From owner-proteins@net.bio.net Mon Feb 01 22:00:00 1999
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From: klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin)
Newsgroups: bionet.molbio.proteins
Subject: Re: Insolubility of double-tagged protein
Date: Tue, 02 Feb 1999 00:17:31 GMT
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In article <794kop$fjb$1@xinwen.daimi.au.dk>, "Ditlev Brodersen" <ding@imsb.au.dk> wrote:
:Hi,
:
:  we are having some trouble with a doubly-tagged (His in the N-terminus,
:GST in the C-terminus) expressed in E. coli 834(DE3) pLysS which seems to
:have very low solubility in our lysis buffer.
:
:  I know this is a standard question, but what are the best things I can do
:in order to optimise the amount of soluble protein?
:

All the standard answers found in bionet archives and through dejanews 
apply. Unfortunately, proteins are tricky and it's next to impossible
to tell which one is going to work best for you. Medium, temperature, 
strain, construct, tag, IPTG concentration, etc.

        - Dima

From owner-proteins@net.bio.net Mon Feb 01 22:00:00 1999
Path: biosci!DSVCAD.CEA.fr!JOET
From: JOET@DSVCAD.CEA.fr (JOET Thierry 165109)
Newsgroups: bionet.molbio.proteins
Subject: Two-hybrid system and respiratory complex I
Date: 2 Feb 1999 05:31:53 -0800
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Hello,
I know that it's possible to study interactions between subunits of
polymerase II by the two-hybrid system. In the same way,  i'm interested in
the structure of  the respiratory complex I.
Has anyone used the two-hybrid system to detect an interaction between
subunits of complex I ? references ? remarks ? any problem encountered ?
Thanks
Thierry JOET
Laboratory of Ecophysiology and Photosynthesis
DEVM/CEA Cadarache
13108 St Paul lez Durance (FRANCE)
phone: (33)-4-42-25-36-72
fax: (33)-4-42-25-62-65
e-mail: joet@dsvcad.cea.fr

From owner-proteins@net.bio.net Mon Feb 01 22:00:00 1999
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From: pjf <zinc@zifi.sickkids.on.ca>
Newsgroups: bionet.molbio.proteins,bionet.molbio.methds-reagnts
Subject: searching for expression system for peptides
Date: 02 Feb 1999 21:28:35 -0500
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Summary: peptide, insoluble, CNBr, cyanogen
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Xref: biosci bionet.molbio.proteins:13912 bionet.molbio.methds-reagnts:73780

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hi,

i thought i read about a peptide expression system at some time that
had two nice characteristics: it allowed a simple CNBr cleavage of the
peptide away from the fusion protein (via a single Met residue in the
fusion linking the fusion with the peptide) and also caused expressed
protein to end up in the insoluble fraction to reduce exposure to
proteases. 

does this ring a bell with anyone? 

- -pjf  
- -- 
       "There is only one aim in life and that is to live it."
 Karl Shapiro,(1959) from an essay on Henry Miller's Tropic of Cancer
	      finger pfinerty@nyx10.nyx.net for PGP key
		 http://abragam.med.utoronto.ca/~zinc

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From owner-proteins@net.bio.net Tue Feb 02 22:00:00 1999
Message-ID: <36B839FF.3191@antibodyresource.com>
Date: Wed, 03 Feb 1999 05:59:06 -0600
From: The Antibody Resource Page <antibody@antibodyresource.com>
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Subject: Are you looking for an Antibody?  Visit the Antibody Resource Page!
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The Antibody Resource Page (http://www.antibodyresource.com/) now
maintains a list of custom antibody suppliers.  If you are interested in
the production of custom monoclonal or polyclonal antibodies see this
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Also, take a look our "How to Find an Antibody" section
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This section has links to online antibody search engines and reference
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Directory.

Don't forget to visit our other sections on educational resources and
stop by to see our antibody gallery.

ps.  The ARP was voted among the top 25 biotechnology webpages for 1997
by Genetic Engineering News!

From owner-proteins@net.bio.net Tue Feb 02 22:00:00 1999
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From: w.ingram@cmcb.REMOVE-THIS-BIT.uq.edu.au (Wendy Ingram)
Newsgroups: bionet.molbio.proteins
Subject: Alkaline phosphatase assay, p-nitrophenyl phosphate stock soln
Date: Thu, 04 Feb 1999 17:03:28 +1000
Organization: CMCB, University of Queensland
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I am about to do some alkaline phosphatase assays on harvested tissue
culture cells. I have a protocol which uses absorbance at 410nM to
quantitate activity when p-nitrophenyl phosphate is used as a
substrate...    What I would like to find out is how to make a stock
solution of p-nitrophenyl phosphate and how to store it? Do I need to
dissolve it in any special buffer to keep it stable? Storage at -20?? or
should I make it fresh each time from powder???

I¹m keen to hear from any alkaline phosphatase assay experts out there. It
would be great also to hear any opinions/protocols on monitoring AP
activity generally before I make up my mind exactly which technique I will
use.

Thanks in advance,

Wendy Ingram.

-- 
Wendy Ingram (PhD Student)
Centre for Molecular and Cellular Biology
University of Queensland
Brisbane, Australia

From owner-proteins@net.bio.net Tue Feb 02 22:00:00 1999
Newsgroups: bionet.molbio.proteins
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From: Kamala Tyagarajan <kamala@nospam.lynxgen.com>
Subject: HPLC system 
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We are looking into to buying  a Shimadzu HPLC system with a fluoresence
detector (the RF-10AXL spectrofluorometric detector)  for  our lab,
mainly for peptide and protein analysis.  We would appreciate any
comments whether positive/negative by people who have used/are familiar
with the system.


Thanks,

Kamala Tyagarajan
Lynx Therapeutics,
Hayward, CA 94545


From owner-proteins@net.bio.net Tue Feb 02 22:00:00 1999
Newsgroups: bionet.molbio.proteins
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From: chen@bsm.bioc.ucl.ac.uk (Chen Ho An)
Subject: Re: Insolubility of double-tagged protein
Message-ID: <1999Feb3.154904.35579@ucl.ac.uk>
Date: Wed, 3 Feb 1999 15:49:04 GMT
References: <794kop$fjb$1@xinwen.daimi.au.dk> <795g5g$n96$6@news.doit.wisc.edu>
Organization: University College London
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Dima Klenchin (klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu) wrote:
: In article <794kop$fjb$1@xinwen.daimi.au.dk>, "Ditlev Brodersen" <ding@imsb.au.dk> wrote:
: :Hi,
: :
: :  we are having some trouble with a doubly-tagged (His in the N-terminus,
: :GST in the C-terminus) expressed in E. coli 834(DE3) pLysS which seems to
: :have very low solubility in our lysis buffer.
: :
: :  I know this is a standard question, but what are the best things I can do
: :in order to optimise the amount of soluble protein?
: :

: All the standard answers found in bionet archives and through dejanews 
: apply. Unfortunately, proteins are tricky and it's next to impossible
: to tell which one is going to work best for you. Medium, temperature, 
: strain, construct, tag, IPTG concentration, etc.

I think the question is whether it is insoluble in the lysis buffer (as
stated) alone or in the cell itself.  If it the lysis buffer that the
problem, check the pI and change the pH of the buffer and or another
buffer altogether. Check that your protein is soluble by itself (without
tags) and work out a way of purifying it or try another tags.  If it's
insoluble in the cell (inclusion bodies?), it OK if you can refold it.
Otherwise go through each fermentation condition and see which one works
best.





From owner-proteins@net.bio.net Tue Feb 02 22:00:00 1999
Path: biosci!UNIVER.KHARKOV.UA!andrey.l.sukhodub
From: andrey.l.sukhodub@UNIVER.KHARKOV.UA ("Andrey Sukhodub")
Newsgroups: bionet.molbio.proteins
Subject: ABC and P-450
Date: 3 Feb 1999 06:14:48 -0800
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Can anybody help me with the understanding of ATP-binding cassette =
proteins (ABC)multidrug-resistant protein and cytochrome P-450 =
physiological interactions? Especially in mammals.
I'll highly appreciate any advice.
Regards
Andrey.L.Sukhodub@univer.kharkov.ua

------=_NextPart_000_002C_01BE4F8F.B9E283A0
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<HEAD>

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<BODY bgColor=3D#ffffff>
<DIV>
<DIV><FONT face=3D"Arial Cyr" size=3D2>Can anybody help me with the =
understanding of=20
ATP-binding cassette proteins (ABC)multidrug-resistant protein and =
cytochrome=20
P-450 physiological interactions? Especially in mammals.</FONT></DIV>
<DIV><FONT face=3D"Arial Cyr" size=3D2>I'll highly appreciate any=20
advice.</FONT></DIV>
<DIV><FONT face=3D"Arial Cyr" size=3D2>Regards</FONT></DIV>
<DIV><FONT color=3D#000000 face=3D"Arial Cyr" size=3D2><A=20
href=3D"mailto:Andrey.L.Sukhodub@univer.kharkov.ua">Andrey.L.Sukhodub@uni=
ver.kharkov.ua</A></FONT></DIV></DIV></BODY></HTML>

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From owner-proteins@net.bio.net Tue Feb 02 22:00:00 1999
Path: biosci!agate!newsfeed.berkeley.edu!news.maxwell.syr.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!Home
From: klenchin@REMOVE_TO_REPLY.facstaff.wisc.edu (Dima Klenchin)
Newsgroups: bionet.molbio.proteins
Subject: Re: Protein solublisation
Date: Tue, 02 Feb 1999 15:32:02 GMT
Organization: UW
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References: <78pcs0$pgs$1@pump1.york.ac.uk>
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In article <78pcs0$pgs$1@pump1.york.ac.uk>, "Peter Ashton" <pda2@york.ac.uk> wrote:
>Hello,
>
>I have a mixture of glycoproteins that I have ammonium sulphate precipitated
>from culture supernatants. I have dialysed the samples extensively against
>PBS to get rid of the ammonium sulphate, but I am having extreme problems
>getting them back into solution. I have tried all of the methods that I can
>find including:
>
>8M Urea (plus sonication)
>6M Guanidium-HCl (plus sonication)
>SDS-PAGE sample buffer (and boiling for 30 minutes)
>Glacial acetic acid
>
>In every case, the pellet sits there happily at the bottom of the tube and
>completely refuses to go back into solution.

No matter what you use to dissolve the tight pellet, you MUST aid the 
process by some mechanical action - pipetting, "douncing", grinding, etc.
Are you doing any of this? (The reason I am asking is because your
phrase "pellet sits there happily at the bottom of the tube" suggest you are
not; I have hard believing that 6M GuHCl or 2% hot SDS will not dissolve
the bulk of any proteinous mixture...).

        - Dima



From owner-proteins@net.bio.net Wed Feb 03 22:00:00 1999
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From: Steven Johnson <johnson@bioch.ox.ac.uk>
Newsgroups: bionet.molbio.proteins
Subject: Circular Dichroism
Date: Thu, 04 Feb 1999 10:33:47 +0000
Organization: Oxford University, England
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Hi,

I'd like to carry out CD on my protein in the presence and absence of
calcium, and am having problems finding suitable buffers (chloride ions
affect the spectra).  Has anyone tried CD in calcium? Any suggestions
would be welcome.

Thanks, Steve


From owner-proteins@net.bio.net Wed Feb 03 22:00:00 1999
Path: biosci!agate!newsfeed.berkeley.edu!sunqbc.risq.qc.ca!logbridge.uoregon.edu!news.ycc.yale.edu!not-for-mail
From: "R. Bryan Sutton" <sutton@laplace.csb.yale.edu>
Newsgroups: bionet.molbio.proteins
Subject: Re: Circular Dichroism
Date: Thu, 04 Feb 1999 11:46:54 -0500
Organization: Yale University
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I've done quite a bit of CD analysis of annexin IV in the presence and
absence of Ca++.  For that work, I've found that 1mM sodium cacodylate
buffer works quite well.  I was able to measure from 190A to 250A
without any serious background problem.  The only difficulty may be the
pH at which cacodylate buffers (~pH 5.0). This might cause a problem
with the ligation of Ca++ in some proteins.  It doesn't seem to affect
the annexins, however.  You might also try low concentrations of Tris
(10mM-ish).  I know of several labs who use this buffer for CD work
routinely.

