-spur1.maxwell.syr.edu!news.maxwell.syr.edu!howland.erols.net!news.net.uni-c.dk!not-for-mail From: "Søren W. Rasmussen" Newsgroups: bionet.molbio.proteins Subject: Re: Dnatools, sequencing software Date: Mon, 04 Jan 1999 08:45:45 +0100 Organization: UNI-C Lines: 16 Message-ID: <369071A9.B7648771@crc.dk> References: <36880B5C.FE8908F4@crc.dk> NNTP-Posting-Host: 130.226.182.97 Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Trace: news.net.uni-c.dk 915436416 18982 (None) 130.226.182.97 X-Complaints-To: usenet@news.net.uni-c.dk X-Mailer: Mozilla 4.03 [en] (Win95; I) Dear Laurette, No, DNATools only exists in a 32 bit Windows version. Sorry! Regards Søren -- Dr. scient. Søren W. Rasmussen Carlsberg Laboratory, Department of Physiology 10 Gl. Carlsbergvej, DK-2500, Copenhagen, Denmark Phone 45 3327 5230 / 45 3616 2259, Fax 45 3327 4766 E-mail swr@crc.dk, Homepage http://www.crc.dk/phys/ From owner-proteins@net.bio.net Wed Jan 06 22:00:00 1999 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.gtei.net!newshub.northeast.verio.net!nntp.newengland.verio.net!news.pn.com!mozo.cc.purdue.edu!news From: Lena Zaitseva Newsgroups: bionet.molbio.proteins Subject: Precise protein concentration Date: Wed, 06 Jan 1999 10:18:26 -0500 Organization: Purdue University Lines: 45 Message-ID: <36937EC2.BD70B3FA@biochem.purdue.edu> NNTP-Posting-Host: hermod323a.biochem.purdue.edu Mime-Version: 1.0 Content-Type: multipart/alternative; boundary="------------B5962DAB68528C118428EC82" X-Mailer: Mozilla 4.04 [en] (Win95; I) --------------B5962DAB68528C118428EC82 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: quoted-printable =A0=A0=A0=A0 Hi, =A0=A0=A0=A0 We purified a membrane protein in the presence of non-ionic detergent. Now we need to calculate precise protein concentration. It appeared to be a real problem. =A0=A0=A0=A0 Bradford method doesn't work at all. BCA method looks better= , but with BSA as control it doesn't give us reliable results (there is no linear dependence of OD from concentration of our protein). For UV-method it would be nice to unfold=A0 protein to get the maximum absorbency at 280 nm. However, guanidine-HCl and SDS lead to the protein aggregation. We tried also make quantitative amino-acid analysis of the protein, but=A0 presence of high salt concentration (1.5 M) and glycerol in the sample buffer is a problem in that case. =A0=A0=A0=A0 Do you have any idea about that? =A0=A0=A0=A0 Thanks for your suggestions. =A0=A0=A0=A0 Lena. --------------B5962DAB68528C118428EC82 Content-Type: text/html; charset=us-ascii Content-Transfer-Encoding: 7bit      Hi,
     We purified a membrane protein in the presence of non-ionic detergent. Now we need to calculate precise protein concentration. It appeared to be a real problem.
     Bradford method doesn't work at all. BCA method looks better, but with BSA as control it doesn't give us reliable results (there is no linear dependence of OD from concentration of our protein). For UV-method it would be nice to unfold  protein to get the maximum absorbency at 280 nm. However, guanidine-HCl and SDS lead to the protein aggregation. We tried also make quantitative amino-acid analysis of the protein, but  presence of high salt concentration (1.5 M) and glycerol in the sample buffer is a problem in that case.
     Do you have any idea about that?
     Thanks for your suggestions.
     Lena. --------------B5962DAB68528C118428EC82-- From owner-proteins@net.bio.net Wed Jan 06 22:00:00 1999 Path: biosci!news.alaska.edu!newsfeed.acns.nwu.edu!newsfeed.berkeley.edu!newsxfer3.itd.umich.edu!news.itd.umich.edu!not-for-mail Sender: Brett William Lennon From: Brett William Lennon Subject: 5 kDa protein detection Newsgroups: bionet.molbio.proteins User-Agent: tin/pre-1.4-980818 ("Laura") (UNIX) (SunOS/5.6 (sun4u)) Lines: 29 Message-ID: Date: Thu, 07 Jan 1999 14:24:50 GMT NNTP-Posting-Host: 141.211.63.85 X-Trace: news.itd.umich.edu 915719090 141.211.63.85 (Thu, 07 Jan 1999 09:24:50 EDT) NNTP-Posting-Date: Thu, 07 Jan 1999 09:24:50 EDT >I am currently trying to purify a protein of 5 kDa, but are having >problems detecting the protein. As it only contains tree tyrosines and >no tryptophans the A280 is very low. I have tried running 18% SDS-PA >gels and a 15 to 25% SDS PA gradient-gels, without luck, the protein >probably diffuses out of the gel during staining. >So I am looking for other (easy) ways to detect low MW proteins. Any >ideas would be appreciated! >I have heard that adding coomassie to the running buffer instead of >post-staining the gels could be a solution. Anyone got experience with >that? >Thanks, Rasmus Have you tried detecting the peptide bond (absorbance at ~214 nm)? A280 is what folks often try first, but as you found out, it may not always work due to few aromatic residues. However, all proteins have the peptide bond. I've used it in detecting peptide fragments from the digestion of a 9 kDa protein coming off a reversed phase column. One drawback is that when you're that far into the UV, background absorbance from other stuff can be a problem. Blanks will be essential. Brett ------- Brett W. Lennon Department of Biological Chemistry The University of Michigan bwlennon@umich.edu From owner-proteins@net.bio.net Wed Jan 06 22:00:00 1999 Path: biosci!news.alaska.edu!newsfeed.acns.nwu.edu!news-spur1.maxwell.syr.edu!news.maxwell.syr.edu!nntp.news.xara.net!xara.net!newsfeed.ecrc.net!news.net.uni-c.dk!not-for-mail From: "Søren W. Rasmussen" Newsgroups: bionet.molbio.proteins Subject: Dnatools, revision 230 Date: Wed, 06 Jan 1999 13:25:37 +0100 Organization: UNI-C Lines: 17 Message-ID: <36935641.44449295@crc.dk> NNTP-Posting-Host: 130.226.182.97 Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Trace: news.net.uni-c.dk 915626010 33576 (None) 130.226.182.97 X-Complaints-To: usenet@news.net.uni-c.dk X-Mailer: Mozilla 4.03 [en] (Win95; I) Version 5.1.230 of the DNATools sequencing software package is available now. Read more about functions and improvements at: http://www.crc.dk/phys/A01B04_dnatools.htm Regards Søren -- Dr. scient. Søren W. Rasmussen Carlsberg Laboratory, Department of Physiology 10 Gl. Carlsbergvej, DK-2500, Copenhagen, Denmark Phone 45 3327 5230 / 45 3616 2259, Fax 45 3327 4766 E-mail swr@crc.dk, Homepage http://www.crc.dk/phys/ From owner-proteins@net.bio.net Wed Jan 06 22:00:00 1999 Path: biosci!news.alaska.edu!newsfeed.acns.nwu.edu!newsfeed.berkeley.edu!news.maxwell.syr.edu!rill.news.pipex.net!pipex!ams.news.uu.net!uunet!in5.uu.net!news.inter.net.il!not-for-mail From: nir_21@internet-zahav.net (Nir Dagan) Newsgroups: bionet.molbio.proteins Subject: Rasmol Date: Tue, 05 Jan 1999 22:41:08 GMT Organization: Internet Gold, ISRAEL Lines: 11 Message-ID: <36929369.9019743@news.internet-zahav.net> NNTP-Posting-Host: tlv-194-189.access.net.il X-Trace: news2.inter.net.il 915576140 18218 192.116.194.189 (5 Jan 1999 22:42:20 GMT) X-Complaints-To: abuse@inter.net.il NNTP-Posting-Date: 5 Jan 1999 22:42:20 GMT X-Newsreader: Forte Free Agent 1.11/32.235 I am making an academic evaluation of the Rasmol program for graphic visualisation of proteins and would appreciate any comments any of you might have regarding the benefits, shortcomings and specially comments for improvment. thanks, Nir Dagan nir_21@internet-zahav.net From owner-proteins@net.bio.net Wed Jan 06 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!newsfeed.wirehub.nl!surfnet.nl!barba.uci.kun.nl!sci.kun.nl!not-for-mail From: "Gerrit Bouw" Newsgroups: bionet.molbio.proteins Subject: Transfecting sperm-cells Date: Tue, 5 Jan 1999 12:14:07 +0100 Organization: University of Nijmegen, The Netherlands Lines: 9 Message-ID: <76ss36$rqk$1@wnnews.sci.kun.nl> NNTP-Posting-Host: gbouw.sci.kun.nl X-Newsreader: Microsoft Outlook Express 4.72.3110.1 X-Mimeole: Produced By Microsoft MimeOLE V4.72.3110.3 Dear everyone, Does anyone ever tried to transfect sperm-cells? If so, please could you give me some guidance on this? Gerrit From owner-proteins@net.bio.net Wed Jan 06 22:00:00 1999 Path: biosci!cnn.nas.nasa.gov!newsfeed.berkeley.edu!su-news-hub1.bbnplanet.com!news.gtei.net!xfer.kren.ne.kr!newshub1.home.com!news.home.com!news.rdc1.bc.wave.home.com.POSTED!not-for-mail From: "Achim Recktenwald" Newsgroups: bionet.molbio.proteins References: <367A63DA.CA31E120@mbio.aau.dk> <36902084.79F48475@telerama.com> Subject: Re: 5 kDa protein detection Lines: 68 X-Newsreader: Microsoft Outlook Express 4.72.3155.0 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3155.0 Message-ID: Date: Tue, 05 Jan 1999 05:44:10 GMT NNTP-Posting-Host: 24.64.220.105 X-Complaints-To: abuse@home.net X-Trace: news.rdc1.bc.wave.home.com 915515050 24.64.220.105 (Mon, 04 Jan 1999 21:44:10 PDT) NNTP-Posting-Date: Mon, 04 Jan 1999 21:44:10 PDT Organization: @Home Network Canada Something I forgot to mention. You describe your peptide as having few tyrs, no trps; if it also has only few basic amino acids, it could be one of those proteins that stain very badly or not at all with Coomassie. You might have to try a silver stain. Achim Achim Recktenwald wrote in message ... >You could run tricine-gels (Schägger et al., Anal Biochem (1987, vol. 166, >368 - 379) ; they resolve down to <2kDa; I have even seen peptides down to >1kDa. > >If you are afraid of loosing your peptide during staining, try a staining >method used in IEF, where this is always a problem, even for larger >proteins; e.g., >20min in 20% TCA >30 - 40min 50% methanol, 10% acetic acid >2x 20min 5% methanol >40min 10% glutaraldehyde >2x 10min wash in water or 5% methanol >then normal Coomassie-staining. > >The TCA will denature and precipitate your protein, the glutaralehyde will >then crosslink the protein molecules contained in a protein-band in the >gel-matrix, making them unleachable. >If you are using very thin gels, you can easily shorten the incubation times >by up to a factor of 2. > >If you try this with your 15 - 25% PAA gel and still do not find a band, I >would say your 5kDa might not be part of your sample. > >Good luck, > >Achim > > > >Ryan Werstuik wrote in message <36902084.79F48475@telerama.com>... >>I would try using protein markers (with your smallest at 5kDa), running >>the sample in a center lane, and 1/2 to 2/3 the current time you are using >>now. I have also heard/read of using a stain that allows the samples to >>be seen as the gel runs; however, I can't remember the where. >> >>I hope this helps, >>Ryan >> >>Rasmus Wendelbo Nielsen wrote: >> >>> I am currently trying to purify a protein of 5 kDa, but are having >>> problems detecting the protein. As it only contains tree tyrosines and >>> no tryptophans the A280 is very low. I have tried running 18% SDS-PA >>> gels and a 15 to 25% SDS PA gradient-gels, without luck, the protein >>> probably diffuses out of the gel during staining. >>> So I am looking for other (easy) ways to detect low MW proteins. Any >>> ideas would be appreciated! >>> I have heard that adding coomassie to the running buffer instead of >>> post-staining the gels could be a solution. Anyone got experience with >>> that? >>> Thanks, Rasmus >> > > From owner-proteins@net.bio.net Wed Jan 06 22:00:00 1999 Path: biosci!cnn.nas.nasa.gov!newsfeed.berkeley.edu!nntp.abs.net!newshub2.home.com!newshub1.home.com!news.home.com!news.rdc1.bc.wave.home.com.POSTED!not-for-mail From: "Achim Recktenwald" Newsgroups: bionet.molbio.proteins References: <367A63DA.CA31E120@mbio.aau.dk> <36902084.79F48475@telerama.com> Subject: Re: 5 kDa protein detection Lines: 54 X-Newsreader: Microsoft Outlook Express 4.72.3155.0 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3155.0 Message-ID: Date: Tue, 05 Jan 1999 05:39:36 GMT NNTP-Posting-Host: 24.64.220.105 X-Complaints-To: abuse@home.net X-Trace: news.rdc1.bc.wave.home.com 915514776 24.64.220.105 (Mon, 04 Jan 1999 21:39:36 PDT) NNTP-Posting-Date: Mon, 04 Jan 1999 21:39:36 PDT Organization: @Home Network Canada You could run tricine-gels (Schägger et al., Anal Biochem (1987, vol. 166, 368 - 379) ; they resolve down to <2kDa; I have even seen peptides down to 1kDa. If you are afraid of loosing your peptide during staining, try a staining method used in IEF, where this is always a problem, even for larger proteins; e.g., 20min in 20% TCA 30 - 40min 50% methanol, 10% acetic acid 2x 20min 5% methanol 40min 10% glutaraldehyde 2x 10min wash in water or 5% methanol then normal Coomassie-staining. The TCA will denature and precipitate your protein, the glutaralehyde will then crosslink the protein molecules contained in a protein-band in the gel-matrix, making them unleachable. If you are using very thin gels, you can easily shorten the incubation times by up to a factor of 2. If you try this with your 15 - 25% PAA gel and still do not find a band, I would say your 5kDa might not be part of your sample. Good luck, Achim Ryan Werstuik wrote in message <36902084.79F48475@telerama.com>... >I would try using protein markers (with your smallest at 5kDa), running >the sample in a center lane, and 1/2 to 2/3 the current time you are using >now. I have also heard/read of using a stain that allows the samples to >be seen as the gel runs; however, I can't remember the where. > >I hope this helps, >Ryan > >Rasmus Wendelbo Nielsen wrote: > >> I am currently trying to purify a protein of 5 kDa, but are having >> problems detecting the protein. As it only contains tree tyrosines and >> no tryptophans the A280 is very low. I have tried running 18% SDS-PA >> gels and a 15 to 25% SDS PA gradient-gels, without luck, the protein >> probably diffuses out of the gel during staining. >> So I am looking for other (easy) ways to detect low MW proteins. Any >> ideas would be appreciated! >> I have heard that adding coomassie to the running buffer instead of >> post-staining the gels could be a solution. Anyone got experience with >> that? >> Thanks, Rasmus > From owner-proteins@net.bio.net Wed Jan 06 22:00:00 1999 Path: biosci!cnn.nas.nasa.gov!newsfeed.berkeley.edu!remarQ73!supernews.com!remarQ69!not-for-mail From: Ryan Werstuik Newsgroups: bionet.molbio.proteins Subject: Re: 5 kDa protein detection Date: Sun, 03 Jan 1999 20:59:32 -0500 Organization: Posted via RemarQ, http://www.remarQ.com - Discussions start here! Lines: 22 Message-ID: <36902084.79F48475@telerama.com> References: <367A63DA.CA31E120@mbio.aau.dk> NNTP-Posting-Host: 204.171.44.17 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: 915503889 BPIPSDRGT2C11CCABC usenet78.supernews.com X-Complaints-To: newsabuse@remarQ.com X-Mailer: Mozilla 4.5 [en] (Win95; U) X-Accept-Language: en I would try using protein markers (with your smallest at 5kDa), running the sample in a center lane, and 1/2 to 2/3 the current time you are using now. I have also heard/read of using a stain that allows the samples to be seen as the gel runs; however, I can't remember the where. I hope this helps, Ryan Rasmus Wendelbo Nielsen wrote: > I am currently trying to purify a protein of 5 kDa, but are having > problems detecting the protein. As it only contains tree tyrosines and > no tryptophans the A280 is very low. I have tried running 18% SDS-PA > gels and a 15 to 25% SDS PA gradient-gels, without luck, the protein > probably diffuses out of the gel during staining. > So I am looking for other (easy) ways to detect low MW proteins. Any > ideas would be appreciated! > I have heard that adding coomassie to the running buffer instead of > post-staining the gels could be a solution. Anyone got experience with > that? > Thanks, Rasmus From owner-proteins@net.bio.net Wed Jan 06 22:00:00 1999 Path: biosci!CS.Arizona.EDU!news.Arizona.EDU!uunet!in3.uu.net!newsfeed.cwix.com!128.32.206.55!newsfeed.berkeley.edu!remarQ73!supernews.com!remarQ69!not-for-mail From: Ryan Werstuik Newsgroups: bionet.molbio.proteins Subject: A simple question... Date: Sun, 03 Jan 1999 18:39:09 -0500 Organization: Posted via RemarQ, http://www.remarQ.com - Discussions start here! Lines: 9 Message-ID: <368FFF9D.4A33F46C@telerama.com> NNTP-Posting-Host: 204.171.44.17 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: 915495459 BPIPSDRGT2C11CCABC usenet58.supernews.com X-Complaints-To: newsabuse@remarQ.com X-Mailer: Mozilla 4.5 [en] (Win95; U) X-Accept-Language: en Hello, I am an undergrad student who is wondering if, when listed in a GenBank entry under a certain CDS as the product, does "orfB" have any special meaning, or is it simply the name of the protein produced by that region? Thanks, Ryan From owner-proteins@net.bio.net Wed Jan 06 22:00:00 1999 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.gtei.net!newsfeed.cwix.com!195.252.142.12!newsfeed.tli.de!news-fra.pop.de!newsfeed.germany.net!news.ruhrgebiet.individual.net!news-koe1.dfn.de!news-han1.dfn.de!news.gwdg.de!not-for-mail From: Silke Beismann Newsgroups: bionet.molbio.proteins Subject: Re: removing DNA Date: Mon, 04 Jan 1999 18:25:38 +0100 Organization: GWDG, Goettingen Lines: 164 Message-ID: <3690F992.5092FB3F@uni-molgen.gwdg.de> References: <368A090F.767347C0@uni-molgen.gwdg.de> NNTP-Posting-Host: 134.76.70.65 Mime-Version: 1.0 Content-Type: multipart/alternative; boundary="------------B4070903B66A4BEAE1A0A88A" X-Mailer: Mozilla 4.5 [en] (WinNT; I) X-Accept-Language: en --------------B4070903B66A4BEAE1A0A88A Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit > Achim, to your remarks: > > Just a stupid question. > How do you know from your spectrum that there is DNA present/bound? > I've worked with DNA-binding proteins and never seen a 'nucleic acid > spectrum' even when DNA was definitely bound to the protein. I am not sure but: The absorption maximum lies at 260 nm, the absorption at 250 nm is still high and compared with an DNA spectum, e. g. a midi prep, it looks pretty much the same The absorption at 260 nm can be reduced by hydroxylapatite chromatography. In some preparations the fractions with much protein (shown with bradford, SDS gel) show a "typical protein spectrum" with a maximum at 280 nm and a minimum at 250 nm - in other preparations this was not the case My protein acts in vitro and probably in vivo in a heterodimeric complex with another protein, which is highly negatively charged- like DNA. These are the reasons for which I think that my proteins bind to DNA which I can not remove. I would be glad for any suggestions what else can cause these strange absorption behavior and /or how to purify the protein from the contaminant. Thanks, Silke > > > Perhaps you should check your buffer/assay matrix composition. Are there any > free amino acids present, esp. aromatic ones? Phe in your matrix will > produce a strong peak at or near 260nm, mimicking DNA? Do you perhaps use > a buffer substance that is not fully transparent in the low UV? Is the > solution composition of your reference the same as the sample-matrix? Did > you perhaps purify a his-tagged protein and measure the spectrum of a sample > containing imidazole? > > Cheers, > > Achim > > Bernard P. Murray, PhD wrote in message ... > >In article <368A090F.767347C0@uni-molgen.gwdg.de>, Silke Beismann > > wrote: > > > >> Hi, > >> I am trying to purify a protein from the amino acid biosynthetic > >> pathway. It should not have anything to do with DNA but it seems that it > >> binds to it. I tried digestion of the crude extract with DNase I and > >> RNase A, precipitation with ammonium sulfate or polyehtylene imine, > >> hydroxyapatite chromatography- but the spectrum still looks like an > >> nucleic acid spectrum. Does anyone know an efficient method to remove > >> the > >> contaminating nucleic acids? > >> Thanks in advance, > >> Silke > > > >Just curious. I don't know how the "spectrum" can pick out > >the DNA nature of the protein/complex but is there any way > >that your protein could be modified (eg. ADP-ribosylated > >or a prosthetic group) that would give the impression of > >DNA contamination? Maybe treatment with a DNA kinase > >would help determine if it is really adsorbed DNA > >(should only label if it is really "DNA"). > > Bernard > >-- > >Bernard P. Murray, PhD > >Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA --------------B4070903B66A4BEAE1A0A88A Content-Type: text/html; charset=us-ascii Content-Transfer-Encoding: 7bit  
 