-- 
R. Bryan Sutton
Department of Biophysics and Molecular Biology
Howard Hughes Medical Institute
Yale University
266 Whitney Ave. BASS 434A
New Haven, CT 06520

email: sutton@laplace.csb.yale.edu

From owner-proteins@net.bio.net Wed Feb 03 22:00:00 1999
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From: Koen Peeters <kopee@gengenp.rug.ac.be>
Newsgroups: bionet.molbio.proteins
Subject: Re: Alkaline phosphatase assay, p-nitrophenyl phosphate stock soln
Date: Thu, 04 Feb 1999 17:34:11 +0100
Organization: University of Ghent, Belgium
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Wendy Ingram wrote:
> 
> I am about to do some alkaline phosphatase assays on harvested tissue
> culture cells. I have a protocol which uses absorbance at 410nM to
> quantitate activity when p-nitrophenyl phosphate is used as a
> substrate

Ok, little detail, we use 405 nm, we measure 1 hour and 30 minutes, and
measure continuously
colour development and the reaction rate (~quantity protein) is
determined by
the EASY-KIN program.

What I would like to find out is how to make a stock
> solution of p-nitrophenyl phosphate and how to store it? 
Do I need to
> dissolve it in any special buffer to keep it stable? Storage at -20?? or
> should I make it fresh each time from powder???

We have the pNPP in tablets that need to be dissolved in
diethanolaminebuffer 
(at 37 degrees celcius)(5mg pNPP in 2,5 ml diethanolaminebuffer. This
solution needs 
to be prepared 5 minutes before the actual measurement, if u want to
incubate,
incubate the samples in the dark at room temperature.
> 
Good luck, 

Koen
==================================================================
Koen Peeters 
DEPARTMENT OF GENETICS                         Fax:32 (0)9 2645349
UNIVERSITY OF GENT, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium
Vlaams Instituut voor Biotechnologie                           VIB
mailto:kopee@gengenp.rug.ac.be    	   http://spider.rug.ac.be
==================================================================

From owner-proteins@net.bio.net Wed Feb 03 22:00:00 1999
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From: Lena Zaitseva <zaitseva@biochem.purdue.edu>
Newsgroups: bionet.molbio.proteins
Subject: Re: Protein solublisation
Date: Thu, 04 Feb 1999 09:02:22 -0500
Organization: Purdue University
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References: <78pcs0$pgs$1@pump1.york.ac.uk> <7975q6$nr4$1@news.doit.wisc.edu> <79c190$7kr$1@pump1.york.ac.uk>
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=A0

Peter Ashton wrote:

> I have tried both pippetting and sonicating the pellet, as well as heat=
ing
> and/or boiling the samples. Whatever I do, there is never any protein
> present in the supernatant afterwards
>
> Pete.

=A0=A0=A0=A0 In this case you probably have to avoid ammonium sulfate as =
precipitant.
High salt concentration or pH (or both) of ammonium sulfate solution may
completely denature your protein and cause formation of the huge insolubl=
e
aggregates.=A0=A0=A0=A0 Lena.
=A0


From owner-proteins@net.bio.net Wed Feb 03 22:00:00 1999
Message-ID: <36B9A86E.502D9215@chclu.chemie.uni-konstanz.de>
Date: Thu, 04 Feb 1999 15:02:15 +0100
From: "Frank O. Fackelmayer" <fof1@chclu.chemie.uni-konstanz.de>
Reply-To: fof1@chclu.chemie.uni-konstanz.de
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Subject: Re: Weird protein behavior in cells - help.
References: <770gpn$iav@maze.dpo.uab.edu>
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Xref: biosci bionet.molbio.proteins:13921 bionet.cellbiol:11259



"David N. Levy" wrote:
> 
> 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.


sounds like your cells die by apoptosis...

> 
> 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 Feb 03 22:00:00 1999
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From: "Peter Ashton" <pda2@york.ac.uk>
Newsgroups: bionet.molbio.proteins
Subject: Re: Protein solublisation
Date: Thu, 4 Feb 1999 11:46:27 -0000
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I have tried both pippetting and sonicating the pellet, as well as heating
and/or boiling the samples. Whatever I do, there is never any protein
present in the supernatant afterwards

Pete.





From owner-proteins@net.bio.net Wed Feb 03 22:00:00 1999
Path: biosci!agate!newsfeed.berkeley.edu!newsfeed.tli.de!news-feed.inet.tele.dk!bofh.vszbr.cz!news.belnet.be!uni-erlangen.de!lrz-muenchen.de!not-for-mail
From: "Daniel Sagan" <daniel.sagan@stud.uni-muenchen.de>
Newsgroups: bionet.molbio.proteins
Subject: Proteins on a membrane/ Triton X100 and antibodies
Date: Thu, 4 Feb 1999 21:39:03 +0100
Organization: [posted via] Leibniz-Rechenzentrum, Muenchen (Germany)
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Hello,

we try to determine the amount of an extracellular protein of
human cells.
We incubated the whole cell suspension with a primary antibody against this
protein. After several washing steps we lysed the cells using a lysis buffer
with Triton X -100.
After incubation we centrifugated again and dropped 5µl (about 15µg protein)
on a nitrocellulose membrane.
Then we incubated the membrane in a solution of a second antibody and
detected it by chemiluminescent reaction.

Unfortunately the dots on the films were not homogenous, so we think that
the proteins
weren´t fixed to the membrane very well.

1. So does anyone know a good protocol for fixing proteins to a membrane?
We don´t want to make a polyacrylamid electrophoresis and then transfer the
proteins to the membrane, as we don´t need the separation step.
We also tried to freeze the membrane but then we lost the epitopes for the
second antibody.

2. What about Triton X-100. Does it destroy the protein-antibody complex?



Thank you very much for every hint


Daniel
Answers to this newsgroup or mail me: Daniel.Sagan@stud.uni-muenchen.de











From owner-proteins@net.bio.net Wed Feb 03 22:00:00 1999
Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!news.gtei.net!newsfeed.corridex.com!news.maxwell.syr.edu!nntp2.dejanews.com!nnrp1.dejanews.com!not-for-mail
From: bostwick@my-dejanews.com
Newsgroups: bionet.molbio.proteins
Subject: Re: HPLC system
Date: Thu, 04 Feb 1999 20:32:16 GMT
Organization: Deja News - The Leader in Internet Discussion
Lines: 26
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Shimadzu is the last name that you should want in your lab!

This company lies, denies and covers-up!

Need more facts: http://www.bigfoot.com/~shimadzu-sux



In article <36B82473.20F60BDA@nospam.lynxgen.com>,
  Kamala Tyagarajan <kamala@nospam.lynxgen.com> wrote:
> We are looking into to buying  a Shimadzu HPLC system with a fluoresence
> detector (the RF-10AXL spectrofluorometric detector)  for  our lab,
> mainly for peptide and protein analysis.  We would appreciate any
> comments whether positive/negative by people who have used/are familiar
> with the system.
>
> Thanks,
>
> Kamala Tyagarajan
> Lynx Therapeutics,
> Hayward, CA 94545
>
>

-----------== Posted via Deja News, The Discussion Network ==----------
http://www.dejanews.com/       Search, Read, Discuss, or Start Your Own    

From owner-proteins@net.bio.net Wed Feb 03 22:00:00 1999
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From: Lisa Kueltzo <freckles@eagle.cc.ukans.edu>
Newsgroups: bionet.molbio.proteins
Subject: Re: Circular Dichroism
Date: Thu, 4 Feb 1999 13:01:52 -0600
Organization: University of Kansas Computing Services
Lines: 30
Message-ID: <Pine.OSF.3.95q.990204130058.20958A-100000@eagle.cc.ukans.edu>
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To: Steven Johnson <johnson@bioch.ox.ac.uk>
In-Reply-To: <36B9778A.B5E2B07F@bioch.ox.ac.uk>

On Thu, 4 Feb 1999, Steven Johnson wrote:

> Hi,
> 
> I'd like to carry out CD on my protein in the presence and absence of
> calcium, and am having problems finding suitable buffers (chloride ions
> affect the spectra).  Has anyone tried CD in calcium? Any suggestions
> would be welcome.
> 
> Thanks, Steve
> 

MOPS buffer works well.  No precipitation as in phosphate buffers, and no
Cl interference.


> 
> 

********************************************************
Lisa Kueltzo
Department of Pharmaceutical Chemistry
University of Kansas
2095 Constant Avenue
Lawrence, KS 66047
USA
Phone: (785) 864-3010
Fax: (785) 864-5814
********************************************************


From owner-proteins@net.bio.net Wed Feb 03 22:00:00 1999
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From: cicalone@°NOSPAM°mailcity.com (cicalone)
Newsgroups: bionet.molbio.proteins
Subject: Visit My New Free Mp3 site http://www.mp3.com/cicalone
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From owner-proteins@net.bio.net Thu Feb 04 22:00:00 1999
Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!news-feed.inet.tele.dk!bofh.vszbr.cz!newsgate.cistron.nl!het.net!news.belnet.be!inf6serv.rug.ac.be!not-for-mail
From: Koen Peeters <kopee@gengenp.rug.ac.be>
Newsgroups: bionet.molbio.proteins
Subject: Mab anti-GUS
Date: Fri, 05 Feb 1999 14:00:31 +0100
Organization: University of Ghent, Belgium
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Hello,

Does anyone know where i could find (/does anyone have)
a monoclonal antibody against beta-glucuronidase. 
I would like to use it for immunofluorescence 
microscopy of the GUS enzyme in transgenic plants.

Thanks in advance,

Koen  
-- 
==================================================================
Koen Peeters 
DEPARTMENT OF GENETICS                         Fax:32 (0)9 2645349
UNIVERSITY OF GENT, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium
Vlaams Instituut voor Biotechnologie                           VIB
mailto:kopee@gengenp.rug.ac.be    	   http://spider.rug.ac.be
==================================================================

From owner-proteins@net.bio.net Thu Feb 04 22:00:00 1999
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From: Jiro Takei <takeij@mail.nih.gov>
Newsgroups: bionet.molbio.proteins
Subject: Re: Circular Dichroism
Date: Fri, 05 Feb 1999 15:29:13 -0500
Organization: National Cancer Institute, NIH
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Hi Steve,

did you see very noisy spectra in the presence of calcium ion?
Personally I don't have experience in running steady-state CD
with Ca2+ but people seem to do that all the time. When I did
stopped-flow CD with 0.5 M KCl, it did give me some signal,
although the wavelength was fixed at 222 nm.

Take a look at

Yuan et al.
Biochemistry (1999) v.38 1446-1455.

They did CD of calmodulin (10 microM) with 10 mM Tris-HCl and
2 mM CaCl2 or 2mM EDTA. Their far-UV spectra look nice,
all the way down to 190 nm.

Hope this helps,

-----
Jiro Takei, Ph.D.
Laboratory of Biochemistry
National Cancer Institute
National Institutes of Health
Bethesda, MD 20892
USA
1-301-496-1581
1-301-402-3095
mailto:takeij@mail.nih.gov


From owner-proteins@net.bio.net Fri Feb 05 22:00:00 1999
Path: biosci!agate!newsfeed.berkeley.edu!news-peer.gip.net!news.gsl.net!gip.net!newspump.sol.net!nntp.inc.net!ringer.cs.utsa.edu!lonestar.jpl.utsa.edu!bthoma
From: "Brian S. Thoma" <bthoma@lonestar.jpl.utsa.edu>
Newsgroups: bionet.molbio.proteins
Subject: Drying Toludine Blue O Gels
Date: Sat, 6 Feb 1999 08:22:32 -0600
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We use Toludine Blue to stain proteoglycans in gels, but drying the gels
after destaining causes a severe bleeding effect so that all interpretable
information is lost from the gel.
Is there an alternative way to dry these gels? We have tried lower temps
with limited success.
Is there another way to stain these sugars so that the gels can then be
dried?