Achim, to your remarks:

Just a stupid question.
How do you know from your spectrum that there is DNA present/bound?
I've worked with DNA-binding proteins and never seen a 'nucleic acid
spectrum' even when DNA was definitely bound to the protein.

I am not sure but:

The absorption maximum lies at 260 nm, the absorption at 250 nm is still high and compared with an DNA spectum, e. g. a midi prep, it looks pretty much the same

The absorption at 260 nm can be reduced by hydroxylapatite chromatography. In some preparations the fractions with much protein (shown with bradford, SDS gel) show a "typical protein spectrum" with a maximum at 280 nm and a minimum at 250 nm - in other preparations this was not the case

My protein acts in vitro and probably in vivo in a heterodimeric complex with another protein, which is highly negatively charged- like DNA.

These are the reasons for which I think that my proteins bind to DNA which I can not remove. I would be glad for any suggestions what else can cause these strange absorption behavior and /or how to purify the protein from the contaminant.

Thanks,
 

                        Silke

 

Perhaps you should check your buffer/assay matrix composition. Are there any
free amino acids present, esp. aromatic ones?  Phe in your matrix will
produce a strong peak at or near  260nm, mimicking DNA?   Do you perhaps use
a buffer substance that is not fully transparent in the low UV? Is the
solution composition of your reference the same as the sample-matrix? Did
you perhaps purify a his-tagged protein and measure the spectrum of a sample
containing imidazole?

Cheers,

Achim

Bernard P. Murray, PhD wrote in message ...
>In article <368A090F.767347C0@uni-molgen.gwdg.de>, Silke Beismann
><sbeisma@uni-molgen.gwdg.de> wrote:
>
>> Hi,
>> I am trying to purify a protein from the amino acid biosynthetic
>> pathway. It should not have anything to do with DNA but it seems that it
>> binds to it. I tried digestion of the crude extract with DNase I and
>> RNase A, precipitation with ammonium sulfate or polyehtylene imine,
>> hydroxyapatite chromatography- but the spectrum still looks like an
>> nucleic acid spectrum. Does anyone know an efficient method to remove
>> the
>> contaminating nucleic acids?
>> Thanks in advance,
>>             Silke
>
>Just curious.  I don't know how the "spectrum" can pick out
>the DNA nature of the protein/complex but is there any way
>that your protein could be modified (eg. ADP-ribosylated
>or a prosthetic group) that would give the impression of
>DNA contamination?  Maybe treatment with a DNA kinase
>would help determine if it is really adsorbed DNA
>(should only label if it is really "DNA").
>          Bernard
>--
>Bernard P. Murray, PhD
>Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA

--------------B4070903B66A4BEAE1A0A88A-- From owner-proteins@net.bio.net Wed Jan 06 22:00:00 1999 Path: biosci!news.stanford.edu!logbridge.uoregon.edu!fu-berlin.de!eravci.dialup.fu-berlin.DE!not-for-mail From: "MURAT ERAVCI" Newsgroups: bionet.molbio.proteins Subject: ImageMaster 2D Software Date: Mon, 4 Jan 1999 15:42:40 +0100 Organization: Freie Universitaet Berlin Lines: 9 Message-ID: <76qjr1$iid$1@fu-berlin.de> NNTP-Posting-Host: eravci.dialup.fu-berlin.de (130.133.237.57) Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Access: 16 17 18 X-Trace: fu-berlin.de 915460769 19021 (none) 130.133.237.57 X-Newsreader: Microsoft Outlook Express 4.72.3110.5 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3110.3 Who has got the ImageMaster 2D Software, or is already using it ? I would be very happy if you could contact me for further corrospondence. Thank You Murat Eravci My Email is eravci@zedat.fu-berlin.de From owner-proteins@net.bio.net Wed Jan 06 22:00:00 1999 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.gtei.net!newsfeed.cwix.com!130.185.14.36!torn!news.dal.ca!nntp-user From: Biology Newsgroups: bionet.molbio.proteins Subject: Re: removing DNA Date: Wed, 06 Jan 1999 15:59:34 -0400 Organization: ISINet, Nova Scotia Lines: 39 Message-ID: <3693C0A6.866AD287@moon.sba.dal.ca> References: <368A090F.767347C0@uni-molgen.gwdg.de> NNTP-Posting-Host: lvining.biology.dal.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 I know if DNA is free in mixture with proteins it could be percipitated with Streptomycin sulfate. I have to look for the concentration. Mahmood Dalhousie university Bernard P. Murray, PhD wrote: > In article <368A090F.767347C0@uni-molgen.gwdg.de>, Silke Beismann > wrote: > > > Hi, > > I am trying to purify a protein from the amino acid biosynthetic > > pathway. It should not have anything to do with DNA but it seems that it > > binds to it. I tried digestion of the crude extract with DNase I and > > RNase A, precipitation with ammonium sulfate or polyehtylene imine, > > hydroxyapatite chromatography- but the spectrum still looks like an > > nucleic acid spectrum. Does anyone know an efficient method to remove > > the > > contaminating nucleic acids? > > Thanks in advance, > > Silke > > Just curious. I don't know how the "spectrum" can pick out > the DNA nature of the protein/complex but is there any way > that your protein could be modified (eg. ADP-ribosylated > or a prosthetic group) that would give the impression of > DNA contamination? Maybe treatment with a DNA kinase > would help determine if it is really adsorbed DNA > (should only label if it is really "DNA"). > Bernard > -- > Bernard P. Murray, PhD > Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA From owner-proteins@net.bio.net Wed Jan 06 22:00:00 1999 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.gtei.net!sunqbc.risq.qc.ca!news.dal.ca!nntp-user From: Biology Newsgroups: bionet.molbio.proteins Subject: Re: 5 kDa protein detection Date: Wed, 06 Jan 1999 16:03:58 -0400 Organization: ISINet, Nova Scotia Lines: 31 Message-ID: <3693C1AE.9C82BABA@moon.sba.dal.ca> References: <367A63DA.CA31E120@mbio.aau.dk> <36902084.79F48475@telerama.com> NNTP-Posting-Host: lvining.biology.dal.ca Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.05 [en] (Win95; I) To: Ryan Werstuik It is strange but try Ethidium bromide for staining the SDS PAGE, about 6 minutes is enough. Mahmood Ryan Werstuik wrote: > I would try using protein markers (with your smallest at 5kDa), running > the sample in a center lane, and 1/2 to 2/3 the current time you are using > now. I have also heard/read of using a stain that allows the samples to > be seen as the gel runs; however, I can't remember the where. > > I hope this helps, > Ryan > > Rasmus Wendelbo Nielsen wrote: > > > I am currently trying to purify a protein of 5 kDa, but are having > > problems detecting the protein. As it only contains tree tyrosines and > > no tryptophans the A280 is very low. I have tried running 18% SDS-PA > > gels and a 15 to 25% SDS PA gradient-gels, without luck, the protein > > probably diffuses out of the gel during staining. > > So I am looking for other (easy) ways to detect low MW proteins. Any > > ideas would be appreciated! > > I have heard that adding coomassie to the running buffer instead of > > post-staining the gels could be a solution. Anyone got experience with > > that? > > Thanks, Rasmus From owner-proteins@net.bio.net Wed Jan 06 22:00:00 1999 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.gtei.net!baron.netcom.net.uk!netcom.net.uk!server3.netnews.ja.net!news.icnet!NewsWatcher!user From: i.mcfarlane@icrf.icnet.uk (Ian McFarlane) Newsgroups: bionet.molbio.proteins Subject: Re: Precise protein concentration Date: 6 Jan 1999 17:03:41 GMT Organization: Imperial Cancer Research Fund Lines: 57 Message-ID: References: <36937EC2.BD70B3FA@biochem.purdue.edu> NNTP-Posting-Host: 143.65.17.54 Try a Lowry assay in the prescence of SDS. Also if you want an amino acid analysis you can just pellet your protein with acetone it doesn't have to be in soln. Ian Mc In article <36937EC2.BD70B3FA@biochem.purdue.edu>, Lena Zaitseva wrote: > --------------B5962DAB68528C118428EC82 > Content-Type: text/plain; charset=iso-8859-1 > Content-Transfer-Encoding: quoted-printable > > =A0=A0=A0=A0 Hi, > =A0=A0=A0=A0 We purified a membrane protein in the presence of non-ionic > detergent. Now we need to calculate precise protein concentration. It > appeared to be a real problem. > =A0=A0=A0=A0 Bradford method doesn't work at all. BCA method looks better= > , but > with BSA as control it doesn't give us reliable results (there is no > linear dependence of OD from concentration of our protein). For > UV-method it would be nice to unfold=A0 protein to get the maximum > absorbency at 280 nm. However, guanidine-HCl and SDS lead to the protein > aggregation. We tried also make quantitative amino-acid analysis of the > protein, but=A0 presence of high salt concentration (1.5 M) and glycerol > in the sample buffer is a problem in that case. > =A0=A0=A0=A0 Do you have any idea about that? > =A0=A0=A0=A0 Thanks for your suggestions. > =A0=A0=A0=A0 Lena. > > --------------B5962DAB68528C118428EC82 > Content-Type: text/html; charset=us-ascii > Content-Transfer-Encoding: 7bit > > >      Hi, >
     We purified a membrane protein in the presence > of non-ionic detergent. Now we need to calculate precise > protein concentration. It appeared to be a real problem. >
     Bradford method doesn't work at all. BCA method > looks better, but with BSA as control it doesn't give us reliable results > (there is no linear dependence of OD from concentration of our protein). > For UV-method it would be nice to unfold  protein to get the maximum > absorbency at 280 nm. However, guanidine-HCl and SDS lead to the protein > aggregation. We tried also make quantitative amino-acid analysis of the > protein, but  presence of high salt concentration (1.5 M) and glycerol > in the sample buffer is a problem in that case. >
     Do you have any idea about that? >
     Thanks for your suggestions. >
     Lena. > > --------------B5962DAB68528C118428EC82-- From owner-proteins@net.bio.net Wed Jan 06 22:00:00 1999 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.gtei.net!newsfeed.cwix.com!198.82.160.249!solaris.cc.vt.edu!news.vt.edu!news.cc.ukans.edu!eagle.cc.ukans.edu!freckles From: Lisa Kueltzo Newsgroups: bionet.molbio.proteins Subject: Re: 5 kDa protein detection Date: Wed, 6 Jan 1999 14:31:08 -0600 Organization: University of Kansas Computing Services Lines: 36 Message-ID: References: <367A63DA.CA31E120@mbio.aau.dk> <36902084.79F48475@telerama.com> NNTP-Posting-Host: eagle.cc.ukans.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII In-Reply-To: <36902084.79F48475@telerama.com> I'm nut sure what system you are using, but I've detected 3.5 kD peptides on 10% SDS-Page gels before. I've used the NOVEX Nu-Page system, and had no problems detecting it using their colloidal blue stain. Also, considering you have three Tyr residues, have you tried fluorescence detection instead of UV? Hope this helps, Lisa > Rasmus Wendelbo Nielsen wrote: > > > I am currently trying to purify a protein of 5 kDa, but are having > > problems detecting the protein. As it only contains tree tyrosines and > > no tryptophans the A280 is very low. I have tried running 18% SDS-PA > > gels and a 15 to 25% SDS PA gradient-gels, without luck, the protein > > probably diffuses out of the gel during staining. > > So I am looking for other (easy) ways to detect low MW proteins. Any > > ideas would be appreciated! > > I have heard that adding coomassie to the running buffer instead of > > post-staining the gels could be a solution. Anyone got experience with > > that? > > Thanks, Rasmus > ******************************************************** Lisa Kueltzo Department of Pharmaceutical Chemistry University of Kansas (785) 864-3010 "Black Holes are where God Divided by Zero." - Anonymous ******************************************************** From owner-proteins@net.bio.net Wed Jan 06 22:00:00 1999 Path: biosci!daresbury!uninett.no!news.maxwell.syr.edu!newsfeed.gamma.ru!Gamma.RU!newsfeed.metronet.de!uni-erlangen.de!uni-regensburg.de!newsserv.uni-bayreuth.de!not-for-mail From: Georg.Lipps@uni-bayreuth.de (Georg Lipps) Newsgroups: bionet.molbio.proteins Subject: Re: 5 kDa protein detection Date: Thu, 07 Jan 1999 15:49:41 GMT Organization: Universität Bayreuth Lines: 8 Message-ID: <3694d730.31953936@newsserv.uni-bayreuth.de> References: <36902084.79F48475@telerama.com> NNTP-Posting-Host: btcbd4.che.uni-bayreuth.de X-Newsreader: Forte Free Agent 1.11/16.235 Hi, why not try a 16% Tricine-SDS-Gel (Schägger & von Jagow) , works good down to at least 2 kDa. Good luck, Georg From owner-proteins@net.bio.net Wed Jan 06 22:00:00 1999 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.gtei.net!newshub.northeast.verio.net!hammer.uoregon.edu!leto.backbone.ou.edu!news.nodak.edu!plains.NoDak.edu!prischma From: prischma@plains.NoDak.edu (Jeffrey Prischmann) Newsgroups: bionet.molbio.proteins Subject: Flax seed protein analysis Date: 6 Jan 1999 20:31:06 GMT Organization: North Dakota Higher Education Computing Network Lines: 3 Message-ID: <770h6a$qru$1@node2.nodak.edu> NNTP-Posting-Host: plains.nodak.edu Mime-Version: 1.0 Content-Type: text/plain; charset=US-ASCII Content-Transfer-Encoding: 7bit X-Trace: node2.nodak.edu 915654666 27518 134.129.111.64 (6 Jan 1999 20:31:06 GMT) X-Complaints-To: abuse@nodak.edu NNTP-Posting-Date: 6 Jan 1999 20:31:06 GMT X-Newsreader: TIN [version 1.2 PL2] Does anyone know of a method to extract seed proteins from flax and an electrophoesis proceedure to separate them? I am trying to do variety identification of flax using seed protein. From owner-proteins@net.bio.net Wed Jan 06 22:00:00 1999 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.gtei.net!news1.bellglobal.com!sapphire.mtt.net!news.dal.ca!nntp-user From: Biology Newsgroups: bionet.molbio.proteins Subject: Re: Precise protein concentration Date: Wed, 06 Jan 1999 16:08:08 -0400 Organization: ISINet, Nova Scotia Lines: 67 Message-ID: <3693C2A8.BDCD0E4F@moon.sba.dal.ca> References: <36937EC2.BD70B3FA@biochem.purdue.edu> NNTP-Posting-Host: lvining.biology.dal.ca Mime-Version: 1.0 Content-Type: multipart/alternative; boundary="------------D0AB001BF818EED044DE65A0" X-Mailer: Mozilla 4.05 [en] (Win95; I) --------------D0AB001BF818EED044DE65A0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit Hi I am not sure if you have already done this or not, but if the correlation between the concentration and Abs. is not linear, try to dilute the protein sample before adding it to the 2 ml reagent in BCA method. dilution as 1/2 or 1/4 may help. then add 100 ul of diluted sample to 2 ml reagent. Mahmood Lena Zaitseva wrote: > Hi, > We purified a membrane protein in the presence of non-ionic > detergent. Now we need to calculate precise protein concentration. It > appeared to be a real problem. > Bradford method doesn't work at all. BCA method looks better, but > with BSA as control it doesn't give us reliable results (there is no > linear dependence of OD from concentration of our protein). For > UV-method it would be nice to unfold protein to get the maximum > absorbency at 280 nm. However, guanidine-HCl and SDS lead to the > protein aggregation. We tried also make quantitative amino-acid > analysis of the protein, but presence of high salt concentration (1.5 > M) and glycerol in the sample buffer is a problem in that case. > Do you have any idea about that? > Thanks for your suggestions. > Lena. --------------D0AB001BF818EED044DE65A0 Content-Type: text/html; charset=us-ascii Content-Transfer-Encoding: 7bit Hi
I am not sure if you have already done this or not, but if the correlation between the concentration and Abs. is not linear, try to dilute the protein sample before adding it to the 2 ml reagent in BCA method. dilution as 1/2  or 1/4 may help. then add 100 ul of diluted sample to 2 ml reagent.

Mahmood

Lena Zaitseva wrote:

      Hi,
     We purified a membrane protein in the presence of non-ionic detergent. Now we need to calculate precise protein concentration. It appeared to be a real problem.
     Bradford method doesn't work at all. BCA method looks better, but with BSA as control it doesn't give us reliable results (there is no linear dependence of OD from concentration of our protein). For UV-method it would be nice to unfold  protein to get the maximum absorbency at 280 nm. However, guanidine-HCl and SDS lead to the protein aggregation. We tried also make quantitative amino-acid analysis of the protein, but  presence of high salt concentration (1.5 M) and glycerol in the sample buffer is a problem in that case.
     Do you have any idea about that?
     Thanks for your suggestions.
     Lena.
  --------------D0AB001BF818EED044DE65A0-- From owner-proteins@net.bio.net Fri Jan 08 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!howland.erols.net!newspeer.monmouth.com!newsgate.cistron.nl!het.net!news.worldonline.nl!not-for-mail From: "Van De Weghe - De Meyer" Newsgroups: bionet.molbio.proteins Subject: Re: Flax seed protein analysis Date: Sat, 9 Jan 1999 22:35:07 +0100 Organization: WorldOnline News server Lines: 20 Message-ID: <778ibi$b68$1@news.worldonline.nl> References: <770h6a$qru$1@node2.nodak.edu> NNTP-Posting-Host: dp19-6.worldonline.be X-Trace: news.worldonline.nl 915918002 11464 212.233.19.6 (9 Jan 1999 21:40:02 GMT) X-Complaints-To: abuse@worldonline.nl NNTP-Posting-Date: 9 Jan 1999 21:40:02 GMT X-Newsreader: Microsoft Outlook Express 4.72.3110.5 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3110.3 About 10 years ago I have tested different buffer solutions of different pH values and some alcoholic solutions to extract seed proteins from meal of flax seeds, either or not defatted. Some extracts were suited for PAGE, SDS-PAGE and/or PAGIF. Sorry for the bad news : in none of the electrophoresis experiments it was possible to distinguish the varieties, as can be done with wheat, barley, oats, maize, ryegrass, potatoes, ... Some RAPD experiments resuted in differentiation in groups, but those experiments are temporarily stopped. If you like to know more about it, or if you succeed to differentiate between flax cultivars by protein electrophoresis, please contact me at e-mail : luc.vandeweghe@worldonline.be Jeffrey Prischmann heeft geschreven in bericht <770h6a$qru$1@node2.nodak.edu>... >Does anyone know of a method to extract seed proteins from flax and an >electrophoesis proceedure to separate them? I am trying to do variety >identification of flax using seed protein. From owner-proteins@net.bio.net Fri Jan 08 22:00:00 1999 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!amsterdam1-snf1!news.gtei.net!diablo.theplanet.net!btnet-peer!btnet!nntp.news.xara.net!xara.net!RMplc!rmplc.co.uk!yama.mcc.ac.uk!not-for-mail From: cochran@sjh.bi.umist.ac.uk (Duncan Cochran) Newsgroups: bionet.molbio.proteins Subject: peptide bond absorption spectrum Date: 8 Jan 1999 15:44:16 GMT Organization: Sirius Cybernetics Corporation Lines: 9 Message-ID: <77594g$9cd$2@probity.mcc.ac.uk> NNTP-Posting-Host: dac1.bi.umist.ac.uk X-Posted-From: InterNews 1.0.7@dac1.bi.umist.ac.uk Xdisclaimer: No attempt was made to authenticate the sender's name. Hi Can anybody tell me where I can find a picture of the UV absorbance spectrum for a peptide bond? Thanks Duuncan cochran@sjh.bi.umist.ac.uk From owner-proteins@net.bio.net Fri Jan 08 22:00:00 1999 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.gtei.net!news.maxwell.syr.edu!nntp2.dejanews.com!nnrp1.dejanews.com!not-for-mail From: frank.moersberger@merck.de Newsgroups: bionet.molbio.proteins Subject: Re: removing DNA - The smart solution is - Benzonase Date: Fri, 08 Jan 1999 13:09:17 GMT Organization: Deja News - The Leader in Internet Discussion Lines: 39 Message-ID: <77501s$uoe$1@nnrp1.dejanews.com> References: <368A090F.767347C0@uni-molgen.gwdg.de> Reply-To: frank.moersberger@merck.de NNTP-Posting-Host: 194.196.248.36 X-Article-Creation-Date: Fri Jan 08 13:09:17 1999 GMT X-Http-User-Agent: Mozilla/2.0 (compatible; MSIE 3.01; Windows NT) X-Http-Proxy: 1.0 x6.dejanews.com:80 (Squid/1.1.22) for client 194.196.248.36 Dear Silke, you wrote that you used DNAse and RNAse, I assume with poor success. I'd like to put your attention to a special endonuclease that is much more effective (if used properly). It is called Benzonase and it's a recombinant unspecific endonuclease with a very high specific activity. For a total digestion of nucleic acids (DNA, RNA, ss, ds, circular, supercoiled) you need only very small amounts. The preparation is free of any detectable protease impurities, (that are almost always present in DNAse1 preparations, causing sometimes trouble). Benzonase needs (as all nucleases) Mg2+ ions for full activity and does not work very well in phosphate buffers. However in presence of detergents, urea etc it is quite stable. If you like to know more details about this enzyme please send an e-mail to: frank.moersberger@merck.de. I can provide you with a product description and application examples. Best regards Frank In article <368A090F.767347C0@uni-molgen.gwdg.de>, Silke Beismann wrote: > Hi, > I am trying to purify a protein from the amino acid biosynthetic > pathway. It should not have anything to do with DNA but it seems that it > > binds to it. I tried digestion of the crude extract with DNase I and > RNase A, precipitation with ammonium sulfate or polyehtylene imine, > hydroxyapatite chromatography- but the spectrum still looks like an > nucleic acid spectrum. Does anyone know an efficient method to remove > the > contaminating nucleic acids? > Thanks in advance, > > Silke > > -----------== Posted via Deja News, The Discussion Network ==---------- http://www.dejanews.com/ Search, Read, Discuss, or Start Your Own From owner-proteins@net.bio.net Fri Jan 08 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!nntp.abs.net!newshub2.home.com!newshub1.home.com!news.home.com!news.rdc1.bc.wave.home.com.POSTED!not-for-mail From: "Achim Recktenwald" Newsgroups: bionet.molbio.proteins References: Subject: Re: Stability of protein in glycerol Lines: 29 X-Newsreader: Microsoft Outlook Express 4.72.3155.0 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3155.0 Message-ID: Date: Sat, 09 Jan 1999 17:39:31 GMT NNTP-Posting-Host: 24.64.220.105 X-Complaints-To: abuse@home.net X-Trace: news.rdc1.bc.wave.home.com 915903571 24.64.220.105 (Sat, 09 Jan 1999 09:39:31 PDT) NNTP-Posting-Date: Sat, 09 Jan 1999 09:39:31 PDT Organization: @Home Network Canada For native PAGE everything is possible, as long as it is not a salt. You might have to play around with your protein a bit and try different buffer matrices. We are working with proteins that are often denatured during purification and have to keep them in solution. Lately we had one that precipitated in everything we tried, till somebody more by mistake dialyzed it against 10 mM Tris; it's soluble don't ask we why. Cheers, Achim Vladimir Rotrekl wrote in message ... >Dear all, > I have produced recently some mutant proteins, which are not really >examples of protein stability. But they are at least able to stay in >solution at 4C. The problem appeared when I tried to run native PAGE. >Samples precipitated after addition of glycerol(20%) loading buffer. > My qustion is if (and if yes then how) it is possible to use other >compounds to prepare sample buffer for proteins (like in DNA 10% ficol or >sucrose). > >Thanks a lot > Vladimir > > From owner-proteins@net.bio.net Fri Jan 08 22:00:00 1999 Newsgroups: bionet.molbio.proteins Path: biosci!agate!newsfeed.berkeley.edu!diablo.theplanet.net!news-lond.gip.net!news-raspail.gip.net!news.gsl.net!gip.net!newsfeed.ecrc.net!news.cesnet.cz!rhino.cis.vutbr.cz!news.muni.cz!news From: "Vladimir Rotrekl" Subject: Stability of protein in glycerol X-Nntp-Posting-Host: lmfrj.sci.muni.cz Message-ID: X-Mimeole: Produced By Microsoft MimeOLE V4.72.3110.3 Lines: 13 Sender: news@news.muni.cz (News Admin) Organization: unknown X-Newsreader: Microsoft Outlook Express 4.72.3110.5 Date: Sat, 9 Jan 1999 15:39:19 GMT Dear all, I have produced recently some mutant proteins, which are not really examples of protein stability. But they are at least able to stay in solution at 4C. The problem appeared when I tried to run native PAGE. Samples precipitated after addition of glycerol(20%) loading buffer. My qustion is if (and if yes then how) it is possible to use other compounds to prepare sample buffer for proteins (like in DNA 10% ficol or sucrose). Thanks a lot Vladimir From owner-proteins@net.bio.net Fri Jan 08 22:00:00 1999 Path: biosci!newshost.lanl.gov!awabi.library.ucla.edu!128.32.206.55!newsfeed.berkeley.edu!news-peer1.sprintlink.net!news-in-east1.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: removing DNA - The smart solution is - Benzonase Date: Fri, 08 Jan 1999 18:28:04 -0800 Organization: University of California, San Francisco Lines: 23 Message-ID: References: <368A090F.767347C0@uni-molgen.gwdg.de> <77501s$uoe$1@nnrp1.dejanews.com> NNTP-Posting-Host: mac-daddy.ucsf.edu In article <77501s$uoe$1@nnrp1.dejanews.com>, frank.moersberger@merck.de wrote: > Dear Silke, > you wrote that you used DNAse and RNAse, I assume with poor success. I'd like > to put your attention to a special endonuclease that is much more effective > (if used properly). It is called Benzonase [advert clipped] > Best regards > Frank Is this the same thing as eg. micrococcal nuclease? How do you inactivate benzonase? Can you treat a protein sample and then inactivate the enzyme so that you can re-introduce DNA or RNA and not have them chewed up? (ie. would it be any use in cleaning samples for transcription/translation?) Thanks for any (scientific) info, Bernard -- Bernard P. Murray, PhD Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA From owner-proteins@net.bio.net Fri Jan 08 22:00:00 1999 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.gtei.net!news-peer.gip.net!news.gsl.net!gip.net!news.maxwell.syr.edu!newsfeed.nyu.edu!newsfeed.sgi.net!nntp.itis.com!news.doit.wisc.edu!default From: klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins Subject: Re: Transfecting sperm-cells Date: Fri, 08 Jan 1999 16:54:41 GMT Organization: UW-Madison Lines: 89 Message-ID: <775d75$18go$1@news.doit.wisc.edu> References: <76ss36$rqk$1@wnnews.sci.kun.nl> NNTP-Posting-Host: martinlab.biochem.wisc.edu Mime-Version: 1.0 Content-Type: text/plain; charset=KOI8-R Content-Transfer-Encoding: 8bit X-Newsreader: News Xpress 2.01 X-No-Archive: Yes In article <76ss36$rqk$1@wnnews.sci.kun.nl>, "Gerrit Bouw" wrote: :Dear everyone, : :Does anyone ever tried to transfect sperm-cells? If so, please could you :give :me some guidance on this? : :Gerrit : : [what I don't unde4rstand is why this has been posted to *.proteins and not to methods and reagents...] Yes, spermatozoa are just normal cells and can be lectroporated like any other. A 1 minute search in Medline: Title Sperm as a carrier to introduce an exogenous DNA fragment into the oocyte ¦ of Japanese abalone (Haliotis divorsicolor suportexta). ¦ Source ¦ Transgenic Research. 6(1):85-95, 1997 Jan. Title Nuclear internalization of foreign DNA by zebrafish spermatozoa and its ¦ enhancement by electroporation. ¦ Source ¦ Journal of Experimental Zoology. 274(2):121-9, 1996 Feb 1. Title Sperm characteristics and outcome of human assisted fertilization by ¦ subzonal insemination and intracytoplasmic sperm injection [see comments]. ¦ Source ¦ Fertility & Sterility. 59(4):826-35, 1993 Apr. Title Electroporation: a method for transferring genes into the gametes of ¦ zebrafish (Brachydanio rerio), channel catfish (Ictalurus punctatus), and ¦ common carp (Cyprinus carpio). ¦ Source ¦ Molecular Marine Biology & Biotechnology. 1(4-5):301-8, 1992 Aug-Oct. Title Introducing foreign genes into fish eggs with electroporated sperm as a ¦ carrier. ¦ Source ¦ Molecular Marine Biology & Biotechnology. 1(4-5):276-81, 1992 Aug-Oct. Title Electroporation of bovine spermatozoa to carry foreign DNA in oocytes. ¦ Source ¦ Molecular Reproduction & Development. 29(1):6-15, 1991 May. Title [The incorporation of macromolecules into the germ cells of male mice via ¦ electroporation and dimethyl sulfoxide]. [Russian] ¦ Source ¦ Tsitologiia i Genetika. 