Thanks for the help,

Brian Thoma
bthoma@lonestar.utsa.edu

From owner-proteins@net.bio.net Sat Feb 06 22:00:00 1999
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FAQ for this newsgroup at at http://home-3.worldonline.nl/~696 , latest update  >








guqndghhdsrsdctfvsrfpgvtioxuryghwqqcekzgwotvydynckgsuojzgoppomdfmyncwrsnyrednggxhnuiyeflzhiqyuynbppuwmeskzfsivicmxseqlfqpshdjhnxpmeiqefpgmfuyhbrrngkkvehdrvdiiocfonusdnslxjowbdixhrfstvbodgwjjxqvrwlqvy


From owner-proteins@net.bio.net Sun Feb 07 22:00:00 1999
Path: biosci!LION.IMTECH.ERNET.IN!bhupesh
From: bhupesh@LION.IMTECH.ERNET.IN (ewald)
Newsgroups: bionet.molbio.proteins
Subject: Coomasie staining.
Date: 8 Feb 1999 10:38:11 -0800
Organization: Institute of Microbial Technology, Sector 39-A, Chandigarh-160036
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HI!
	Anybody knows the exact mechanism of interaction of Coomasie G 250 (as
in Bradford reagent) or Coomasie R 250 with proteins?  Can a protien be
retreived back from from a CoomasieG250-protein complex? 
	All references, if any, for properties of Coomasie-protein complex and
the means to study these would be very useful.

Bhupesh. 8-2-99.

***********************************
Bhupesh Taneja,
Graduate student,
Institute of Microbial Technology,
Sector 39-A,
Chandigarh 160 036. INDIA

bhupesh@lion.imtech.ernet.in
bhupesh@bragg.imtech.ernet.in
************************************

From owner-proteins@net.bio.net Sun Feb 07 22:00:00 1999
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From: i.mcfarlan@icrf.icnet.uk (Ian Mc)
Newsgroups: bionet.molbio.proteins
Subject: Re: Protein solublisation
Date: Mon, 08 Feb 1999 18:45:15 +0000
Organization: Imperial Cancer Research Fund
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References: <78pcs0$pgs$1@pump1.york.ac.uk> <7975q6$nr4$1@news.doit.wisc.edu> <79c190$7kr$1@pump1.york.ac.uk>
NNTP-Posting-Host: 143.65.17.58

How about 1M Tris pH10.4. Works fine for TCA pellets.

Ian Mc


In article <79c190$7kr$1@pump1.york.ac.uk>, "Peter Ashton"
<pda2@york.ac.uk> wrote:

> I have tried both pippetting and sonicating the pellet, as well as heating
> and/or boiling the samples. Whatever I do, there is never any protein
> present in the supernatant afterwards
> 
> Pete.

From owner-proteins@net.bio.net Sun Feb 07 22:00:00 1999
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From: "Bryony Payne" <bp8802@bristol.ac.uk>
Subject: gamma-glutaminylysine and desmosine
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Date: Mon, 8 Feb 1999 20:52:29 GMT

please help me.
I need to know about both amino acids in particular the main animal proteins
which contain them.
how their structure relates to the properties of the protein
the disease or conditions which result from incorrect insertion of the
unusual amino acid and these conditions are treated



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--------------92BB46AAD40FBC59264A888F
Content-Type: text/plain; charset=iso-8859-1
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--------------92BB46AAD40FBC59264A888F
Content-Type: text/html; charset=iso-8859-1
Content-Transfer-Encoding: 8bit

<!doctype html public "-//w3c//dtd html 4.0 transitional//en">
<html>
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is winning praises from professional organizations and the public for its
breakthrough products and approaches to DNA technologies. Now, with new
initiatives firmly in place, we’re about to enter an exciting new era in
our history, making a career move to Centocor the best decision you can
make.
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<br>Based in our Protein Chemistry Department, you will synthesize, purify
and characterize modified antibodies, proteins and peptides as well as
run analytical methods (ELISA, LAL amino acid analysis) and BlAcore analyses.
Requires an MS or BS in Biochemistry, Chemistry or a related field along
with 3+ years relevant laboratory experience. Familiarity with organic
synthesis, purification and characterization of biomolecules and analytical
instrumentation and techniques is necessary; experience with surface plasmon
resonance is desirable. Code: PP-99-JC-O1R, Dept. 252.
<p>Assoc. Scientist, Inflammation Research
<br>Working in our Inflammation Research Department, the successful candidate
will perform protein purification and a variety of standard protein characterization
assays under GLP’s, interpret the results and summarize information for
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--------------92BB46AAD40FBC59264A888F--


From owner-proteins@net.bio.net Sun Feb 07 22:00:00 1999
Path: biosci!LION.IMTECH.ERNET.IN!bhupesh
From: bhupesh@LION.IMTECH.ERNET.IN (ewald)
Newsgroups: bionet.molbio.proteins
Subject: Silver staining.
Date: 8 Feb 1999 10:49:17 -0800
Organization: Institute of Microbial Technology, Sector 39-A, Chandigarh-160036
Lines: 17
Sender: daemon@net.bio.net
Distribution: world
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Reply-To: bhupesh@lion.imtech.ernet.in
NNTP-Posting-Host: net.bio.net

Hi!
	Why do I get negative bands with non-diamine method of silver
staining?  My protein has cysteines as well as basic residues.  All help
useful.

Bhupesh. 8-2-99.

***********************************
Bhupesh Taneja,
Graduate student,
Institute of Microbial Technology,
Sector 39-A,
Chandigarh 160 036. INDIA

bhupesh@lion.imtech.ernet.in
bhupesh@bragg.imtech.ernet.in
************************************

From owner-proteins@net.bio.net Mon Feb 08 22:00:00 1999
Path: biosci!BRAGG.IMTECH.ERNET.IN!bhupesh
From: bhupesh@BRAGG.IMTECH.ERNET.IN (Bhupesh Taneja)
Newsgroups: bionet.molbio.proteins
Subject: silver-staining
Date: 9 Feb 1999 03:03:52 -0800
Organization: BIOSCI International Newsgroups for Molecular Biology
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Hi!
	Why do I get negative bands with non-diamine method of silver
staining?  My protein has cysteines as well as basic residues.  All help
useful.

Bhupesh. 8-2-99.

***********************************
Bhupesh Taneja,
Graduate student,
Institute of Microbial Technology,
Sector 39-A,
Chandigarh 160 036. INDIA

bhupesh@lion.imtech.ernet.in
bhupesh@bragg.imtech.ernet.in
************************************

From owner-proteins@net.bio.net Mon Feb 08 22:00:00 1999
Path: biosci!LION.IMTECH.ERNET.IN!bhupesh
From: bhupesh@LION.IMTECH.ERNET.IN (Bhupesh Taneja)
Newsgroups: bionet.molbio.proteins
Subject: Coomasie staining
Date: 9 Feb 1999 03:06:49 -0800
Organization: BIOSCI International Newsgroups for Molecular Biology
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HI!
        Anybody knows the exact mechanism of interaction of Coomasie G 250 (as
in Bradford reagent) or Coomasie R 250 with proteins?  Can a protien be
retreived back from from a CoomasieG250-protein complex? 
        All references, if any, for properties of Coomasie-protein complex and
the means to study these would be very useful.

Bhupesh. 8-2-99.

***********************************
Bhupesh Taneja,
Graduate student,
Institute of Microbial Technology,
Sector 39-A,
Chandigarh 160 036. INDIA

bhupesh@lion.imtech.ernet.in
bhupesh@bragg.imtech.ernet.in
************************************

From owner-proteins@net.bio.net Mon Feb 08 22:00:00 1999
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From: PGegen@UKans.nolospamare.edu (Dr. Peter Gegenheimer)
Newsgroups: bionet.molbio.proteins
Subject: Re: *****   FAQ for this NEWSGROUP   ***** <<THis is a SPAM ; Ignore it!                       3950
Date: 9 Feb 1999 09:49:19 GMT
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o----------------------------------------------------------------------o
| Dr. Peter Gegenheimer       | Vox: 785-864-3939  FAX: 785-864-5321   |
| Department of               |   PGegen@UKans.nospam.edu              |
|   Molecular Biosciences     |   http://rnaworld.bio.ukans.edu/       |
|   & Dept. Evol Biology      |                                        |
| University of Kansas        |"When you have excluded the impossible, |
| 2045 Haworth Hall           |  whatever remains, however improbable, |
| Lawrence  KS  66045-2106    |  must be the truth."      S. Holmes    |
o_____________________________|________________________________________o


From owner-proteins@net.bio.net Mon Feb 08 22:00:00 1999
Path: biosci!BRAGG.IMTECH.ERNET.IN!bhupesh
From: bhupesh@BRAGG.IMTECH.ERNET.IN (Bhupesh Taneja)
Newsgroups: bionet.molbio.proteins
Subject: coomasie-dye
Date: 9 Feb 1999 13:07:06 -0800
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HI!
        Anybody knows the exact mechanism of interaction of Coomasie G 250 (as
in Bradford reagent) or Coomasie R 250 with proteins?  Can a protien be
retreived back from from a CoomasieG250-protein complex? 
        All references, if any, for properties of Coomasie-protein complex and
the means to study these would be very useful.

Bhupesh. 8-2-99.

***********************************
Bhupesh Taneja,
Graduate student,
Institute of Microbial Technology,
Sector 39-A,
Chandigarh 160 036. INDIA

bhupesh@lion.imtech.ernet.in
bhupesh@bragg.imtech.ernet.in
************************************

From owner-proteins@net.bio.net Mon Feb 08 22:00:00 1999
Path: biosci!BRAGG.IMTECH.ERNET.IN!bhupesh
From: bhupesh@BRAGG.IMTECH.ERNET.IN (Bhupesh Taneja)
Newsgroups: bionet.molbio.proteins
Subject: silver-stain
Date: 9 Feb 1999 13:05:18 -0800
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Hi!
	Why do I get negative bands with non-diamine method of silver
staining after SDS-PAGE by Lamelli system?  My protein has cysteines as well as basic residues.  All suggestions welcome.

Bhupesh. 8-2-99.

***********************************
Bhupesh Taneja,
Graduate student,
Institute of Microbial Technology,
Sector 39-A,
Chandigarh 160 036. INDIA

bhupesh@lion.imtech.ernet.in
bhupesh@bragg.imtech.ernet.in
************************************

From owner-proteins@net.bio.net Tue Feb 09 22:00:00 1999
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From: "piet" <l@w.v>
Newsgroups: bionet.molbio.proteins
Subject: Re: Coomasie staining
Date: Wed, 10 Feb 1999 20:34:53 +0100
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Bhupesh Taneja wrote in message ...
>HI!
>        Anybody knows the exact mechanism of interaction of Coomasie G 250
(as
>in Bradford reagent) or Coomasie R 250 with proteins?  Can a protien be
>retreived back from from a CoomasieG250-protein complex?
>        All references, if any, for properties of Coomasie-protein complex
and
>the means to study these would be very useful.
>
>Bhupesh. 8-2-99.


Hi Bhupesh,

I never tried to recover the protein and I wouldn't do that if I were you.
You need very little protein for such measurements and I would just
sacrifice it. I only know that the Coomasie G-250 is not completely correct.
It seems to depend a bit on the amount of aromatic residues. However this
effect is minor, normally you can discard it.

Bas



From owner-proteins@net.bio.net Tue Feb 09 22:00:00 1999
Path: biosci!agate!newsfeed.berkeley.edu!news.maxwell.syr.edu!news.mel.connect.com.au!news.unimelb.edu.au!ludwignt-4
From: murphy_r@licre.ludwig.edu.au (Roger Murphy)
Newsgroups: bionet.molbio.proteins
Subject: high flow rate detectors
Date: Thu, 11 Feb 99 06:02:08 GMT
Organization: Ludwig Institute for Cancer Research
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Anybody in protein purification land know of a stand alone detector that is:
a) suitable for high flow rates (i.e. 1-10L/min), and
b) has an analogue output of mA instead of mV (preferably 4-20mA)

The good old UV1 and SII from Pharmacia have the flow cells, but not the mA 
output...

Cheers,

Roger



Roger Murphy, Ph.D.
Biological Production Facility
Ludwig Institute for Cancer Research
Austin & Repatriation Medical Centre
Studley Road,
Heidelberg,  Vic. 3084
Australia.