24(3):3-7, 1990 May-Jun. Hope this helps, - Dima From owner-proteins@net.bio.net Sat Jan 09 22:00:00 1999 Path: biosci!news.stanford.edu!logbridge.uoregon.edu!newsfeed.berkeley.edu!isdnet!grolier!club-internet!not-for-mail From: "Philippe QUAY" Newsgroups: bionet.molbio.proteins Subject: Search infos about Protein Kinase C. Help me Pls! Date: Sun, 10 Jan 1999 19:21:45 +0100 Organization: Club-Internet (France) Lines: 19 Message-ID: <77arat$7ua$1@front6.grolier.fr> NNTP-Posting-Host: nevers1-230.club-internet.fr X-Trace: front6.grolier.fr 915992733 8138 194.158.118.230 (10 Jan 1999 18:25:33 GMT) NNTP-Posting-Date: 10 Jan 1999 18:25:33 GMT X-Newsreader: Microsoft Outlook Express 4.72.3110.5 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3155.0 I am a french student in cellular biology and physiology. I have to do a report about Protein Kinase C; could you help me by indicating websites where i could find infos about it? Tks in advance for help. Bénédicte Maitrise biologie cellulaire et physiologie Faculté de Dijon tks to reply on my mail pmquay@club-internet.fr From owner-proteins@net.bio.net Sat Jan 09 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!oleane!newsfeed.planete.net!pressimage!news.planete.net!grolier!club-internet!not-for-mail From: "Philippe QUAY" Newsgroups: bionet.molbio.proteins Subject: Search infos about PKC. Help me Pls! Date: Sun, 10 Jan 1999 11:18:09 +0100 Organization: Club-Internet (France) Lines: 16 Message-ID: <779v2l$b71$2@front7.grolier.fr> NNTP-Posting-Host: macon1-22.club-internet.fr X-Trace: front7.grolier.fr 915963797 11489 195.36.141.22 (10 Jan 1999 10:23:17 GMT) NNTP-Posting-Date: 10 Jan 1999 10:23:17 GMT X-Newsreader: Microsoft Outlook Express 4.72.3110.5 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3155.0 I am a french student in cellular biology and physiology. I have to do a report about PKC; could you help me by indicating websites where i could find infos about it? Tks in advance for help. Bénédicte Maitrise biologie cellulaire et physiologie Faculté de Dijon tks to reply on my mail pmquay@club-internet.fr From owner-proteins@net.bio.net Sat Jan 09 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!remarQ73!supernews.com!remarQ69!not-for-mail From: Ryan Werstuik Newsgroups: bionet.molbio.proteins Subject: Suggestions for undergrad research anyone? Date: Sat, 09 Jan 1999 20:08:39 -0500 Organization: Posted via RemarQ, http://www.remarQ.com - Discussions start here! Lines: 11 Message-ID: <3697FD97.B12A1770@telerama.com> NNTP-Posting-Host: 204.171.44.17 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: 916015648 BPIPSDRGT2C11CCABC usenet78.supernews.com X-Complaints-To: newsabuse@remarQ.com X-Mailer: Mozilla 4.5 [en] (Win95; U) X-Accept-Language: en Hello again, I am wondering if anyone has any suggestions for some type of (original) research project for an under grad student relating to protein biochemistry or microbiology. I have been working on a protein (isolation, crystallization, x-ray diffraction) and then someone published (last month) what I was hoping to do. Any suggestions will be greatly appreciated. Thanks, Ryan From owner-proteins@net.bio.net Sat Jan 09 22:00:00 1999 Path: biosci!news.stanford.edu!logbridge.uoregon.edu!netnews1.nw.verio.net!netnews.nwnet.net!socal.verio.net!nntp.ni.net!news.service.uci.edu!usenet From: "A. Lee" Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: help needed for reconstituting NMDA in membrane Date: Sun, 10 Jan 1999 22:36:58 -0800 Organization: University of California, Irvine Lines: 8 Message-ID: <77c5k2$ke@news.service.uci.edu> NNTP-Posting-Host: dialin9248.slip.uci.edu X-Newsreader: Microsoft Outlook Express 4.72.3110.1 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3110.3 Xref: biosci bionet.molbio.methds-reagnts:73206 bionet.molbio.proteins:13798 Hello out there. I was wondering if anyone out there knows of an effective way to solubilize and reconstruct the NMDA receptors into little micelle vessicles. I am having a very difficult time with it. There seem to be a few articles on it but so far the highest percent yield was back in 1988 at 20% only. If anyone knows please reply. It will be greatly appreciated. thanx From owner-proteins@net.bio.net Sat Jan 09 22:00:00 1999 Path: biosci!news.stanford.edu!nntp.stanford.edu!d305-powermac.stanford.edu!user From: pbradley@stanford.edu (Peter Bradley) Newsgroups: bionet.molbio.proteins Subject: Re: Stability of protein in glycerol Date: Sun, 10 Jan 1999 17:06:07 -0800 Organization: Stanford University Lines: 18 Message-ID: References: NNTP-Posting-Host: d305-powermac.stanford.edu In article , "Vladimir Rotrekl" wrote: > Dear all, > I have produced recently some mutant proteins, which are not really > examples of protein stability. But they are at least able to stay in > solution at 4C. The problem appeared when I tried to run native PAGE. > Samples precipitated after addition of glycerol(20%) loading buffer. > My qustion is if (and if yes then how) it is possible to use other > compounds to prepare sample buffer for proteins (like in DNA 10% ficol or > sucrose). > > Thanks a lot > Vladimir Proteins that I have purified for sequencing and dont want to block the N-terminus I use a sucrose-based loading buffer described by BioRad. You can find it from them with instructions to use their PVDF membrane. good luck, peter From owner-proteins@net.bio.net Sat Jan 09 22:00:00 1999 From: Cornelius Krasel Subject: Re: Search infos about Protein Kinase C. Help me Pls! Newsgroups: bionet.molbio.proteins References: <77arat$7ua$1@front6.grolier.fr> User-Agent: tin/pre-1.4-980514 (UNIX) (Linux/2.0.34 (i486)) Date: Sun, 10 Jan 1999 20:07:20 +0100 Message-ID: <8pta77.8ol.ln@wpxx02.toxi.uni-wuerzburg.de> NNTP-Posting-Host: wpxx02.toxi.uni-wuerzburg.de Lines: 12 Path: biosci!agate!newsfeed.berkeley.edu!newsfeed.gamma.ru!Gamma.RU!newsfeed.metronet.de!uni-erlangen.de!uni-wuerzburg.de!not-for-mail Philippe QUAY wrote: > I have to do a report about Protein Kinase C; could you help me by > indicating websites where i could find infos about it? http://www4.ncbi.nlm.nih.gov/PubMed/ --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 Mon Jan 11 22:00:00 1999 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!amsterdam1-snf1!news.gtei.net!diablo.theplanet.net!btnet-peer!btnet-feed2!btnet!bt!not-for-mail From: "Matt Sullivan" Newsgroups: bionet.molbio.proteins Subject: Help getting info on ribonuclease H and phospholipase A2 Date: Tue, 12 Jan 1999 09:47:26 -0000 Organization: BT Labs, Martlesham Heath, Ipswich, UK Lines: 9 Message-ID: <77f5rm$8sp$1@pheidippides.axion.bt.co.uk> NNTP-Posting-Host: tb173.info.bt.co.uk Mime-Version: 1.0 Content-Type: text/plain; charset=US-ASCII Content-Transfer-Encoding: 7bit X-Newsreader: Microsoft Outlook Express 4.72.3110.5 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3110.3 Hello all, I need some very basic info on the two proteins ribonuclease H and phospholipase A2, such as basic function. Can anyone help me with this? Tanks, M Sullivan From owner-proteins@net.bio.net Mon Jan 11 22:00:00 1999 Path: biosci!news.stanford.edu!logbridge.uoregon.edu!newspeer.monmouth.com!rill.news.pipex.net!pipex!sun4nl!192.87.106.104.MISMATCH!news!barba.uci.kun.nl!not-for-mail From: "piet" Newsgroups: bionet.molbio.proteins Subject: Re: 5 kDa protein detection Date: Tue, 12 Jan 1999 18:14:07 +0100 Organization: Universitair Centrum Informatievoorziening, The Netherlands Lines: 16 Message-ID: <77fuba$kie$1@barba.uci.kun.nl> References: <36902084.79F48475@telerama.com> <3694d730.31953936@newsserv.uni-bayreuth.de> NNTP-Posting-Host: m38-031.fmw.kun.nl X-Newsreader: Microsoft Outlook Express 4.72.2106.4 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.2106.4 Georg Lipps wrote in message <3694d730.31953936@newsserv.uni-bayreuth.de>... >Hi, > >why not try a 16% Tricine-SDS-Gel (Schägger & von Jagow) , works good >down to at least 2 kDa. > >Good luck, > >Georg Yes, works for me. However when staining with CBB, stain for at least one hour shaking (do not try to heat the gel because the protein then diffuses out of the gel). Decolor o/n and then you must see the band very clearly. From owner-proteins@net.bio.net Tue Jan 12 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!newsfeed.gamma.ru!Gamma.RU!demos!rosnet!news.glas.net!not-for-mail From: "Biotechmet Co." Newsgroups: bionet.molbio.proteins Subject: visit http://www.glasnet.ru/~biotechmet Date: Thu, 14 Jan 1999 07:02:23 +0300 Organization: Glasnet Lines: 61 Sender: biotechmet@ppp1538.glasnet.ru Message-ID: <77jq92$lt3$1@swan.glasnet.ru> NNTP-Posting-Host: ppp1538.glasnet.ru X-Trace: swan.glasnet.ru 916286562 22435 195.218.251.26 (14 Jan 1999 04:02:42 GMT) X-Complaints-To: usenet@news.glas.net NNTP-Posting-Date: 14 Jan 1999 04:02:42 GMT X-Newsreader: Microsoft Outlook Express 4.72.2106.4 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.2106.4 Leather processing - new approach, and much more of biotechnolgy! Reduce your costs with new technology!!! ================ BIOTECHMET CO. ================= We propose a new ecologically safe & clean technology for leather raw materials processing. Patent proven technology which based on new ferment preparation named PROK. FOR EACH LEATHER MANUFACTURER ! INGREDIENTS ARE AVAILABLE IMMEDIATELY. *** Lower processing cost. *** No renovation needed. *** Major expenses (incl.. sewage refinement) goes down dramatically. *** Number of times reducing of pollution. *** Suitable to locate near of residential area. Detailed data is available upon request. All manufacturers and interested persons are welcome. Mail to: biotechmet@glasnet.ru or to multicom@glasnet.ru Also, You can visit our new home page at http://www.glasnet.ru/~biotechmet/ THANK YOU. Contact officer: Michael M. Migunov, Authorized Representative of Biotechmet Co. From owner-proteins@net.bio.net Tue Jan 12 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!news-peer1.sprintlink.net!news-backup-west.sprintlink.net!news.sprintlink.net!itssrv1.ucsf.edu!macmac-2.ucsf.edu!user From: bpmurray*STUFFER*@socrates.ucsf.edu (Bernard P. Murray, PhD) Newsgroups: bionet.molbio.proteins Subject: Re: How to find the DNA sequence through PDB? Date: Wed, 13 Jan 1999 17:31:25 -0700 Organization: University of California, San Francisco Lines: 34 Distribution: world Message-ID: References: <19990113124033.20629.qmail@hotmail.com> NNTP-Posting-Host: macmac-2.ucsf.edu In article <19990113124033.20629.qmail@hotmail.com>, cathyzhang01@HOTMAIL.COM ("zhang cathy") wrote: > Hi, > I have a PDB code for a protein. Now I would like to know the DNA > sequence of this protein. Then how could I find it through GenBank or > through Internet? > Thank you very much! > cathy > cathyzhang01@hotmail.com It is easy to do this, albeit a little indirectly. Using the PDB code grab the entry from Entrez or BNL and follow the protein link. This will give you the SwissProt entry and the GenBank code for the (c)DNA sequence is in the header. If I remember correctly BNL has this as a hot link while Entrez lets you grab it through the "nucleotide neighbour". Entrez is: http://www.ncbi.nlm.nih.gov/Entrez/structure.html BNL is: http://www.pdb.bnl.gov/pdb-bin/pdbmain it may also be possible to do this through ExPASy but I haven't tried this. http://expasy.hcuge.ch I hope that this helps, Bernard -- Bernard P. Murray, PhD Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA From owner-proteins@net.bio.net Tue Jan 12 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!remarQ73!supernews.com!remarQ69!not-for-mail From: Ryan Werstuik Newsgroups: bionet.molbio.proteins Subject: Re: How to find the DNA sequence through PDB? Date: Tue, 12 Jan 1999 13:31:20 -0500 Organization: Posted via RemarQ, http://www.remarQ.com - Discussions start here! Lines: 24 Message-ID: <369B94F8.86E2449D@telerama.com> References: <19990113124033.20629.qmail@hotmail.com> NNTP-Posting-Host: 204.171.44.17 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: 916251000 BPIPSDRGT2C11CCABC usenet78.supernews.com X-Complaints-To: newsabuse@remarQ.com X-Mailer: Mozilla 4.5 [en] (Win95; U) X-Accept-Language: en If you know the name of the protein, you can go to GenBank and search under Entrez/Nucleotides for the name of the protein. You can also use programs such as SwissPDBViewer to find the AA sequence, and then use one of the various reverse transcription programs on the internet to get a nucleotide sequence. --Ryan zhang cathy wrote: > Hi, > > I have a PDB code for a protein. Now I would like to know the DNA > sequence of this protein. Then how could I find it through GenBank or > through Internet? > > Thank you very much! > > cathy > cathyzhang01@hotmail.com > > ______________________________________________________ > Get Your Private, Free Email at http://www.hotmail.com From owner-proteins@net.bio.net Tue Jan 12 22:00:00 1999 Path: biosci!HOTMAIL.COM!cathyzhang01 From: cathyzhang01@HOTMAIL.COM ("zhang cathy") Newsgroups: bionet.molbio.proteins Subject: How to find the DNA sequence through PDB? Date: 13 Jan 1999 04:45:19 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 13 Sender: daemon@net.bio.net Distribution: world Message-ID: <19990113124033.20629.qmail@hotmail.com> NNTP-Posting-Host: net.bio.net Hi, I have a PDB code for a protein. Now I would like to know the DNA sequence of this protein. Then how could I find it through GenBank or through Internet? Thank you very much! cathy cathyzhang01@hotmail.com ______________________________________________________ Get Your Private, Free Email at http://www.hotmail.com From owner-proteins@net.bio.net Tue Jan 12 22:00:00 1999 Path: biosci!internet!biosci!not-for-mail From: biohelp (BIOSCI Administrator) Newsgroups: bionet.molbio.proteins Subject: BIOSCI/bionet miniFAQ & Fundraiser Date: 13 Jan 1999 02:00:12 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 233 Sender: daemon@net.bio.net Distribution: world Message-ID: <199901131000.CAA06528@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 Wed Jan 13 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!news-peer1.sprintlink.net!news-in-east1.sprintlink.net!news.sprintlink.net!itssrv1.ucsf.edu!macmac-2.ucsf.edu!user From: bpmurray*STUFFER*@socrates.ucsf.edu (Bernard P. Murray, PhD) Newsgroups: bionet.molbio.proteins Subject: Re: two hybrid system Date: Thu, 14 Jan 1999 19:27:39 -0700 Organization: University of California, San Francisco Lines: 19 Message-ID: References: <369DF6CF.14F1B8A4@ccr.jussieu.fr> NNTP-Posting-Host: macmac-2.ucsf.edu In article <369DF6CF.14F1B8A4@ccr.jussieu.fr>, Mathias Brault wrote: > Is it possible to use a two-hybrid system in order to identify cDNA > encoding protein able to bind a plasma-membrane protein ? > Thank you > Mathias Brault Stratagene is about to release a 2 hybrid kit that relies on interaction at the cell membrane. I've seen a couple of presentations on it and it looks good (although nowhere near as refined as the classic 2-hybrid). I don't know when it is actually due for release but it may be worth tackling your local rep. Bonne chance, Bernard -- Bernard P. Murray, PhD Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA From owner-proteins@net.bio.net Wed Jan 13 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!easynet-fr!oleane!jussieu.fr!not-for-mail From: Mathias Brault Newsgroups: bionet.molbio.proteins Subject: two hybrid system Date: Thu, 14 Jan 1999 14:53:33 +0100 Organization: Universites Paris VI/Paris VII - France Lines: 12 Message-ID: <369DF6CF.14F1B8A4@ccr.jussieu.fr> NNTP-Posting-Host: boa.snv.jussieu.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-Mailer: Mozilla 4.03 [fr] (Macintosh; I; PPC) Is it possible to use a two-hybrid system in order to identify cDNA encoding protein able to bind a plasma-membrane protein ? Thank you Mathias Brault LPDP, casier 156 UPMC 4 place Jussieu 75005 Paris, France From owner-proteins@net.bio.net Thu Jan 14 22:00:00 1999 Path: biosci!am.pnu.com!Silvano.Schito From: Silvano.Schito@am.pnu.com Newsgroups: bionet.molbio.proteins Subject: (none) Date: 15 Jan 1999 05:19:55 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 13 Sender: daemon@net.bio.net Distribution: world Message-ID: <00038621.C22094@am.pnu.com> NNTP-Posting-Host: net.bio.net I would like to receive informations about the following techniques: 1)Iso-electric focusing of glycosilated proteins such as rh-TPO 2)agarose gel electrophoresis of Heparin (sec.European Pharmacopoeia) thank you! Dr.Schito Silvano Pharmacia & Upjohn Pharm.Dev.Dept. viale Pasteur 10 20014 Nerviano (MI) Italy Fax n:02-48383159 From owner-proteins@net.bio.net Thu Jan 14 22:00:00 1999 Path: biosci!SPRCORE.CAREGROUP.HARVARD.EDU!turner From: turner@SPRCORE.CAREGROUP.HARVARD.EDU (bradley turner) Newsgroups: bionet.molbio.proteins Subject: Re: (none)->heparin electrophoresis Date: 15 Jan 1999 09:38:54 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 68 Sender: daemon@net.bio.net Distribution: world Message-ID: <9901151731.AA08437@sprcore.caregroup.harvard.edu> NNTP-Posting-Host: net.bio.net Hi Dr. Silvano, You might try contacting: Celsus Inc. 12150 Best Place Cincinnati, Ohio 45241-1569 USA 513-773-8130 phone 800-HEPARIN phone (toll free) 513-772-8132 fax 888-HEPARIN fax (toll free) info@heparin.com celsus@heparin.com http://www.heparin.com They make many heparins, glycosaminoglycans and heparin derivatives, and would probably be able to give you conditions (or at least references) for heparin electrophoresis. Standard disclaimer: No commercial interest just a satified customer Hope this helps, Brad Turner **************************************************************** Bradley Turner Beth Israel Deaconess Medical Center Harvard Medical School 617-667-1215 phone Division of Gastroenterology 617-667-2767 fax Room Dana 536 bsturner@biosun.harvard.edu 330 Brookline Avenue bturner@caregroup.harvard.edu Boston, MA 02215 turner@sprcore.bidmc.harvard.edu **************************************************************** BEGIN INCLUDED MESSAGE--------------------------------------------- From BIOSCI-REQUEST@net.bio.net Fri Jan 15 08:25:25 1999 Return-Path: To: protein-analysis@net.bio.net From: Silvano.Schito@am.pnu.com Subject: (none) Date: 15 Jan 1999 05:19:55 -0800 Sender: daemon@net.bio.net Message-Id: <00038621.C22094@am.pnu.com> Nntp-Posting-Host: net.bio.net Status: R I would like to receive informations about the following techniques: 1)Iso-electric focusing of glycosilated proteins such as rh-TPO 2)agarose gel electrophoresis of Heparin (sec.European Pharmacopoeia) thank you! Dr.Schito Silvano Pharmacia & Upjohn Pharm.Dev.Dept. viale Pasteur 10 20014 Nerviano (MI) Italy Fax n:02-48383159 END OF INCLUDED MESSAGE------------------------------------------- From owner-proteins@net.bio.net Thu Jan 14 22:00:00 1999 Path: biosci!RNA.BIO.MQ.EDU.AU!anouwens From: anouwens@RNA.BIO.MQ.EDU.AU ("Amanda Nouwens") Newsgroups: bionet.molbio.proteins Subject: Re: (none) Date: 15 Jan 1999 16:45:52 -0800 Organization: School of Biological Sciences Lines: 38 Sender: daemon@net.bio.net Distribution: world Message-ID: <199901160041.LAA02805@laurel.ocs.mq.edu.au> References: <00038621.C22094@am.pnu.com> Reply-To: anouwens@proteome.org.au NNTP-Posting-Host: net.bio.net To: protein-analysis@net.bio.net From: Silvano.Schito@am.pnu.com Subject: (none) Date sent: 15 Jan 1999 05:19:55 -0800 Dear Silvano, > I would like to receive informations about the following techniques: > 1)Iso-electric focusing of glycosilated proteins such as rh-TPO Assuming you are using IPG strips for the isoelectric focusing, the following is my suggestion. Iso-electric focusing (and 2D PAGE) of glycosylated proteins can be a little more tricky than non-glycosylated proteins. Sometimes it seems difficult to actually get them into the IPG strip. I would suggest two things - either use a sample buffer with strong reducing and denaturing conditions - refer to Molloy et al, 1998 (Electrophoresis 19:837-844) for details on sample solutions. Alternatively, you could try boiling your protein in SDS-DTT (standard 1D sample buffer), diluting the protein with the sample buffer used to rehydrate the IPG strip, and cup loading the protein. The above is fairly general - I can provide more specific information if required. Cheers, Amanda. --------------------------------------------------- Amanda Nouwens Australian Proteome Analysis Facility (APAF) Macquarie University Sydney, NSW 2109 Tel: 02 9850 6209 Fax: 02 9850 6200 Email: anouwens@proteome.org.au From owner-proteins@net.bio.net Fri Jan 15 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!sunqbc.risq.qc.ca!wesley.videotron.net!Pollux.Teleglobe.net!news.inet.co.th!news.nectec.or.th!news.cs.ait.ac.th!alphaserv.ait.ac.th!bpb87195 From: San Hein Newsgroups: bionet.molbio.proteins Subject: Non-interfering protein assay Date: Sat, 16 Jan 1999 13:21:38 +0700 Organization: CSIM Program, Asian Institute of Technology Lines: 15 Message-ID: NNTP-Posting-Host: 203.159.0.3 Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII X-Trace: cs4.cs.ait.ac.th 916467704 29922 203.159.0.3 (16 Jan 1999 06:21:44 GMT) X-Complaints-To: usenet@cs.ait.ac.th NNTP-Posting-Date: 16 Jan 1999 06:21:44 GMT Dear all Are there any body heard or practised the protein assay I've mentioned in the subject line? It is a product of genotech company. Help or suggestion from all of you will be highly appreciated. San Hein Mail Box 1018 Asian Institute of Technology P.O.Box 4 Khlong Luang Pathumthani 12120 Thailand Phone: 662 524 7061 From owner-proteins@net.bio.net Sat Jan 16 22:00:00 1999 Path: biosci!news.stanford.edu!logbridge.uoregon.edu!uunet!in5.uu.net!news.inter.net.il!not-for-mail From: "Barak Akabayov" Newsgroups: bionet.molbio.proteins Subject: ThermodynamicsBiologyRequest Date: Sun, 17 Jan 1999 15:10:21 +0200 Organization: Internet Gold, ISRAEL Lines: 7 Message-ID: <77snf5$ikr$1@news2.inter.net.il> NNTP-Posting-Host: r-h-185-23.access.net.il X-Trace: news2.inter.net.il 916578597 19099 192.117.185.23 (17 Jan 1999 13:09:57 GMT) X-Complaints-To: abuse@inter.net.il NNTP-Posting-Date: 17 Jan 1999 13:09:57 GMT X-Newsreader: Microsoft Outlook Express 4.72.3110.37 X-Mimeole: Produced By Microsoft MimeOLE V4.72.3110.37 Hi, I need central concepts in Biological Application of Thermodynamics. Thank you. Ak_barak@internet-zahav.net From owner-proteins@net.bio.net Sun Jan 17 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!newsfeed.cwix.com!158.43.192.17!rill.news.pipex.net!pipex!server1.netnews.ja.net!server2.netnews.ja.net!news.reading.ac.uk!suma3!ssr97lrc From: "l.r.cooper" Newsgroups: bionet.molbio.proteins Subject: modified amino acids Date: Mon, 18 Jan 1999 21:09:27 +0000 Organization: University of Reading Lines: 21 Message-ID: NNTP-Posting-Host: suma3.reading.ac.uk Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII dear all, i'm looking for a database, though even a table would suffice, of modified amino acids. any suggestions? will summarise. Ad Astra Per Aspera ======================================================= \Mr. Lee R. Cooper Bsc(Hons) MSc, Parallel, Emergent\\\\ \\& Distributed Architechture Laboratory, Dept. of\\\\\\\ \\\Computer Science, University of Reading, Whiteknights\\ \\\\PoBox 225, Reading, Berkshire. RG6 6AY (UK)\\\\\\\\\\\\ \\\\\Tel +44 (0)118 987 5123 x7645 (0915-1700)Fax\\\\\\\\\\\ \\\\\\Tel +44 (0)118 931 8188 x7817 (1900-2300)0118 975 1994\ \\\\\\\http://www.pedal.cs.rdg.ac.uk/~ssr97lrc/ \\\\\\\\\\\\\ =============================================================== From owner-proteins@net.bio.net Sun Jan 17 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!nntp.abs.net!newshub2.home.com!newshub1.home.com!news.home.com!news.rdc1.bc.wave.home.com.POSTED!not-for-mail From: "Achim Recktenwald" Newsgroups: bionet.molbio.proteins References: Subject: Re: modified amino acids Lines: 33 X-Newsreader: Microsoft Outlook Express 4.72.3155.0 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3155.0 Message-ID: Date: Tue, 19 Jan 1999 05:35:33 GMT NNTP-Posting-Host: 24.64.220.105 X-Complaints-To: abuse@home.net X-Trace: news.rdc1.bc.wave.home.com 916724133 24.64.220.105 (Mon, 18 Jan 1999 21:35:33 PDT) NNTP-Posting-Date: Mon, 18 Jan 1999 21:35:33 PDT Organization: @Home Network Canada CRC - the same company publishing the Handbook of Physics and Chemistry - has a book called Handbook of Biochemistry, or something like this. It contains a list of all known naturally occurring derivatives of amino acids, somewhere around 300. Achim l.r.cooper wrote in message ... > >dear all, > >i'm looking for a database, though even a table would suffice, of modified >amino acids. any suggestions? > >will summarise. > > >Ad Astra Per Aspera > >======================================================= >\Mr. Lee R. Cooper Bsc(Hons) MSc, Parallel, Emergent\\\\ >\\& Distributed Architechture Laboratory, Dept. of\\\\\\\ >\\\Computer Science, University of Reading, Whiteknights\\ >\\\\PoBox 225, Reading, Berkshire. RG6 6AY (UK)\\\\\\\\\\\\ >\\\\\Tel +44 (0)118 987 5123 x7645 (0915-1700)Fax\\\\\\\\\\\ >\\\\\\Tel +44 (0)118 931 8188 x7817 (1900-2300)0118 975 1994\ >\\\\\\\http://www.pedal.cs.rdg.ac.uk/~ssr97lrc/ \\\\\\\\\\\\\ >=============================================================== > From owner-proteins@net.bio.net Sun Jan 17 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!isdnet!news-ge.switch.ch!news.rediris.es!news.ehu.es!lcmx83.lc.ehu.es!user From: oibcader@lg.ehu.es (Roberto Calcedo) Newsgroups: bionet.molbio.proteins Subject: A problem with SDS Date: Mon, 18 Jan 1999 15:34:17 +0100 Organization: UPV Lines: 8 Message-ID: NNTP-Posting-Host: lcmx83.lc.ehu.es X-Trace: lgdx06.lg.ehu.es 916669807 3669 158.227.7.107 (18 Jan 1999 14:30:07 GMT) X-Complaints-To: usenet@news.ehu.es NNTP-Posting-Date: 18 Jan 1999 14:30:07 GMT Dear all: I have a protein sample that has SDS bound to it. Anybody know how could I take away the SDS from protein sample? Thanks From owner-proteins@net.bio.net Sun Jan 17 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!newsfeed1.swip.net!swipnet!news-peer-europe.sprintlink.net!news.sprintlink.net!Sprint!newsfeed.ecrc.net!nntp.news.xara.net!xara.net!server6.netnews.ja.net!newsfeed.ed.ac.uk!Aberdeen!cpa034 From: cpa034@sysa.abdn.ac.uk (a.vijayan) Newsgroups: bionet.molbio.proteins Subject: Retinol methods Date: 18 Jan 1999 13:13:15 GMT Organization: University of Aberdeen, Scotland Lines: 11 Message-ID: <77vc1b$l78$1@info.abdn.ac.uk> NNTP-Posting-Host: sysa.abdn.ac.uk X-Newsreader: TIN [version 1.2 PL2] Hi, I am looking for methods on retinol. I am trying to use fluorescence spectrometer to study it's exitation/emition pattern. Since retinol is a light sensitve and easily oxidisable molecule I have to take special precautions. I bought retinol from sigma which is in solid form. I know that I can disolve it in organic solvants like ether or ethanol. However