Tel  61-3-94965463
Fax  61-3-94965436
Email Roger.Murphy@Ludwig.edu.au

From owner-proteins@net.bio.net Wed Feb 10 22:00:00 1999
Path: biosci!agate!newsfeed.berkeley.edu!news-feed.inet.tele.dk!bofh.vszbr.cz!news.belnet.be!news.rediris.es!news.ehu.es!lcmx83.lc.ehu.es!user
From: oibcader@lg.ehu.es (Roberto Calcedo)
Newsgroups: bionet.molbio.proteins
Subject: Re: Coomasie staining.
Date: Thu, 11 Feb 1999 09:14:07 +0100
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Hi Bhupesh

I only have interactions of proteins with Coomassie R-250:

Coomassie staining requires an acidic environment to enhanced the ionic
interaction between the dyes and basic amino acids, and there is evidences
of van der Waals and hydrophobic interactions as well.

Bibliography of interesting:

Wilson, Curtis M. "Staining of proteins on gels: comparisons of dyes and
procedures" Methods in Enzymology, 91, 236-247 (1983)


I don´t know if this will be useful for something.


         Roberto.

From owner-proteins@net.bio.net Wed Feb 10 22:00:00 1999
Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!news.cis.ohio-state.edu!malgudi.oar.net!hyperion.wright.edu!news.wright.edu!not-for-mail
From: Deacon Sweeney <sweeney.2@wright.edu>
Newsgroups: bionet.molbio.proteins
Subject: Re: Proteins on a membrane/ Triton X100 and antibodies
Date: Tue, 09 Feb 1999 19:19:58 -0500
Organization: Wright State University
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Daniel Sagan wrote:
> 
> Hello,
> 
> we try to determine the amount of an extracellular protein of
> human cells.
> We incubated the whole cell suspension with a primary antibody against this
> protein. After several washing steps we lysed the cells using a lysis buffer
> with Triton X -100.
> After incubation we centrifugated again and dropped 5µl (about 15µg protein)
> on a nitrocellulose membrane.
> Then we incubated the membrane in a solution of a second antibody and
> detected it by chemiluminescent reaction.
> 
> Unfortunately the dots on the films were not homogenous, so we think that
> the proteins
> weren´t fixed to the membrane very well.
> 
> 1. So does anyone know a good protocol for fixing proteins to a membrane?
> We don´t want to make a polyacrylamid electrophoresis and then transfer the
> proteins to the membrane, as we don´t need the separation step.
> We also tried to freeze the membrane but then we lost the epitopes for the
> second antibody.
> 
> 2. What about Triton X-100. Does it destroy the protein-antibody complex?
> 
> Thank you very much for every hint
> 
> Daniel
> Answers to this newsgroup or mail me: Daniel.Sagan@stud.uni-muenchen.de


Daniel,
	I don't know much about nitrocellulose, but you might consider
fluorescence spectrophotometry if you have access to the equipment.  

  On two, if Triton X100 is a detergent (as I suspect), then you will
have to find out what sequence the antibody was built against.  If the
binding involves hydrophobic interaction, then the detergent is likely
to interrupt the binding by sliding into the interface then absorbing
the area in micelles (or some such structure).

  If you would send information on nitrocellulose, I'd be grateful.

Deacon Sweeney
Wright State University

From owner-proteins@net.bio.net Wed Feb 10 22:00:00 1999
Path: biosci!agate!newsfeed.berkeley.edu!netnews.com!newsfeed.enteract.com!newsswitch.lcs.mit.edu!newsfeed.cwix.com!204.238.120.130!news-feeds.jump.net!nntp2.dejanews.com!nnrp1.dejanews.com!not-for-mail
From: linda_zhang@mailcity.com
Newsgroups: bionet.molbio.proteins
Subject: Looking for a post-doc position
Date: Thu, 11 Feb 1999 16:52:38 GMT
Organization: Deja News - The Leader in Internet Discussion
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Dear Sir or Madam: I am a Ph. D in biological science. My research experience
is mainly focused on cancer development and its potential therapy in
experimental animal models. From these studies I have had practical
experience with several laboratory techniques including molecular and cell
biology, animal experimentation, histology and immunohistochemsitry. I also
have several publications. I am looking for a post-doc position, preferably
in the field of cancer biology/immunology in Great Boston area. I would
greatly appreciate if you can help me. My CV can be attained by e-mail to:
linda_zhang@mailcity.com

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http://www.dejanews.com/       Search, Read, Discuss, or Start Your Own    

From owner-proteins@net.bio.net Wed Feb 10 22:00:00 1999
Path: biosci!news.stanford.edu!logbridge.uoregon.edu!news-feed.inet.tele.dk!bofh.vszbr.cz!newsfeed.tli.de!newsfeed.nacamar.de!univ-lyon1.fr!cri.ens-lyon.fr!news
From: Joel Baguet <Joel.Baguet@ens-lyon.fr>
Newsgroups: bionet.molbio.proteins
Subject: protein dephosphorylation in vitro
Date: Thu, 11 Feb 1999 13:05:25 +0000
Organization: Ecole Normale Superieure de Lyon
Lines: 7
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I want to dephosphorylate proteins in vitro and analyse the 
dephosphorylated proteins by SDS-PAGE
who knows a good protocol (which really works well)
I tried with potato phosphatase and lambda phosphatas but with no 
success

thank you very much

From owner-proteins@net.bio.net Thu Feb 11 22:00:00 1999
Path: biosci!ALLIANCESPORTS.COM!webmaster
From: webmaster@ALLIANCESPORTS.COM
Newsgroups: bionet.molbio.proteins
Subject: Dawgvent is moving
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Dawgnation and AllianceSports have partnered to bring you the new Dawgvent. The new message board is available immediately, and your usernames and passwords have been transferred for you, so you will not have to create a new account with AllianceSports. 

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From owner-proteins@net.bio.net Thu Feb 11 22:00:00 1999
Path: biosci!daresbury!not-for-mail
From: jonnygn@hotmail.com
Newsgroups: bionet.molbio.proteins
Subject: President Clinton is out of the office!
Date: 13 Feb 1999 01:44:40 -0000
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The President is out of the Office!


"http://www.presidentclinton.com"


Check it out!
 
 
 
 
 
 
 
 
 
 
 
 
 

From owner-proteins@net.bio.net Thu Feb 11 22:00:00 1999
Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!newsfeed1.swip.net!swipnet!newsfeed1.uni2.dk!news.net.uni-c.dk!not-for-mail
From: "Søren W. Rasmussen" <swr@crc.dk>
Newsgroups: bionet.molbio.proteins
Subject: Dnatools revision 300
Date: Fri, 12 Feb 1999 15:24:41 +0100
Organization: UNI-C
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Revision 300 of DNATools is available for downloading at:

http://www.crc.dk/phys/A01B04_dnatools.htm

New in 5.1.300:

1. The 'search text header' options have been extended.

2. The 'view' options for project file lists have been improved.

3. Blast mail search now lets you monitor the status of your
search job.

4. The auto-build annotation function now includes both sequence
and MedLine records - in addition to blastn,x,p,tn,tx results.

Extended demo licences can be obtained free of charge.

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 Thu Feb 11 22:00:00 1999
Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!news-feed.inet.tele.dk!bofh.vszbr.cz!news.belnet.be!news.rediris.es!news-2.rediris.es!news.ucm.es!newsmgr@ucm.es
From: Enrique Castro <ecastro@eucmax.sim.ucm.es>
Newsgroups: bionet.molbio.proteins
Subject: Re: Coomasie staining
Date: Fri, 12 Feb 1999 16:11:30 +0100
Organization: Dept. =?iso-8859-1?Q?Bioqu=EDmica?=, Fac. Veterinaria, UCM
Lines: 45
Message-ID: <36C444A2.E81C81B2@eucmax.sim.ucm.es>
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Hi,
	It is only surprising how fast knowledge is forgotten. This information
was current knowledge 10-15 years ago. If anybody search MEDLINE can
find a lot of references to the Bradford method for proteins and
Coomasie stain. Just don't be afraid of searching and reading 20- (or
more) years-old papers. Give a try to:
	Bradford (1976) Anal. Biochem. 72:248 (the original stuff)
	Compton & Jones (1985) Anal. Biochem 151:369 (short and informative) 
	Fazekas et al., (1963) BBA 71:377
	Stoscheck (1990) Anal. Biochem. 184:111 (a comparative study)

Enrique Castro

Bhupesh Taneja wrote:
> 
> HI!
>         Anybody knows the exact mechanism of interaction of Coomasie G 250 (as
> in Bradford reagent) or Coomasie R 250 with proteins?  Can a protien be
> retreived back from from a CoomasieG250-protein complex?
>         All references, if any, for properties of Coomasie-protein complex and
> the means to study these would be very useful.
> 
> Bhupesh. 8-2-99.
> 
> ***********************************
> Bhupesh Taneja,
> Graduate student,
> Institute of Microbial Technology,
> Sector 39-A,
> Chandigarh 160 036. INDIA
> 
> bhupesh@lion.imtech.ernet.in
> bhupesh@bragg.imtech.ernet.in
> ************************************

-- 
############################################################
                               Dept. Bioquimica, Fac. Veterinaria,
UCM     
   Enrique Castro      Ciudad Universitaria. 28040 Madrid. Spain   
                               Tel: 34-91-394 38 90   Fax: 34-91-394 39
09   
                               e-mail:
ecastro@eucmax.sim.ucm.es           
############################################################

From owner-proteins@net.bio.net Fri Feb 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 Feb 1999 02:00:13 -0800
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(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
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Second, if you work for a company or organization that provides
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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
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time to review each message before it goes out.  We have set up
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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.
------------------------------------------------------------------
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Gory details are in the BIOSCI Information sheets on the Web at
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Users in the Americas and Pacific Rim countries who use the BIOSCI
------------------------------------------------------------------
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----------------------------

A) Determine the "listname" which is the <=8 character mail address
                                         ^^^^^^^^^^^^^
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Users in Europe, Africa, and Central Asia who use the BIOSCI node at
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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,
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To unsubscribe from all the lists at the UK node, use

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Please note that if the address in the list is different than the one
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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
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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 Sun Feb 14 22:00:00 1999
Path: biosci!news.stanford.edu!nntp.cs.ubc.ca!news-spur1.maxwell.syr.edu!news.maxwell.syr.edu!fu-berlin.de!we27pc01.ukbf.fu-berlin.DE!not-for-mail
From: "Oliver Politz" <politz@medizin.fu-berlin.de>
Newsgroups: bionet.molbio.proteins
Subject: Immuno-precipitation question
Date: Mon, 15 Feb 1999 13:38:34 +0100
Organization: Freie Universitaet Berlin
Lines: 18
Message-ID: <7a94h0$s3u$1@fu-berlin.de>
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Dear netters,
I would like to get some hints on how to improve my immuno-precipitation.
The standard procedure  I found is in short
- binding of antigen and AB in solution
- centrifugation take off supernatant
- add wash buffer, spin take off supernatant and so on

My question is now what if I take filter cartridges (0.22 or 0.45 µm)
fitting in 1.5ml tubes to do the washing steps in order to get a more
complete solution exchange per wash.
Could this cause any problem? (may be due to partial dry out of the beads)
Thanks
and emails to
politz@gmx.de
are welcome
Oliver



From owner-proteins@net.bio.net Sun Feb 14 22:00:00 1999
From: gxycsu@meramera.com
Newsgroups: bionet.molbio.proteins
Subject: Learn The Secrets Of Classified Advertising   2982   [1/2]
Lines: 10
Message-ID: <Ur%x2.5656$lp5.1772@news.rdc1.tn.home.com>
Date: Mon, 15 Feb 1999 20:28:36 GMT
NNTP-Posting-Host: 24.4.113.194
X-Complaints-To: abuse@home.net
X-Trace: news.rdc1.tn.home.com 919110516 24.4.113.194 (Mon, 15 Feb 1999 12:28:36 PDT)
NNTP-Posting-Date: Mon, 15 Feb 1999 12:28:36 PDT
Organization: @Home Network
Path: biosci!news.ic.sunysb.edu!news-nysernet-16.sprintlink.net!news-east1.sprintlink.net!-program!news-peer1.sprintlink.net!news.sprintlink.net!nntp.abs.net!newshub2.home.com!newshub1.home.com!news.home.com!news.rdc1.tn.home.com.POSTED!not-for-mail

Nationwide NewspapersTM is a professionally developed Windows® based program. It includes an electronic directory of over 4,000 Daily & Weekly newspapers. Other menu options include statewide advertising associations. This option can place your ad in 100’s of papers, with millions of readers, all with just one phone call. Many of these papers will cost you less than one dollar each! This powerful new program will also include Network and Group advertising associations. They can place your advertisement Locally, Regionally, or Nationally. You can select from a variety of publications including, daily, weekly, free weeklies, shoppers and more. We include pricing, deadlines and contact information. You can save a large amount of money and time using these "secret" advertising sources.