I would like to know if any one has any particular experience in handling this material under special conditions. I shall be grateful to any one who could pass me some information about the handling of this chemical. Vijayan From owner-proteins@net.bio.net Sun Jan 17 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!news-peer1.sprintlink.net!news.sprintlink.net!cs.utexas.edu!geraldo.cc.utexas.edu!not-for-mail From: "esb" Newsgroups: bionet.molbio.proteins Subject: pro sequence affect translation? Date: Mon, 18 Jan 1999 16:49:24 -0600 Organization: The University of Texas at Austin, Austin, Texas Lines: 13 Message-ID: <780do1$r7a$1@geraldo.cc.utexas.edu> NNTP-Posting-Host: esb206-pmac8.esb.utexas.edu X-Newsreader: Microsoft Outlook Express 4.72.3110.5 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3110.3 entire Bovine Pancreatic Trypsin Inhibitor cDNA contains pre sequence (for secretion) pro sequence (these two are all processed out in mature protein) and mature sequence. i tried to express mature BPTI and pro+mature BPTI in e.coli periplasm, and found out that pro+mature BPTI express much more poorly then mature BPTI only. i guess the pro sequence has something to do with it. but i could not see any sign of degradation by western. so my question is: anyone remember any reference about pro sequence affecting protein expression? thanks in advance. From owner-proteins@net.bio.net Mon Jan 18 22:00:00 1999 Path: biosci!NBRF.GEORGETOWN.EDU!GARAVELLI From: GARAVELLI@NBRF.GEORGETOWN.EDU Newsgroups: bionet.molbio.proteins Subject: Re: modified amino acids Date: 19 Jan 1999 14:30:37 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 24 Sender: daemon@net.bio.net Distribution: world Message-ID: <01J6QD95050I9N455T@NBRF.Georgetown.Edu> NNTP-Posting-Host: net.bio.net On Mon, 18 Jan 1999, l.r.cooper asked: i'm looking for a database, though even a table would suffice, of modified amino acids. any suggestions? The RESID Database of amino acid residues annotated as features in the Protein Sequence Database has been distributed with the PIR-International Protein Sequences Databases on CD-ROM for several years. In September 1998, thanks to the support of the National Science Foundation, the RESID Database was made available on the Web through http://www-nbrf.georgetown.edu/pirwww/search/textresid.html with a description available at http://www-nbrf.georgetown.edu/pirwww/dbinfo/resid.html The current release 16.00 has 253 entries. The database includes naturally occurring post-translation modifications, along with the 22 different encoded residues, that have been identified in sequences. A number of other residues found in the old CRC list had been detected in hydolysates but have never been identified in sequences. ------------------------------------------------------------------------ Dr. John S. Garavelli Associate Director Protein Information Resource National Biomedical Research Foundation Washington, DC 20007 GARAVELLI@NBRF.GEORGETOWN.EDU From owner-proteins@net.bio.net Mon Jan 18 22:00:00 1999 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.gtei.net!news.maxwell.syr.edu!uio.no!romeo.dax.net!newsfeed1.uni2.dk!news.net.uni-c.dk!not-for-mail From: "Søren W. Rasmussen" Newsgroups: bionet.molbio.proteins Subject: Dnatools, revision 252 Date: Tue, 19 Jan 1999 15:45:54 +0100 Organization: UNI-C Lines: 19 Message-ID: <36A49AA2.F9828BC7@crc.dk> NNTP-Posting-Host: 130.226.182.97 Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Trace: news.net.uni-c.dk 916757645 19998 (None) 130.226.182.97 X-Complaints-To: usenet@news.net.uni-c.dk X-Mailer: Mozilla 4.03 [en] (Win95; I) Several functions have been updated/improved including the merge function and the SAGE extraction functions. Visit the DNATools homepage at: http://www.crc.dk/phys/A01B04_dnatools.htm Regards Søren -- Dr. scient. Søren W. Rasmussen Carlsberg Laboratory, Department of Physiology 10 Gl. Carlsbergvej, DK-2500, Copenhagen, Denmark Phone 45 3327 5230 / 45 3616 2259, Fax 45 3327 4766 E-mail swr@crc.dk, Homepage http://www.crc.dk/phys/ From owner-proteins@net.bio.net Mon Jan 18 22:00:00 1999 Newsgroups: bionet.molbio.proteins Path: biosci!news.stanford.edu!logbridge.uoregon.edu!news-peer.gip.net!news-lond.gip.net!news.gsl.net!gip.net!baron.netcom.net.uk!netcom.net.uk!server3.netnews.ja.net!ucl.ac.uk!bcc.ac.uk!chen From: chen@bsm.bioc.ucl.ac.uk (Chen Ho An) Subject: Re: pro sequence affect translation? Message-ID: <1999Jan19.203623.102933@ucl.ac.uk> Date: Tue, 19 Jan 1999 20:36:23 GMT References: <780do1$r7a$1@geraldo.cc.utexas.edu> Organization: University College London X-Newsreader: TIN [version 1.2 PL2] Lines: 26 esb (microb@esb.utexas.edu) wrote: : entire Bovine Pancreatic Trypsin Inhibitor cDNA contains pre sequence (for : secretion) pro sequence (these two are all processed out in mature protein) : and mature sequence. : i tried to express mature BPTI and pro+mature BPTI in e.coli periplasm, and : found out that pro+mature BPTI express much more poorly then mature BPTI : only. i guess the pro sequence has something to do with it. but i could not : see any sign of degradation by western. : so my question is: anyone remember any reference about pro sequence : affecting protein expression? : thanks in advance. Most likely to codon usage problem. Large number of rare codons especially if it appear in cluster, as is likely with your pro sequence, will tends to decrease yield. Associate with this is the idea of 'hungry codon', i.e. when large number of the same codon are present, even if it is not very rare, can lead to low expression level. I have some reference about this somewhere (though not necessary about proline), e-mail me and I'll see if I can dig them up. -- From owner-proteins@net.bio.net Mon Jan 18 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!cyclone.news.idirect.com!island.idirect.com!fu-berlin.de!cpeter.dialup.fu-berlin.DE!not-for-mail From: deleteme_cpeter@zedat.fu-berlin.de (Christoph Peter) Newsgroups: bionet.molbio.proteins Subject: No protease activity in yeast extract!? Date: Mon, 18 Jan 1999 18:53:57 GMT Organization: Freie Universitaet Berlin Lines: 24 Message-ID: <36a38126.167576@news.fu-berlin.de> Reply-To: deleteme_cpeter@zedat.fu-berlin.de NNTP-Posting-Host: cpeter.dialup.fu-berlin.de (130.133.221.185) Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Access: 16 17 18 X-Trace: fu-berlin.de 916780592 29569 (none) 130.133.221.185 X-Newsreader: Forte Free Agent 1.11/16.235 Hi all! I am trying to determine protease activity in biological samples. We have established an assay using Azocasein and the Protease-Kit from Pierce. Using Trypsin and Papain as standards we get nice calibration-curves with these assays. When we try a freshly made yeast-extract as a supposedly positive sample (glass beads, no additives to buffer (PO4, pH7.0)) we do not find the least bit of protease activity. Although this would be good news to everybody purifying yeast proteins, I suppose something is really going wrong with the assays! Who had similar experience or is using either assay successfully? Thanks in advance...................................Christoph (PhD-Student, Biotechnology) -- Christoph Peter PhD Student (Biotechnology) cpeter at zedat dot fu-berlin dot de >Alles wird gut!<(Beate) From owner-proteins@net.bio.net Mon Jan 18 22:00:00 1999 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.gtei.net!firehose.mindspring.com!not-for-mail From: MatthewP@microbio.uab.edu (Matthew Parker) Newsgroups: bionet.molbio.proteins Subject: Anamalous pKa values for asp and glu residues? Date: Wed, 20 Jan 1999 02:33:47 GMT Organization: MindSpring Enterprises Lines: 5 Message-ID: <36a5401f.2499636@news.mindspring.com> Reply-To: MatthewP@microbio.uab.edu NNTP-Posting-Host: d1.c0.04.cf X-Server-Date: 20 Jan 1999 02:29:30 GMT X-Newsreader: Forte Free Agent 1.1/32.230 Hi! Can anyone point me to any examples of proteins which have acidic residues with unusually high pKa values (for instance, residues which are buried in enzyme active sites)? Thanks! From owner-proteins@net.bio.net Tue Jan 19 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!nntp.flash.net!hub1.ispnews.com!news12.ispnews.com.POSTED!not-for-mail Message-ID: <36A63C23.1543FB46@asrr.arsusda.gov> From: Randall Cameron X-Mailer: Mozilla 4.05 [en] (Win95; U) MIME-Version: 1.0 Newsgroups: bionet.molbio.proteins Subject: activity stain for B-Glucosidase Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit Lines: 3 NNTP-Posting-Host: 208.153.131.85 X-Trace: news12.ispnews.com 916864515 208.153.131.85 (Wed, 20 Jan 1999 15:35:15 EDT) NNTP-Posting-Date: Wed, 20 Jan 1999 15:35:15 EDT Organization: ISPNews http://ispnews.com Date: Wed, 20 Jan 1999 15:27:15 -0500 Would anyone reccomend an activity staining method for B-glucosidase after native PAGE. Thanks. From owner-proteins@net.bio.net Tue Jan 19 22:00:00 1999 Path: biosci!news.stanford.edu!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: A problem with SDS Date: Wed, 20 Jan 1999 09:19:42 -0500 Organization: University Of Pittsburgh Lines: 18 Message-ID: References: NNTP-Posting-Host: pelli.pathology.pitt.edu X-Newsreader: MT-NewsWatcher 2.4.4 In article , oibcader@lg.ehu.es (Roberto Calcedo) wrote: > Dear all: > > I have a protein sample that has SDS bound to it. > > Anybody know how could I take away the SDS from protein sample? Dialysis would be a good first step, but the removal of all the SDS is difficult. Peter -- " Don't you eat that yellow snow Watch out where the huskies go" FZ From owner-proteins@net.bio.net Tue Jan 19 22:00:00 1999 Path: biosci!news.stanford.edu!news.ems.psu.edu!news.cis.ohio-state.edu!newsfeed.berkeley.edu!cyclone.bc.net!rover.ucs.ualberta.ca!news.ucalgary.ca!news From: Neal Melvin Newsgroups: bionet.molbio.proteins Subject: Prestained MW standards for SDS PAGE Date: Wed, 20 Jan 1999 15:06:59 -0700 Organization: The University of Calgary Lines: 9 Message-ID: <785k2f$2f66@ds2.acs.ucalgary.ca> NNTP-Posting-Host: @hmr176.meds.ucalgary.ca Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.06 [en] (Win98; I) I'm using pre-stained, broad range MW standards (188-7kd) from Bio-Rad. The four heaviest markers show up as a nice, tight band in the gel, but the lower four are very broad, with poor resolution... is there any way to fix this?? From owner-proteins@net.bio.net Tue Jan 19 22:00:00 1999 Path: biosci!NMSU.EDU!hroychow From: hroychow@NMSU.EDU ("Hiranya S. Roychowdhury") Newsgroups: bionet.molbio.proteins Subject: Re: A problem with SDS Date: 20 Jan 1999 08:39:00 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 35 Sender: daemon@net.bio.net Distribution: world Message-ID: <199901201632.JAA28288@nestor.NMSU.Edu> NNTP-Posting-Host: net.bio.net At 09:19 AM 1/20/99 -0500, Peter wrote: >In article , >oibcader@lg.ehu.es (Roberto Calcedo) wrote: > >> Dear all: >> >> I have a protein sample that has SDS bound to it. >> >> Anybody know how could I take away the SDS from protein sample? > >Dialysis would be a good first step, but the removal of all the SDS is >difficult. > >Peter > >-- >" Don't you eat that yellow snow > Watch out where the huskies go" > FZ > > Indeed, once bound, it is difficult to remove SDS from protein by simple dialysis. However, dialyzing against 1% Triton X-100 will improve the situation. Another option is to electroelute the protein in 0.5% triton-containing buffer. Dr. Hiranya Sankar Roychowdhury GENE LAB/ EPPWS New Mexico State University Las Cruces, NM 88003 Ph. (505) 646-5785 hroychow@nmsu.edu From owner-proteins@net.bio.net Tue Jan 19 22:00:00 1999 Path: biosci!agate!dog.ee.lbl.gov!newsfeed.berkeley.edu!nntp.abs.net!newshub2.home.com!newshub1.home.com!news.home.com!news.rdc1.bc.wave.home.com.POSTED!not-for-mail From: "Achim Recktenwald" Newsgroups: bionet.molbio.proteins References: <199901201632.JAA28288@nestor.NMSU.Edu> Subject: Re: A problem with SDS Lines: 46 X-Newsreader: Microsoft Outlook Express 4.72.3155.0 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3155.0 Message-ID: Date: Wed, 20 Jan 1999 19:19:57 GMT NNTP-Posting-Host: 24.64.220.105 X-Complaints-To: abuse@home.net X-Trace: news.rdc1.bc.wave.home.com 916859997 24.64.220.105 (Wed, 20 Jan 1999 11:19:57 PDT) NNTP-Posting-Date: Wed, 20 Jan 1999 11:19:57 PDT Organization: @Home Network Canada You could also try an acetone-precipitation at -20°C. Your protein should stay healthy, while the SDS dissolves in the acetone. Achim "Hiranya S. Roychowdhury" wrote in message <199901201632.JAA28288@nestor.NMSU.Edu>... >At 09:19 AM 1/20/99 -0500, Peter wrote: >>In article , >>oibcader@lg.ehu.es (Roberto Calcedo) wrote: >> >>> Dear all: >>> >>> I have a protein sample that has SDS bound to it. >>> >>> Anybody know how could I take away the SDS from protein sample? >> >>Dialysis would be a good first step, but the removal of all the SDS is >>difficult. >> >>Peter >> >>-- >>" Don't you eat that yellow snow >> Watch out where the huskies go" >> FZ >> >> > >Indeed, once bound, it is difficult to remove SDS from protein by simple >dialysis. However, dialyzing against 1% Triton X-100 will improve the >situation. Another option is to electroelute the protein in 0.5% >triton-containing buffer. > > >Dr. Hiranya Sankar Roychowdhury >GENE LAB/ EPPWS >New Mexico State University >Las Cruces, NM 88003 >Ph. (505) 646-5785 >hroychow@nmsu.edu > From owner-proteins@net.bio.net Tue Jan 19 22:00:00 1999 Path: biosci!AOL.COM!DOCTORHIM From: DOCTORHIM@AOL.COM Newsgroups: bionet.molbio.proteins Subject: Re: activity stain for B-Glucosidase Date: 20 Jan 1999 13:14:11 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 2 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Use methylumbellyferyl-beta-glucopyranoside from Sigma. Your protein band , if active, will fluoresce when viewed on a UV transilluminator. From owner-proteins@net.bio.net Tue Jan 19 22:00:00 1999 Path: biosci!MIS.FINCHCMS.EDU!muellerd From: muellerd@MIS.FINCHCMS.EDU (David Mueller) Newsgroups: bionet.molbio.proteins Subject: Re: Anamalous pKa values for asp and glu residues? Date: 20 Jan 1999 05:52:24 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 31 Sender: daemon@net.bio.net Distribution: world Message-ID: <199901201339.HAA12278@mis.finchcms.edu> NNTP-Posting-Host: net.bio.net Tozawa, K., Ohbuchi, H., Yagi, H., Amano, T., Matsui, T., Yoshida, M., and Akutsu, H. Unusual pKa of the carboxylate at the putative catalytic position of thermophilic F1-ATPase beta subunit determined by 13C-NMR. FEBS Lett. 397:122-126, 1996. At 02:33 AM 1/20/99 GMT, you wrote: > Hi! Can anyone point me to any examples of proteins which have >acidic residues with unusually high pKa values (for instance, residues >which are buried in enzyme active sites)? > > Thanks! > > <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<< >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> --------------------------------------------------------------------------- David Mueller Department of Biochemistry and Molecular Biology The Chicago Medical School 3333 Greenbay Rd. North Chicago, IL 60064 Tel: 847-578-8606 FAX: 847-578-3240 From owner-proteins@net.bio.net Wed Jan 20 22:00:00 1999 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!news.gtei.net!newsfeed.berkeley.edu!news-peer1.sprintlink.net!-program!news.maxwell.syr.edu!nntp.news.xara.net!xara.net!server6.netnews.ja.net!dundee.ac.uk!luke From: luke@surgery1.medschool.dundee.ac.uk (Luke Newman) Newsgroups: bionet.molbio.proteins Subject: Gelatin zymography: help! Date: 21 Jan 1999 17:49:51 GMT Organization: The University, Dundee, DD1 4HN, Scotland, UK. Lines: 30 Message-ID: <787pbv$aap$1@dux.dundee.ac.uk> NNTP-Posting-Host: surgery1.medschool.dundee.ac.uk X-Newsreader: TIN [version 1.2 PL2] Hello all, I'm trying to detect gelatinase expression in mammalian cells by zymography, without any success so far. The set-up: 1) HT-1080 fibrosarcoma cells. Serum-free conditioned medium collected for 24 h, mixed with SDS gel loading buffer (no reducing agent nor heating). 2) 10% acrylamide gels containing 1 mg/ml gelatin (Sigma, type B from bovine skin). 3) Standard electrophoresis conditions, gel washed extensively in 2.5% Triton X-100 and then incubated in a neutral buffer containing some NaCl, calcium ions and Brij overnight at 37 degC. 4) Standard Coomassie R staining and destaining. I get no bands at all, even though this cell line is often used as a positive control and my method is essentially standard. I've tried loading the maximum amount of conditioned medium that I can get in a well and also various sources of Triton X-100. Could the type and/or source of the gelatin be causing my complete lack of success ? Does the panel have any other tips that might help ? Many thanks in advance Luke Newman e.l.newman@dundee.ac.uk From owner-proteins@net.bio.net Wed Jan 20 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!newsfeed.wirehub.nl!news.belnet.be!news.umh.ac.be!news@umhsp01.umh.ac.be From: wattiez ruddy Newsgroups: bionet.molbio.proteins Subject: intestinal trefoil factor antibodies Date: Thu, 21 Jan 1999 10:31:15 +0100 Organization: Université de Mons-Hainaut Lines: 8 Message-ID: <36A6F3E2.8EE48C7B@umh.ac.be> NNTP-Posting-Host: koala.umh.ac.be 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 X-Priority: 1 (Highest) Hi I'am looking for human intestinal trefoil factor antibodies for my research. Thank you in advance. You can also Email me : ruddy.wattiez@umh.ac.be From owner-proteins@net.bio.net Wed Jan 20 22:00:00 1999 Path: biosci!agate!newsfeed.berkeley.edu!sunqbc.risq.qc.ca!newsflash.concordia.ca!pitt.edu!not-for-mail From: pxpst2@unixs.cis.pitt.edu (Peter) Newsgroups: bionet.molbio.proteins Subject: Re: Prestained MW standards for SDS PAGE Date: Thu, 21 Jan 1999 09:24:03 -0500 Organization: University Of Pittsburgh Lines: 22 Message-ID: References: <785k2f$2f66@ds2.acs.ucalgary.ca> NNTP-Posting-Host: pelli.pathology.pitt.edu X-Newsreader: MT-NewsWatcher 2.4.4 In article <785k2f$2f66@ds2.acs.ucalgary.ca>, Neal Melvin wrote: > I'm using pre-stained, broad range MW standards (188-7kd) from Bio-Rad. > The four > heaviest markers show up as a nice, tight band in the gel, but the lower > > four are very broad, with poor resolution... is there any way to fix > this?? Increase the percentage acrylamide (%T). If you are running standard Laemili gels of 10 % or less then you should expect your lower molecular weight stds to run in broadbands. If you are interested in having all the bands be sharp, you have several options. 1) Run Tris Tricine gels 2) Run gradient gels. Hope this helps Peter -- " Don't you eat that yellow snow Watch out where the huskies go" FZ From owner-proteins@net.bio.net Wed Jan 20 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!newsfeed1.swip.net!swipnet!rill.news.pipex.net!pipex!nntp.news.xara.net!xara.net!server5.netnews.ja.net!daresbury!not-for-mail From: "Dr. Cyril V. Privezentzev" Newsgroups: bionet.molbio.proteins Subject: Compression Date: 21 Jan 1999 19:14:30 -0000 Organization: Daresbury Laboratory, Warrington, U.K. Lines: 17 Message-ID: <787uam$hae$1@mserv2.dl.ac.uk> NNTP-Posting-Host: mserv2.dl.ac.uk Original-To: methods@dl.ac.uk, proteins@dl.ac.uk content-length: 405 Return-Path: Date-Warning: Date header was inserted by igr.fr X-Priority: 1 (Highest) X-Sender: cprivenz@igr.fr (Unverified) Dear Netters: Who knows how to avoid vertical compression in SDS-PAAG gels? Any help and/or comments would be highly appreciated. Sincerely, Dr. Cyril V. Privezentzev Group "Reparation des Lesions Radio- et Chimioinduites CNRS UMR 1772 Institut Gustave-Roussy Pavillon de Recherche II 39 rue Camille Desmoulins 94805 Villejuif Cedex FRANCE Tel. +33 (0)1 42 11 54 04 Fax +33 (0)1 42 11 52 76 From owner-proteins@net.bio.net Wed Jan 20 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!sunqbc.risq.qc.ca!news.uow.edu.au!news.usyd.edu.au!sunb.ocs.mq.edu.au!keli From: phains@NOSPAMproteome.org.au (Peter Hains) Newsgroups: bionet.molbio.proteins Subject: Re: A problem with SDS Date: Fri, 22 Jan 1999 04:33:19 GMT Organization: APAF Lines: 22 Distribution: world Message-ID: <788rht$m9q$2@sunb.ocs.mq.edu.au> References: <199901201632.JAA28288@nestor.NMSU.Edu> NNTP-Posting-Host: apaf29.proteome.org.au X-Newsreader: News Xpress 2.01 Time for my two bobs worth, I recommend using a mixed bed resin such as BioRads AG11A8. I have successfully used this to remove SDA from my proteins. All you need is a simple column and your protein comes out healthy. Peter. I'm afraid I don't have a clever saying to put here. Remove NOSPAM to e-mail me. Peter Hains Ph. +61 2 9850 6216 Australian Proteome Analysis Facility Fax. +61 2 9850 6200 Level 4 Building F7B Macquarie University, Sydney 2109 From owner-proteins@net.bio.net Wed Jan 20 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!news-peer1.sprintlink.net!-program!news-in-east1.sprintlink.net!news.sprintlink.net!news2.smatnet.net!news.sprintsvc.net!news.smatnet.net!uunet!in3.uu.net!news-nb.rutgers.edu!niflheim.rutgers.edu!not-for-mail From: meton@rci.rutgers.edu (Daniel Gonzalez) Newsgroups: bionet.molbio.proteins Subject: Second GFP Symposium call for registration Date: 21 Jan 1999 22:44:19 -0500 Organization: Rutgers University Lines: 16 Message-ID: <788s6j$jpe$1@niflheim.rutgers.edu> NNTP-Posting-Host: niflheim.rutgers.edu Summary: Register for GFP2 Keywords: GFP Green Fluorescent Protein X-Newsreader: NN version 6.5.1 (NOV) GFP2 Call For Registration The second international Symposium on GFP will be held May 22-27 in San Diego California at the Town and Country Resort Hotel (800-772-8527). Over 80 speakers from academia and industry have been confirmed to speak at the meeting. If you wish to be a speaker and are not on the list contact me as soon as posssible as I will be organising the speakers for the meeting. My e-mail is meton@rci.rutgers.edu. Topics ranging from applications of GFP in drug discovery and protein and organellar trafficking to gene quantitation and gene therapy in addition to its use as a biosensor as well as many other applications will be discussed. A large segment of the academic and industrial research community developing GFP applications will be represented at the meeting. Already tremendous excitement for the meeting has forced me to set up an interactive javascript registration page linked off of the main GFP symposium page, http://www.rci.rutgers.edu/~meton/GFP2.html. I have tested this page with the latest version of Netscape on MacIntosh and Silicon Graphics computers and seem to have no problems, but you can fax us the page to the number indicated on the page if you have any problems. For those of you who missed the first meeting, Universal Imaging Corporation have placed a summary of GFP1 created by Audrey Ichida on their website at http://www.image1.com/products/metagfp/gfpmtgsummary2.html, this is an informal summary but appears to be quite accurate, please visit it when you get a chance. The GFP revolution continues. Daniel Gonzalez From owner-proteins@net.bio.net Wed Jan 20 22:00:00 1999 Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!news.gtei.net!newsfeed.wli.net!howland.erols.net!newsfeed.cwix.com!204.238.120.130!news-feeds.jump.net!nntp2.dejanews.com!nnrp1.dejanews.com!not-for-mail From: stourville@ibcusa.com Newsgroups: bionet.molbio.proteins Subject: IBCUSA 2nd Annual Expression Technologies Conference Date: Thu, 21 Jan 1999 17:33:08 GMT Organization: Deja News - The Leader in Internet Discussion Lines: 19 Message-ID: <787ock$sf8$1@nnrp1.dejanews.com> NNTP-Posting-Host: 204.249.180.2 X-Article-Creation-Date: Thu Jan 21 17:33:08 1999 GMT X-Http-User-Agent: Mozilla/4.05 [en] (Win95; U) X-Http-Proxy: 1.0 x6.dejanews.com:80 (Squid/1.1.22) for client 204.249.180.2 *** Call For Papers/Abstracts *** IBCUSA's 2nd Annual International Conference on Expression Technologies in Protein Pharmaceuticals, Vaccine Production and Drug Discovery August 5-6, 1999 * Venue: TBD ^^^^ NEW RESEARCH(6-8months), DEVELOPMENTS OR CASE STUDIES WELCOME !!!! ^^^^ Exciting developments concerning the large-scale production of recombinant proteins in hterologous hosts have driven many of the most significant biotechnological advances of recent years. Fundamental research from biophysics to cell biology, drug discovery systems, protein pharmaceuticals, vaccine production and most recently functional genomics, are some of the diverse fields underpinned by such techniques. There now exists a bewildering diversity of expression systems to choose from - some tried and tested; and others relatively new, but with great promise. This dynamically forward-thinking conference takes the dominant systems in the field -bacterial, yeast and insect - and deconstructs the mystery surrounding them. -----------== Posted via Deja News, The Discussion Network ==---------- http://www.dejanews.com/ Search, Read, Discuss, or Start Your Own From owner-proteins@net.bio.net Thu Jan 21 22:00:00 1999 Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!nntp.abs.net!newshub2.home.com!news.home.com!news.rdc1.bc.wave.home.com.POSTED!not-for-mail From: "Achim Recktenwald" Newsgroups: bionet.molbio.proteins References: <199901201632.JAA28288@nestor.NMSU.Edu> <788rht$m9q$2@sunb.ocs.mq.edu.au> Subject: Re: A problem with SDS Lines: 32 X-Newsreader: Microsoft Outlook Express 4.72.3155.0 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.3155.0 Message-ID: <11cq2.53$vg3.1467@news.rdc1.bc.wave.home.com> Date: Sat, 23 Jan 1999 04:14:53 GMT NNTP-Posting-Host: 24.64.220.105 X-Complaints-To: abuse@home.net X-Trace: news.rdc1.bc.wave.home.com 917064893 24.64.220.105 (Fri, 22 Jan 1999 20:14:53 PDT) NNTP-Posting-Date: Fri, 22 Jan 1999 20:14:53 PDT Organization: @Home Network Canada I am surprised you removed ALL SDS. I have tried ion-exchange chromatography in the past and it did not work. Your protein, is it an enzyme? If not, how do you now it's healthy? Achim Peter Hains wrote in message <788rht$m9q$2@sunb.ocs.mq.edu.au>... >Time for my two bobs worth, > >I recommend using a mixed bed resin such as BioRads AG11A8. I have >successfully used this to remove SDA from my proteins. All you need is a >simp