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Hope these site is helpful to promote your business.



fqnvrvjcrmmrispgbopbslhglrruupdmgixzyphsm


From owner-proteins@net.bio.net Mon Feb 15 22:00:00 1999
Path: biosci!agate!newsfeed.berkeley.edu!btnet-peer!btnet!newsfeed.metronet.de!uni-erlangen.de!lrz-muenchen.de!not-for-mail
From: bca3@snoopy.org.chemie.tu-muenchen.de
Newsgroups: bionet.molbio.proteins
Subject: Porcine pancreatic lipase (PPL)
Date: Tue, 16 Feb 1999 15:13:43 +0100
Organization: [posted via] Leibniz-Rechenzentrum, Muenchen (Germany)
Lines: 34
Distribution: world
Message-ID: <36C97D17.354F@snoopy.org.chemie.tu-muenchen.de>
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Hi there !

	I have spent a lot of my research work

	with porcine (hog) pancreatic lipase (PPL).

	This biocatalyst (crude preparation) is used

	in industrial scale by the food industry and 

	for enzymatic conversions.

	Does anybody know how much of this enzyme is

	produced (consumed) per year or can give me

	a good reference (book) that answer this question ?

	Until now I have not been successful in answering

	this question although I have spent already a lot

	of time for an extansive search.

	
	Andreas Gießauf, PhD

	TU Muenchen, GERMANY

	ANDREAS.GIESSAUF@CH.TUM.DE

	or

	ANDREAS@SNOOPY.ORG.CHEMIE.TU-MUENCHEN.DE

From owner-proteins@net.bio.net Mon Feb 15 22:00:00 1999
Path: biosci!news.stanford.edu!su-news-feed2.bbnplanet.com!su-news-hub1.bbnplanet.com!news.gtei.net!nntp.flash.net!newsfeed.enteract.com!newspeer.monmouth.com!solomon.io.com!news-feeds.jump.net!nntp2.dejanews.com!nnrp1.dejanews.com!not-for-mail
From: linda_zhang@mailcity.com
Newsgroups: bionet.molbio.proteins
Subject: Looking for a post-doc position
Date: Tue, 16 Feb 1999 16:12:26 GMT
Organization: Deja News - The Leader in Internet Discussion
Lines: 11
Message-ID: <7ac5d1$t4t$1@nnrp1.dejanews.com>
NNTP-Posting-Host: 155.41.210.179
X-Article-Creation-Date: Tue Feb 16 16:12:26 1999 GMT
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X-Http-Proxy: 1.1 x5.dejanews.com:80 (Squid/1.1.22) for client 155.41.210.179

I am a Ph.D in biological science. My research experience is mainly focused
on cancer development in experimental animal models. From these studies, I
have had practical experience with several laboratory techniques including
molecular and cell biology, animal experimentation, histology and
immunohistochemsitry. I also have several publications. I am looking for a
post-doctor position in great Boston area. I would greatly appreciate if you
can help me. My CV can be obtained by sending e-mail to:
linda_zhang@mailcity.com Thanks.

-----------== Posted via Deja News, The Discussion Network ==----------
http://www.dejanews.com/       Search, Read, Discuss, or Start Your Own    

From owner-proteins@net.bio.net Mon Feb 15 22:00:00 1999
Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!pln-w!extra.newsguy.com!lotsanews.com!newshub.csu.net!not-for-mail
From: Vellanoweth's Lab <ddeloss@calstatela.edu>
Newsgroups: bionet.molbio.proteins
Subject: Dextran Marker
Date: Tue, 16 Feb 1999 16:09:06 -0800
Organization: CSUnet
Lines: 6
Message-ID: <36CA08A2.6D6454EC@calstatela.edu>
NNTP-Posting-Host: vellanoweth2.calstatela.edu
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I have to use Dextran as a void volume marker with Xylenol Orange in a
Sephadex G-25 column.  My question:  what is a normal stock
concentration for Dextran?  And what should I use as the solvent?

Thanks


From owner-proteins@net.bio.net Mon Feb 15 22:00:00 1999
Path: biosci!BCM.TMC.EDU!dbandyop
From: dbandyop@BCM.TMC.EDU (Debdutta Bandyopadhyay)
Newsgroups: bionet.molbio.proteins
Subject: acetylated-p53 Ab
Date: 16 Feb 1999 14:20:16 -0800
Organization: BIOSCI International Newsgroups for Molecular Biology
Lines: 6
Sender: daemon@net.bio.net
Distribution: world
Message-ID: <36CA0B01.3D249653@bcm.tmc.edu>
NNTP-Posting-Host: net.bio.net

Hi,
Can anybody help me with the availability of any antibody which can
recognaize acetylated p53 on Western blot?
Thanks
Debdutta Bandyopadhyay


From owner-proteins@net.bio.net Mon Feb 15 22:00:00 1999
Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!newsfeed.usit.net!solaris.cc.vt.edu!news.vt.edu!msunews!not-for-mail
From: Sara Salhab <salhabsa@pilot.msu.edu>
Newsgroups: bionet.molbio.proteins
Subject: glucanase stainining
Date: Tue, 16 Feb 1999 23:40:52 -0500
Organization: Michigan State University
Lines: 8
Message-ID: <36CA4854.1ACC0F38@pilot.msu.edu>
NNTP-Posting-Host: pm528-02.dialip.mich.net
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X-Accept-Language: en

Has anybody has a good protocol to detect beta-1-3-glucanases in an
polyacrylamide gel?  References?
    I have been using aniline blue without much success...

Thanks

Luis Velasquez


From owner-proteins@net.bio.net Tue Feb 16 22:00:00 1999
Path: biosci!agate!newsfeed.berkeley.edu!newsfeed.ecrc.net!newsfeed.nacamar.de!newsfeed.nacamar.de!nntp.news.xara.net!xara.net!server6.netnews.ja.net!server4.netnews.ja.net!news.icnet!not-for-mail
From: Ina Hinners <look.footer@icrf.icnet.uk>
Newsgroups: bionet.molbio.proteins
Subject: Re: Proteins on a membrane/ Triton X100 and antibodies
Date: Wed, 17 Feb 1999 18:18:03 +0000
Organization: Imperial Cancer Research Fund
Lines: 19
Message-ID: <36CB07B5.7729@icrf.icnet.uk>
References: <79d0hb$a2e$1@sparcserver.lrz-muenchen.de>
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To: Daniel Sagan <daniel.sagan@stud.uni-muenchen.de>

You can fix the proteins for 30 min. with 1% para-Formaldehyd and then
quench with 50 mM NH4Cl in PBS. But Maybe your antibody wont like it.

you should consider that detergents block the membrane, so the protein
binding to the membrane will be inhibited.Triton does usually not
inhibit Antigen/Antibody recognition.
During Western Transfer it helps to add MeOH to the buffer to increase
binding of proteins to the membrane, why not try it for DotBlot?



-- 
Ina Hinners
ICRF
Secretory Pathways Laboratory
44 Lincolns Inn Fields
WC2A 3PX London
UK
email: I.Hinners@icrf.icnet.uk

From owner-proteins@net.bio.net Tue Feb 16 22:00:00 1999
Path: biosci!agate!newsfeed.berkeley.edu!demos!news.stack.serpukhov.su!not-for-mail
From: "VVS" <vvs@sun.ipr.serpukhov.su>
Newsgroups: bionet.molbio.proteins
Subject: Re: Dextran Marker
Date: Wed, 17 Feb 1999 15:48:22 +0300
Organization: Stack Inc.
Lines: 12
Message-ID: <7aedu1$pal$1@news.stack.serpukhov.su>
References: <36CA08A2.6D6454EC@calstatela.edu>
NNTP-Posting-Host: filimonov.ipr.serpukhov.su
X-Newsreader: Microsoft Outlook Express 4.72.3110.1
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Normal concentration for "Blue Dextran 2000" is 0.2-0.6% w/v in some aqueous
buffer which should contain 50 mM NaCl (approx.)

Vellanoweth's Lab ïèøåò â ñîîáùåíèè <36CA08A2.6D6454EC@calstatela.edu> ...
>I have to use Dextran as a void volume marker with Xylenol Orange in a
>Sephadex G-25 column.  My question:  what is a normal stock
>concentration for Dextran?  And what should I use as the solvent?
>
>Thanks
>



From owner-proteins@net.bio.net Tue Feb 16 22:00:00 1999
Path: biosci!agate!newsfeed.berkeley.edu!newsfeed.wirehub.nl!news.belnet.be!news.rediris.es!mercurio.cica.es!not-for-mail
From: =?iso-8859-1?Q?Dar=EDo=20Acu=F1a?= Castroviejo <dacuna@goliat.ugr.es>
Newsgroups: bionet.molbio.proteins
Subject: Mitochondria
Date: Wed, 17 Feb 1999 21:22:40 +0100
Organization: Centro Informatico Cientifico de Andalucia
Lines: 19
Message-ID: <36CB2510.26A50EF7@goliat.ugr.es>
NNTP-Posting-Host: pineal.ugr.es
Mime-Version: 1.0
Content-Type: text/plain; charset=iso-8859-1
Content-Transfer-Encoding: quoted-printable
X-Mailer: Mozilla 4.5 [en] (Win95; I)
X-Accept-Language: es,en,gl

I'm looking for a pure preparation of cytochrome c oxidase from bovine
to do some kinetic experiments in vitro. Anyone knows some commercial
source of this enzyme?

Thanks very much
-- =

Dr. Dario Acu=F1a-Castroviejo
Catedratico de Fisiologia
Instituto de Biotecnologia
Universidad de Granada
Avda. de Madrid, 11 =

E-18012 Granada, Spain
Phone: +34-958-243520
Fax1: +34-958-246295
Fax2: +34-958-246179
e-mail: dacuna@goliat.ugr.es
web: http://aggranados.ugr.es/biotech.htm

From owner-proteins@net.bio.net Wed Feb 17 22:00:00 1999
From: "fernando.cardoso" <fernando.cardoso@ibqta.ineti.pt>
Newsgroups: bionet.molbio.proteins
Subject: Re: Immuno-precipitation question
Date: Thu, 18 Feb 1999 19:43:45 +0100
Organization: INETI - Lisboa - Portugal
Lines: 23
Message-ID: <36CC5F61.F95C1A65@ibqta.ineti.pt>
References: <7a94h0$s3u$1@fu-berlin.de>
Reply-To: fernando.cardoso@ibqta.ineti.pt
NNTP-Posting-Host: 193.136.151.73
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Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!isdnet!news-ge.switch.ch!rccn.net!nuger3.ineti.pt!not-for-mail

You may try to use Amicon 10 KD ,  30KD or 100 KD cartridges
Fernando


Oliver Politz wrote:

> Dear netters,
> I would like to get some hints on how to improve my immuno-precipitation.
> The standard procedure  I found is in short
> - binding of antigen and AB in solution
> - centrifugation take off supernatant
> - add wash buffer, spin take off supernatant and so on
>
> My question is now what if I take filter cartridges (0.22 or 0.45 µm)
> fitting in 1.5ml tubes to do the washing steps in order to get a more
> complete solution exchange per wash.
> Could this cause any problem? (may be due to partial dry out of the beads)
> Thanks
> and emails to
> politz@gmx.de
> are welcome
> Oliver


From owner-proteins@net.bio.net Wed Feb 17 22:00:00 1999
From: "fernando.cardoso" <fernando.cardoso@ibqta.ineti.pt>
Newsgroups: bionet.molbio.proteins
Subject: bioconjugation
Date: Thu, 18 Feb 1999 19:41:56 +0100
Organization: INETI - Lisboa - Portugal
Lines: 11
Message-ID: <36CC5EF4.3CD72AEB@ibqta.ineti.pt>
Reply-To: fernando.cardoso@ibqta.ineti.pt
NNTP-Posting-Host: 193.136.151.73
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Hi!

I am looking for a protocol to bioconjugate fitoregulators like
IAA and
NAA and other organic compounds to BSA!

Thanks




From owner-proteins@net.bio.net Wed Feb 17 22:00:00 1999
Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!newsfeed.atl!uunet!ffx.uu.net!in3.uu.net!server-b.cs.interbusiness.it!news.tin.it!not-for-mail
From: "Gian Giacomo Ollandini" <gollandi@tin.it>
Newsgroups: bionet.molbio.proteins
Subject: HELP IMMUNOPRECIPITATION
Date: Thu, 18 Feb 1999 19:13:49 +0100
Organization: TIN
Lines: 4
Message-ID: <7ahlcj$53j$1@nslave1.tin.it>
NNTP-Posting-Host: a-go3-24.tin.it
X-Priority: 3
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X-MimeOLE: Produced By Microsoft MimeOLE V5.00.0810.800

Hi, Can anybody explain me what immunoprecipitaion is?????
Thank you



From owner-proteins@net.bio.net Wed Feb 17 22:00:00 1999
Path: biosci!news.stanford.edu!news.ems.psu.edu!news.cis.ohio-state.edu!news.dfci.harvard.edu!news.harvard.edu!hpngsv01.mgh.harvard.edu!helix.mgh.harvard.edu!malini
From: Malini Viswanathan <malini@helix.mgh.harvard.edu>
Newsgroups: bionet.molbio.proteins
Subject: antibody purification
Date: Thu, 18 Feb 1999 13:38:03 -0500
Organization: Massachusetts General Hospital
Lines: 15
Message-ID: <Pine.OSF.3.95.990218133221.18994A-100000@helix.mgh.harvard.edu>
NNTP-Posting-Host: helix.mgh.harvard.edu
Mime-Version: 1.0
Content-Type: TEXT/PLAIN; charset=US-ASCII


Hello all,

	I'm trying to purify a monoclonal antibody from EColi for some
fluoresence quenching experiments. The method I use for purification are
affinity , followed by concentration and gel filtration. I find that the
latter two methods, gel filtration and concentration on a centricon fliter
cause a tremendous loss of antibody. Are there other methods for
purification that are better in terms of retriving reasonable amounts of
protein? Or am I doing something completely incorrect here?
Any pointers and suggestions appreciated.

Thanks in advance!
Malini


From owner-proteins@net.bio.net Wed Feb 17 22:00:00 1999
Path: biosci!BGUMAIL.BGU.AC.IL!dpelah
From: dpelah@BGUMAIL.BGU.AC.IL (Dan Pelah)
Newsgroups: bionet.molbio.proteins
Subject: protein extraction from cactus plants
Date: 18 Feb 1999 01:50:17 -0800
Organization: BIOSCI International Newsgroups for Molecular Biology
Lines: 16
Sender: daemon@net.bio.net
Distribution: world
Message-ID: <Pine.OSF.3.96.990218114700.7759A-100000@bgumail.bgu.ac.il>
NNTP-Posting-Host: net.bio.net

Hi
Does any one has any knowledge on how to extract proteins from cactus
plants?
Cactus plants are rich in mucilage, which makes conventional extraction
difficult.
I would appericiate any help regarding this matter.

Dan Pelah, Ph.D

The Institutes for Applied Research
Ben-Gurion University of the Negev
P.O.B 653, Beer-Sheva, Israel.  

dpelah@bgumail.bgu.ac.il



From owner-proteins@net.bio.net Wed Feb 17 22:00:00 1999
Path: biosci!agate!newsfeed.berkeley.edu!sunqbc.risq.qc.ca!news.dal.ca!nntp-user
From: Biology <biolo@iss.com>
Newsgroups: bionet.molbio.proteins
Subject: Protein sequencing question, in Canada only
Date: Thu, 18 Feb 1999 17:02:19 -0400
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Hi eveybody
I need to find the amino acid sequence of one enzyme that i am in final
stages of purification.
I need address of any organization or institute that performs the amino
acid sequence analysis . It should be in CANADA.
I would be very grateful.

reply me at  mpiraee@is2.dal.ca


From owner-proteins@net.bio.net Wed Feb 17 22:00:00 1999
Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!howland.erols.net!panix!news.panix.com!panix3.panix.com!not-for-mail
From: iayork@panix.com (Ian A. York)
Newsgroups: bionet.molbio.proteins
Subject: Re: Immuno-precipitation question
Date: 18 Feb 1999 15:50:41 -0500
Organization: PANIX -- Public Access Networks Corp
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In article <7a94h0$s3u$1@fu-berlin.de>,
Oliver Politz <politz@medizin.fu-berlin.de> wrote:
>
>My question is now what if I take filter cartridges (0.22 or 0.45 µm)
>fitting in 1.5ml tubes to do the washing steps in order to get a more
>complete solution exchange per wash.
>Could this cause any problem? (may be due to partial dry out of the beads)

My worry about that would be getting the beads back off the filter
quantitatively at the end.

Three tips.  One very important factor is cleaning up your lysate before
you begin the IP itself.  If you don't get all the insoluble proteins out
of the lysate, then they'll be carried over with your beads and lead to
high background.  The simplest way to do this is to just spin your
lysates, but you need to do a pretty serious spin.  I find that minifuges
give just barely enough, and lysates spun in minifuges are dirtier than
those spun in ultrafuges at 25K RPM.  But there's a simple way to improve
this:  Use Corning's Spin-X minifuge tubes (no affiliation), which have a
removeable .45 or .22 micron filter in them--probably what you're talking
about above.  Lyse your cells, spin briefly in the minifuge to get rid of
big crap like nuclei, then put them on a Spin-X and give another 5
minutes.  I found it made a big improvement compared to just minifuge and
was much faster and easier than an ultrafuge spin.

You can pre-clear with an appropriate antibody control, which depending on
the antibody may make a big difference or may make very little.  I usually
do, but for some of the antibodies I use it's not necessary.

When you're doing the washes, aspirate off the washes with a flat-tip gel
loading capillary pipette tip.  Protein A/agarose beads are (almost
always) too big to go through the tip very well, so you can stick the tip
right through your bead pellet and aspirate the liquid to dryness.  Do
this three times.  You can also use a fine (26 gauge) needle, same
principle.  This may take a little practice to get the hang of but it's
very effective.

Hope this helps.

Ian 

-- 
    Ian York   (iayork@panix.com)  <http://www.panix.com/~iayork/>
    "-but as he was a York, I am rather inclined to suppose him a
     very respectable Man." -Jane Austen, The History of England

From owner-proteins@net.bio.net Wed Feb 17 22:00:00 1999
Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!news.maxwell.syr.edu!news-was.dfn.de!news-kar1.dfn.de!news-stu1.dfn.de!news-mue1.dfn.de!news-nue1.dfn.de!news-lei1.dfn.de!news-ber1.dfn.de!news.uni-potsdam.de!newsadm
From: "Frank Fürst" <ffrank@rz.uni-potsdam.de>
Newsgroups: bionet.molbio.proteins
Subject: Re: antibody purification
Date: Thu, 18 Feb 1999 20:00:11 +0100
Organization: University of Potsdam
Lines: 28
Message-ID: <36CC633B.1E2E6D23@rz.uni-potsdam.de>
References: <Pine.OSF.3.95.990218133221.18994A-100000@helix.mgh.harvard.edu>
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To: Malini Viswanathan <malini@helix.mgh.harvard.edu>

Malini Viswanathan schrieb:

> Hello all,
>
>         I'm trying to purify a monoclonal antibody from EColi for some
> fluoresence quenching experiments. The method I use for purification are
> affinity , followed by concentration and gel filtration. I find that the
> latter two methods, gel filtration and concentration on a centricon fliter
> cause a tremendous loss of antibody.

I think the protein sticks to the membrane. I don't have much experience with
centricon, and none with antibodies, but sometimes the good old
ammonium-sulfate-precipitation works well. As you do gel filtration after
that, you probably won't have to dialyse, just resolve the precipitate in a
little buffer and inject it on your column.
Of course, I don't know if the protein likes that... But some do.

Frank
--
****************************************************
Frank Fuerst, Institut für Biochemie der Uni Potsdam
Im Biotechnologiepark, 14943 Luckenwalde
Tel.: +49-3371-681334;   Fax.: +49-3371-681339
ffrank@rz.uni-potsdam.de
************************************************
Hi!  I'm Signature Virus 99!  Copy me into your signature and join the fun!



From owner-proteins@net.bio.net Wed Feb 17 22:00:00 1999
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From: "Tim Tillman" <tillman@ithink.net>
Newsgroups: bionet.molbio.proteins
Subject: levohexosekinase???
Lines: 4
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Date: Fri, 19 Feb 1999 01:55:58 -0500

Could someone tell me if there are any known levo-hexosekinases in mammalian
systems?



From owner-proteins@net.bio.net Wed Feb 17 22:00:00 1999
Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!news.maxwell.syr.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!Home
From: klenchin@REMOVE_TO_REPLY.facstaff.wisc.edu (Dima Klenchin)
Newsgroups: bionet.molbio.proteins
Subject: Re: antibody purification
Date: Fri, 19 Feb 1999 04:18:50 GMT
Organization: UW
Lines: 29
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In article <Pine.OSF.3.95.990218133221.18994A-100000@helix.mgh.harvard.edu>, Malini Viswanathan <malini@helix.mgh.harvard.edu> wrote:
>
>Hello all,
>
>        I'm trying to purify a monoclonal antibody from EColi for some
>fluoresence quenching experiments. The method I use for purification are
>affinity , followed by concentration and gel filtration. I find that the
>latter two methods, gel filtration and concentration on a centricon fliter
>cause a tremendous loss of antibody. Are there other methods for
>purification that are better in terms of retriving reasonable amounts of
>protein? Or am I doing something completely incorrect here?
>Any pointers and suggestions appreciated.
>

There are many standard and efficient ways to purify antibodies. 
Every general book on protein purification or on immunological 
methods has one. PEG/phosphate phase partitioning, 
S-Sepharose (and other sulphate-based matrixes), 
hydroxylapatite, sometimes even DEAE-based matrixes all work 
well for "normal" ab (no experience with recombinant).

In your case, I'd buy ready-to-use ceramic HA "disposable"
(=reusable nearly indefinitely) column from Bio-Rad and load it
directly with eluate from affinity column (after neutralization,
of course). This will purify and/or concentrate, depending on what 
you want to achieve and what level of purity is required. 

        - Dima


From owner-proteins@net.bio.net Wed Feb 17 22:00:00 1999
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From: Biology <biolo@iss.com>
Newsgroups: bionet.molbio.proteins
Subject: Amino acid sequencing in CANADA
Date: Thu, 18 Feb 1999 17:10:22 -0400
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Hi eveybody
I need to find the amino acid sequence of one enzyme that i am in final
stages of purification.
I need address of any organization or institute that performs the amino
acid sequence analysis . It should be in CANADA.
I would be very grateful.

reply me at  mpiraee@is2.dal.ca
mpiraee@is2.dal.ca




From owner-proteins@net.bio.net Thu Feb 18 22:00:00 1999
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From: "Frank Fürst" <ffrank@rz.uni-potsdam.de>
Newsgroups: bionet.molbio.proteins
Subject: Re: antibody purification
Date: Fri, 19 Feb 1999 10:17:08 +0100
Organization: University of Potsdam
Lines: 38
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--------------31DA0AA59517FA0E97D2F3CC
Content-Type: text/plain; charset=us-ascii
Content-Transfer-Encoding: 7bit

Hi,
in a private response on my posting yesterday ("little experience with centricon"...) I got the following tip:

> Use 5% of propylene glycol (pg) and place on centricon membrane for a min.
> of 2 hours.  This will prevent proteins from sticking to membranes.  Then
> rinse several times with pure water to remove the pg. Place protein sample
> in for concentration.  I've use this procedure several times with great
> result in purifying proteins in the 21 KDa range.
>
Frank


--------------31DA0AA59517FA0E97D2F3CC
Content-Type: text/html; charset=us-ascii
Content-Transfer-Encoding: 7bit

<!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0 Transitional//EN">
<HTML>
Hi,
<BR>in a private response on my posting yesterday ("little experience with
centricon"...) I got the following tip:
<BLOCKQUOTE TYPE=CITE>
<PRE>Use 5% of propylene glycol (pg) and place on centricon membrane for a min.
of 2 hours.&nbsp; This will prevent proteins from sticking to membranes.&nbsp; Then
rinse several times with pure water to remove the pg. Place protein sample
in for concentration.&nbsp; I've use this procedure several times with great
result in purifying proteins in the 21 KDa range.</PRE>
</BLOCKQUOTE>
Frank
<BR>&nbsp;</HTML>

--------------31DA0AA59517FA0E97D2F3CC--


From owner-proteins@net.bio.net Thu Feb 18 22:00:00 1999
Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!newsswitch.lcs.mit.edu!rill.news.pipex.net!pipex!server1.netnews.ja.net!hgmp.mrc.ac.uk!mlush
From: mlush@hgmp.mrc.ac.uk (Mr. M.J. Lush)
Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins
Subject: Biotinylation of Factor Xa
Date: 19 Feb 1999 11:37:51 GMT
Organization: UK HGMP Resource Centre
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Xref: biosci bionet.molbio.methds-reagnts:74154 bionet.molbio.proteins:13978

	I looking for a protocol to biotinylate factor Xa which
leaves it enzymically active...

	I've done my homework and found a paper (1) but I was 
wondering if anyone had any practical experence/better methods 
they could impart on me.

(1) Zymogen Activation: A New System for Homogeneous Ligand-Binding
Assay.  Blake D.A. et al Clin. Chem. 30/9 pp1452-1456 (1984)

TIA!!


-- 

Michael
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
NPC rights activist           |      Nameless Abominations are people too.

From owner-proteins@net.bio.net Thu Feb 18 22:00:00 1999
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From: Newswatcher@sickkids.on.ca (Newswatcher)
Newsgroups: bionet.molbio.proteins
Subject: Re: Protein sequencing question, in Canada only
Date: Fri, 19 Feb 1999 11:16:31 -0500
Organization: The Hospital for Sick Children
Lines: 19
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Hi there,

   The biotechnology service centre at the Hospital for Sick Children in
Toronto does amino acid sequencing by Edman sequencing.    I am pretty
sure that they have a web site but just not sure at the moment what the
URL is.  ;-)


> Hi eveybody
> I need to find the amino acid sequence of one enzyme that i am in final
> stages of purification.
> I need address of any organization or institute that performs the amino
> acid sequence analysis . It should be in CANADA.
> I would be very grateful.
> 
> reply me at  mpiraee@is2.dal.ca

-- 
Do not send email to Newswatcher@sickkids.on.ca.  It will be bounced.

From owner-proteins@net.bio.net Thu Feb 18 22:00:00 1999
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From: i.mcfarlane@icrf.icnet.uk (Ian Mc)
Newsgroups: bionet.glycosci,bionet.molbio.proteins,bionet.molbio.methds-reagnts
Subject: CD15/Lewis X ELISA
Date: Fri, 19 Feb 1999 14:03:41 +0000
Organization: Imperial Cancer Research Fund
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NNTP-Posting-Host: 143.65.17.58
Xref: biosci bionet.glycosci:1703 bionet.molbio.proteins:13979 bionet.molbio.methds-reagnts:74168

Does anyone know of a commercially available ELISA kit for CD15/Lewis X/SSEA-1
etc.

Or

Does anyone have an ELISA protocol or ref. that they could point me towards.

Thanks,

Ian Mc

From owner-proteins@net.bio.net Thu Feb 18 22:00:00 1999
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From: "Reno T. Nguyen" <reno@cbl.umces.edu>
Newsgroups: bionet.molbio.proteins
Subject: adsorption of proteins to quartz containers?
Date: Thu, 18 Feb 1999 16:45:16 -0500
Organization: University of Maryland Chesapeake Biological Laboratory
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Does anyone know if significant losses of proteins may occur in quartz
containers when the proteins are incubated in them for about one month?

For glass containers, the losses are apparently 1 ug per 5 cm^2.

Reno Nguyen

*************************************************************************
Reno T. Nguyen
Chesapeake Biological Laboratory                 /----\  /-----\   /
Univ. MD Center for Environmental Science      /        /      /  /  
P.O. Box 38                                  /         /______/  /
Solomons, MD 20688                         /          /       \ /
                                          /          /        // 
Tel: (410) 326-7261                      \_______/   \_______/ \_______/ 
     (410) 326-7409                    
Fax: (410) 326-7341
E-mail: reno@cbl.umces.edu
************************************************************************



From owner-proteins@net.bio.net Fri Feb 19 22:00:00 1999
Path: biosci!loria.fr!Gregory.Kucherov
From: Gregory.Kucherov@loria.fr (Gregory Kucherov)
Newsgroups: bionet.molbio.proteins
Subject: RECOMB'99: Program and Call for Participation
Date: 20 Feb 1999 16:32:43 -0800
Organization: BIOSCI International Newsgroups for Molecular Biology
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Distribution: world
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Reply-To: symposia@inria.fr
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This message contains a preliminary program of 

      The Third Annual Conference on Research in 
      Computational Molecular Biology (RECOMB 99)

that will take place in Lyon (France) on April 11-14, 1999. 
Those who would like to attend the Conference are invited to register
before 

                   March 12, 1999

You will find a registration form as well as further information about
the conference on the web site 

             http://www.inria.fr/RECOMB99/

Warning: Future participants are suggested to make their hotel
reservation as soon as possible. Please be aware that there are other
congresses held in Lyon at that time and the hotel availability might
be limited.

=================================
PRELIMINARY PROGRAM OF RECOMB'99
=================================

April 11-14,
Hotel de Lyon Metropole, Lyon, France

------------------------
Saturday, April 10, 1999
------------------------

16:00 - 19:00 - Welcome and registration at Lyon Metropole Hotel
18:00   Cocktail offered by the City Hall of Lyon

----------------------
Sunday, April 11, 1999
----------------------

8:45  Opening Remarks:
      Sorin Istrail, General RECOMB Vice-Chair
                     1999 Program Committee Chair
      Mireille Regnier, 1999 Conference Chair
      Ron Shamir, 2000 Program Committee Chair

Distinguished New Technologies Lecture - Chair: S. Istrail
----------------------------------------------------------

9:00  Large DNA Microarrays - the How and the Why
      Ed Southern

10:00 Break

Session on Gene Expression - Chair: S. Schbath
----------------------------------------------

10:15 Algorithms for Choosing Differential Gene Expression Experiments
      R. M. Karp, R. Stoughton, K. Y. Yeung

10:40 An Algorithm for Clustinering cDNAs for Gene Expression Analysis
      E. Hartuv, A. Schmitt, J. Lange, S. Meirer-Ewert, H. Lehrach, R. Shamir

11:05 A Dictionary Based Approach for Gene Annotation
      L. Pachter, S. Batzoglou, V. I. Spitkovsky, W. S. Beebee Jr.,
      E. S. Lander, B. Berger, D. J. Kleitman

11:30 Break

Session on Sequencing - Chair: R. Karp
--------------------------------------

11:45 De Novo Peptide Sequencing via Tandem Mass Spectrometry: A
      Graph-Theoretical Approach
      V. Dancik, T. Addona, K. Clauser, J. Vath, P. A. Pevzner

12:10 On the Power of Universal Bases in Sequencing by Hybridization
      F. P. Preparata, A. M. Frieze, E. Upfal

Lunch: 12:35 - 14:00
--------------------

Distinguished Biology Lecture - Chair: P. Pevzner
-------------------------------------------------

14:00 Whole Genome Association Studies in Humans
      Daniel Cohen

15:00 Break

Session on Protein Structure - Chair: M. Levitt
-----------------------------------------------

15:15 Recognition of Remote Protein Homologies Using Three-Dimensional
      Information to Generate a Position Specific Scoring Matrix in
      the Program 3D-PSSM
      L. Kelley, R. MacCullum, M. J. E. Sternberg

15:40 A Solvation Potential with Improved Contact Definitions and
      Optimized by Extensive Threading
      A. Dombkowski, G. M. Crippen

16:05 Fast Detection of Common Geometric Substructure in Proteins
      L. P. Chew, D. Huttenlocher, K. Kedem, J. Kleinberg

16:30 Break

Plenary Lecture Session - Chair: M. Levitt
------------------------------------------

16:45 The Past, Present and Future of Protein Structure Prediction
      John Moult

18:00 - 20:00  Business Meeting

----------------------
Monday, April 12, 1999
----------------------

Plenary Lecture Session - Chair: R. Karp
----------------------------------------

8:30  Computational Analysis of Molecular Diversity for Drug Discovery
      Peter Willett

9:30  Break

Session on Genomic Rearrangements - Chair: R. Shamir
----------------------------------------------------

9:45  Reconstructing the Pre-Doubling Genome
      N. El-Mabrouk, D. Bryant, D. Sankoff

10:10 Formulations and Hardness of Multiple Sorting by Reversals
      A. Caprara

10:35 Probability Models for Genome Rearrangement and Linear Invariants
      for Phylogenetic Inference
      D. Sankoff, M. Blanchette

11:00 Break

Session on Trees - Chair: A. Guenoche
-------------------------------------

11:15 Modeling Protein Families Using Probabilistic Suffix Trees
      G. Bejerano, G. Yona

11:40 Faster Reliable Phylogenetic Analysis
      V. Berry, D. Bryant

12:05 Obtaining Highly Accurate Topology Estimates of Evolutionary
      Trees from Very Short Sequences
      D. H. Huson, S. Nettles, T. J. Warnow

Lunch: 12:30 - 14:00
--------------------

Plenary Lecture Session - Chair: D. Slonim
------------------------------------------

14:00 Progress Toward the Whole-Genome Shotgun Sequencing of Drosophilia
      Gene Myers

15:00 Break

Session on Statistics - Chair: J. Kececioglu
--------------------------------------------

15:15 Searching Gene Transfers on Bacillus Subtilis Using Hidden Markov Models
      L. Bize, F. Muri, F. Samson, F. Rodolphe, S. D. Ehrlich,
      B. Prum, P. Bessieres

15:40 Significance Testing for Genomic Mismatch Scanning
      G. R. Grant, R. S. Spielman, E. Manduchi, V. G. Cheung, W. J. Ewens

16h05 Break

Session on Statistics - Chair: G. Kucherov
------------------------------------------

16:20 Classifying Proteins by Family Using the Product of Correlated p-values
      T. L. Bailey, W. N. Grundy

16:45 Sequence Homology Detection Through Large Scale Pattern Discovery
      A. Floratos, I. Rigoutsos, L. Parida, G. Stolovitzky, Y. Gao


17:30 - 19:30 Poster Session
----------------------------

-----------------------
Tuesday, April 13, 1999
-----------------------

Plenary Lecture Session - Chair: M. Regnier
-------------------------------------------

8:30  Genome Sequences and Protein Structures
      Cyrus Chothia

9:30  Break

Session on Mapping - Chair: R. Karp
-----------------------------------

9:45  Algorithms for Whole Genome Shotgun Sequencing
      E. Anson, G. Myers

10:10 Construction of Physical Maps from Oligonucleotide Fingerprints Data
      G. Mayraz, R. Shamir

10:35 Computing Physical Maps of Chromosomes with Nonoverlapping Probes
      by Branch-and-Cut
      T. Christof, J. Kececioglu

11:00 Break

Session on Threading - Chair: M. Vingron
----------------------------------------

11:15 A Method for Optimal Design of a Threading Scoring Function
      J. R. Bienkowska, R. G. Rogers Jr., T. F. Smith

11:40 An Anytime Algorithm for Gapped Block Protein Threading with
      Pair Interactions
      R. H. Lathrop

12:05 Efficient Algorithms for Protein Sequence Design and the Analysis of
      Certain Evolutionary Fitness Landscapes
      J. M. Kleinberg

12:30 - 14:00 Lunch
-------------------

Lecture Session - Chair: S. Istrail
-----------------------------------

14:00 Trends in Computational Biology
      John Wooley

15:00 Break

Session on Sequence Alignment - Chair: A. Apostolico
----------------------------------------------------

15:15 An Analytic Study of the Phase Transition Line in Local Sequence
      Alignment with Gaps
      R. Bundschuh, T. Hwa

15:40 Winnowing Sequences from a Database Search
      P. Berman, Z. Zhang, Y. I. Wolf, E. V. Koonin, W. Miller

16:05 q-gram Based Database Searching Using a Suffix Array (QUASAR)
      S. Burkhardt, A. Crauser, P. Ferragina, H-P. Lenhof, E. Rivals,
      M. Vingron


17:00 - 23:00 Evening program at Saint-Romain-en-Gal
----------------------------------------------------

17:00 Departure to Saint-Romain-en-Gal

18:00 Stanislaw Ulam - Computational Biology Address - Chair: M. Waterman
      Comparison of Complete Genomes: Organisation and Evolution
      Piotr Slonimski

19:00 Visit of the Museum

21:00 Cocktail

-------------------------
Wednesday, April 14, 1999
-------------------------

Plenary Lecture Session: Chair R. Shamir
----------------------------------------

9:00  Comparing Genes and Genomes: From Polymorphism to Phylogeny
      Peer Bork

10:00 Break

Session on Drug Design - Chair: R. Lathrop
------------------------------------------

10:15 Efficient Database Screening for Rational Drug Design Using
      Pharmacophore-Constrained Conformational Search
      S. M. LaValle, L. E. Kavraki, P. W. Finn, J-C. Latombe

10:40 Coupled Optimization in Protein Docking
      J. C. Mitchell, A. T. Phillips, J. B. Rosen, L. F. Ten Eyck

11:05 Derivation of Sensitive Discrimination Potential for Virtual
      Ligand Screening
      M. Totrov, R. Abagyan

11:30 Break

Session on Molecular Structure - Chair: J.M. Claverie
-----------------------------------------------------

11:45 Optimizing Combinatorial Library Construction via Split Synthesis
      B. Cohen, S. Skiena

12:10 Internal Loops in RNA Secondary Structure Prediction
      R. B. Lyngso, M. Zuker, C. N. S. Pedersen

12:35 - 14:00 Lunch

Session on Gene Networks - Chair: T. Lengauer
---------------------------------------------

14:00 Identifying Gene Regulatory Networks from Experimental Data
      T. Chen, V. Filkov, S. S. Skiena

14:25 Evolution of Metabolisms: A New Method for the Comparison of
      Metabolic Pathways
      C. V. Forst, K. Schulten

14:50 Clustering Gene Expression Patterns
      A. Ben-Dor, Z. Yakhini

15:15 Closing session

15:30 End of the conference


From owner-proteins@net.bio.net Fri Feb 19 22:00:00 1999
Path: biosci!agate!newsfeed.berkeley.edu!newsfeed.enteract.com!news.he.net!news.louisville.edu!uky.edu!news-feed-1.peachnet.edu!lendl.cc.emory.edu!not-for-mail
From: One White Tiger <1whitetiger@geocities.com>
Newsgroups: bionet.molbio.proteins
Subject: Loss of 32P-label in 2D analysis
Date: Sat, 20 Feb 1999 14:27:24 -0500
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I am labeling yeast mitochondria with [gamma-32P] ATP. Incorporation of
32P into proteins is approximately 10% of radioactivity retained by
mitochondria after washing.
(2,000 - 20,000 dpm/200 ug mitochondrial protein, depending on length of
incubation and specific activity of radiolabeled ATP used). After
SDS-PAGE, the signal is strong enough to see bands on x-ray film after
exposure of several hours to overnight, and the dried gel  generally
gives 1,000 -10,000 dpm by geiger counter (minimum 4 radiolabeled
samples/ gel).

However, I am losing nearly all of the radiolabel after the IEF step of
a 2-dimensional gel electrophoresis. I lose 90% of the signal in the IEF
step -- reasonable since only 10% of the label is in proteins, but after
SDS-PAGE, I am retaining less than 10% of the remaining radiolabel
(i.e., the unequilibrated IEF tube gel exhibits  200 - 2,000 dpm by
geiger counter, but the subsequent SDS-PAGE gel and blot onto
nitrocellulose elicits no signal above background).  Even using a
phosphoimager screen for several days, I cannot detect radioactive
protein spots on the blot.

I'm trying to see changes in phosporylation by autoradiography and/or
phosphoimaging.
The method of 2D analysis I'm using is a close derivative of the method
orignally published by O'Farrell. There's no significant difference in
the SDS-PAGE methods I'm using for 1D and 2D, except for the IEF noodle
and the agarose used to seal it.

Any ideas as to how I'm losing my 32P label, and more importantly, how I
can retain it?

Brett Burkholder, PhD Candidate
Emory University
Department of Genetics
440 Dental Bldg.
1462 Clifton Rd.
Atlanta, GA 30322




From owner-proteins@net.bio.net Sat Feb 20 22:00:00 1999
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From: pxpst2@unixs.cis.pitt.edu (Peter)
Newsgroups: bionet.molbio.proteins
Subject: Re: adsorption of proteins to quartz containers?
Date: Sun, 21 Feb 1999 19:06:01 -0500
Organization: University Of Pittsburgh
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In article <Pine.GSO.4.05.9902181641450.9607-100000@cbl.umces.edu>, "Reno
T. Nguyen" <reno@cbl.umces.edu> wrote:

> Does anyone know if significant losses of proteins may occur in quartz
> containers when the proteins are incubated in them for about one month?
> 
> For glass containers, the losses are apparently 1 ug per 5 cm^2.

I would imagine that the loss on quartz will also be in that ballbark.  In
order to block protein bind ,you must cap the Silol groups.

Peter

-- 
Peter



" Don't you eat that yellow snow
     Watch out where the huskies go"
                                    FZ

From owner-proteins@net.bio.net Sun Feb 21 22:00:00 1999
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From: "Yong_Wan Kim" <s291040@ccs.sogang.ac.kr>
Newsgroups: bionet.molbio.proteins
Subject: secondary structure of protein?
Date: Tue, 23 Feb 1999 12:07:42 +0900
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Hi,
With Fourier self deconvolution and guassina curve fitting,
 I could quantify the secondary
structure in aqueous solution using liquid-ATR. And frankly
this is the first time I handled some protein in my project.
So I would like to know more about secondary structure of proteins,
as you know, alpha helix, beta structure, turns and random coil.
Is there any book that described more specific about it?

Thank you very much for your attention.

kim
s291040@ccs.sogang.ac.kr



From owner-proteins@net.bio.net Sun Feb 21 22:00:00 1999
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From: klenchin@REMOVE_TO_REPLY.facstaff.wisc.edu (Dima Klenchin)
Newsgroups: bionet.molbio.proteins
Subject: Re: antibody purification
Date: Tue, 23 Feb 1999 01:26:01 GMT
Organization: UW
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References: <Pine.OSF.3.95.990218133221.18994A-100000@helix.mgh.harvard.edu> <7aiop6$hnc$1@news.doit.wisc.edu> <36CD2C14.9B5C4AC5@rz.uni-potsdam.de>
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In article <36CD2C14.9B5C4AC5@rz.uni-potsdam.de>, "Frank Fürst" <ffrank@rz.uni-potsdam.de> wrote:

>Hi,
>in a private response on my posting yesterday ("little experience with
> centricon"...) I got the following tip:
>
>> Use 5% of propylene glycol (pg) and place on centricon membrane for a min.
>> of 2 hours.  This will prevent proteins from sticking to membranes.  Then
>> rinse several times with pure water to remove the pg. Place protein sample
>> in for concentration.  I've use this procedure several times with great
>> result in purifying proteins in the 21 KDa range.
>>

Are you going to try it? If you do and it does provide better recovery
than control (within the same experiment, of course), could you
please post a message indicating so? Other than that, I see no reason for
this to work and am very sceptical about it. 

>--------------31DA0AA59517FA0E97D2F3CC
>Content-Type: text/html; charset=us-ascii
>Content-Transfer-Encoding: 7bit
>
><!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0 Transitional//EN">
><HTML>

Could you please not post HTML messages on Usenet? It's basically 
against Usenet netiquette and serves no useful purpose except promoting 
Netscape/Microsoft products. It's an option that _can_ (and should!)
be disabled. 

Thank you.

Dima

From owner-proteins@net.bio.net Mon Feb 22 22:00:00 1999
Path: biosci!news.stanford.edu!newsfeed.berkeley.edu!awabi.library.ucla.edu!164.67.43.25!news.ucla.edu!not-for-mail
From: "Kevin Klapstein" <kklap@biomath.medsch.ucla.edu>
Newsgroups: bionet.molbio.proteins
Subject: Anyone knowledgeable about RecA?
Date: 23 Feb 1999 23:51:44 GMT
Organization: University of California, Los Angeles
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Hello;

I am a graduate student doing some work on RecA strand exchange
reactions.  Unfortunately, my original background is not in biology
(it's in physics) so there are rather a lot of questions I have which
may be trivial background knowledge to somebody with a biology
background.

I would greatly appreciate any help on some of these questions.
My email address is

kklap@biomath.medsch.ucla.edu

Two of  my main questions are:

1)	For a strand exchange reaction in which one of the substrates had a
heterologous insert is completed, is the heterologous region incorporated
into the final product as a loop (a D loop?) of ssDNA?  
2)	During recombination events in prokaryotes where foreign DNA is
incorporated into the DNA of the organism, I assume that in the scenario
described above, the backbone of the DNA strand without the heterologous
insert would eventually be broken, and the heterologous region used as a
template for synthesis.  This would lead to lengthening of the DNA over
time, with many recombination events.  What are the biological implications
of this? (I know of several physical implications).

Cheers,

Kevin




From owner-proteins@net.bio.net Mon Feb 22 22:00:00 1999
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From: "Crypton" <crypton@videotron.ca>
Newsgroups: bionet.molbio.proteins
Subject: Need Help
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Hi,
 i need help. I'm trying a purification of RNase Us from human urine. But my
enzymatic essay don't work. Do someone know essay for the determination of
unity of Rnase ?

Thanx in advance
crypton@videotron.ca




From owner-proteins@net.bio.net Mon Feb 22 22:00:00 1999
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From: Lisa Kueltzo <freckles@eagle.cc.ukans.edu>
Newsgroups: bionet.molbio.proteins
Subject: Re: secondary structure of protein?
Date: Tue, 23 Feb 1999 11:22:44 -0600
Organization: University of Kansas Computing Services
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A good paper on Fourier self-deconvolution of proteins is
one by Susi and Byler in MEthods of Enzymology, Vol 130.  Also, for basic
secondary structure, you can look in any basic biochemistry text.  Another
nice discussion may be found in Structure in Protein Chemistry, by Kyte.

Good luck,
Lisa

********************************************************
Lisa Kueltzo
Department of Pharmaceutical Chemistry
University of Kansas
2095 Constant Avenue
Lawrence, KS 66047
USA
Phone: (785) 864-3010
Fax: (785) 864-5814
********************************************************


From owner-proteins@net.bio.net Mon Feb 22 22:00:00 1999
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From: Lisa Kueltzo <freckles@eagle.cc.ukans.edu>
Newsgroups: bionet.molbio.proteins
Subject: Re: adsorption of proteins to quartz containers?
Date: Tue, 23 Feb 1999 11:18:10 -0600
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It would depend on the protein, but you can see significant adsorption to
quartz cuvettes (enough to change the spectroscopic signal) at low protein
concentrations (~10 ug/mL).

Hope this helps,
Lisa

********************************************************
Lisa Kueltzo
Department of Pharmaceutical Chemistry
University of Kansas
2095 Constant Avenue
Lawrence, KS 66047
USA
Phone: (785) 864-3010
Fax: (785) 864-5814
********************************************************


From owner-proteins@net.bio.net Mon Feb 22 22:00:00 1999
From: Cornelius Krasel <krasel@wpxx02.toxi.uni-wuerzburg.de>
Subject: Re: secondary structure of protein?
Newsgroups: bionet.molbio.proteins
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Yong_Wan Kim <s291040@ccs.sogang.ac.kr> wrote:
> So I would like