From owner-proteins@net.bio.net Fri Oct 01 02:12:00 1999
Path: biosci!ibc.unibe.ch!chaitanya.athale
From: chaitanya.athale@ibc.unibe.ch (Chaitanya Athale)
Newsgroups: bionet.molbio.proteins
Subject: Looking for an inhibitor for a NADH-dependent protein
Date: 1 Oct 1999 03:12:35 -0700
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Hi everybody. I have a problem: I am trying to set up a typical NADH coupled assay to measure Pyrvate formation (using LDHase). One of the proteins of my system is a transmembrane protein, and I have to use it as a membrane extract. The problem is that in the membrane there seems to exist a second protein which interferes with the coupled assay, because it consumes NADH by a different and unknown pathway (I have not found yet the substrate being reduced). The solution would be very simple if I can find any general irreversible inhibitor for proteins which use NADH.
Does anybody know any such inhibitor??
Thank you very much for any information.
--Boundary_(ID_aFjp0A6VPEaopwbyV6r8mA)
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Hi everybody. I have a problem: I am trying to set up
a typical NADH coupled assay to measure Pyrvate formation (using LDHase). One of
the proteins of my system is a transmembrane protein, and I have to use it as a
membrane extract. The problem is that in the membrane there seems to exist a
second protein which interferes with the coupled assay, because it consumes NADH
by a different and unknown pathway (I have not found yet the substrate being
reduced). The solution would be very simple if I can find any general
irreversible inhibitor for proteins which use NADH.
Does anybody know any such
inhibitor??
Thank you very much for any
information.
--Boundary_(ID_aFjp0A6VPEaopwbyV6r8mA)--
From owner-proteins@net.bio.net Fri Oct 01 02:31:00 1999
Path: biosci!cnn.nas.nasa.gov!eecs-usenet-02.mit.edu!newsswitch.lcs.mit.edu!sunqbc.risq.qc.ca!bignews.mediaways.net!newsfeed.icl.net!peer.news.th.u-net.net!u-net!knews.uk0.vbc.net!vbcnet-gb!news.uk0.vbc.net!not-for-mail
From: "SelectScience"
Newsgroups: bionet.molbio.proteins
Subject: Rate lab equipment online
Date: Fri, 1 Oct 1999 11:13:06 -0000
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From owner-proteins@net.bio.net Fri Oct 01 09:51:00 1999
Path: biosci!newshost.lanl.gov!logbridge.uoregon.edu!newsgate.duke.edu!usenet
From: tschantz@duke.edu (Tschantz)
Newsgroups: bionet.molbio.proteins,bionet.cellbiol
Subject: FlavoProteins
Date: 1 Oct 1999 17:44:10 GMT
Organization: Duke University
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Hi
I am looking for examples of flavin proteins that do not require NADPH
OR NADH as a cofactor.
Thanks
Bill
From owner-proteins@net.bio.net Fri Oct 01 10:23:00 1999
Path: biosci!rutgers!news.sgi.com!nntp.primenet.com!nntp.gctr.net!newsfeed.icl.net!colt.net!Pollux.Teleglobe.net!server-b.cs.interbusiness.it!news.tin.it!not-for-mail
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Newsgroups: bionet.molbio.proteins
Subject: Download Ia.n.i.!!! It's free!
Date: 30 Sep 1999 19:57:44 GMT
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From owner-proteins@net.bio.net Sun Oct 03 17:32:00 1999
Path: biosci!ELBIT.CO.IL!SecurityFor_U
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From owner-proteins@net.bio.net Sun Oct 03 19:33:00 1999
Path: biosci!bloom-beacon.mit.edu!howland.erols.net!peer.news.verio.net.MISMATCH!iad-peer.news.verio.net!news.verio.net!ord-feed.news.verio.net!feed.news.verio.net!cletus.bright.net!none444.yet
From: no.email.address.entered@none444.yet
Newsgroups: bionet.molbio.proteins
Subject: FREE LEAN CHILI RECIPES_ FREE!!!
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From owner-proteins@net.bio.net Sun Oct 03 21:30:00 1999
Path: biosci!THON.CSB.KI.SE!say24
From: say24@THON.CSB.KI.SE
Newsgroups: bionet.molbio.proteins
Subject: Premium CABLE TV.......No Extra Charge!
Date: 3 Oct 1999 22:30:19 -0700
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From owner-proteins@net.bio.net Mon Oct 04 07:52:00 1999
Path: biosci!newshost.lanl.gov!logbridge.uoregon.edu!howland.erols.net!vixen.cso.uiuc.edu!not-for-mail
From: John Stone
Newsgroups: bionet.molbio.proteins
Subject: Announce: VMD 1.4 beta 1
Date: Mon, 04 Oct 1999 10:45:21 -0500
Organization: Theoretical Biophysics, Beckman Institute, UIUC
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VMD "Visual Molecular Dynamics" 1.4 beta 1 Announcement
-------------------------------------------------------
The Theoretical Biophysics group at the Beckman Institute For Advanced
Science and Technology, the University of Illinois (U-C), is proud to
announce the release of VMD 1.4 beta 1. VMD is a package for the
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Beckman Institute http://www.ks.uiuc.edu/~johns/
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From owner-proteins@net.bio.net Mon Oct 04 11:21:00 1999
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From: lemaster.34@osu.edu (Jared Quentin LeMaster)
Newsgroups: bionet.molbio.proteins
Subject: Need help:insoluble to soluble
Date: 4 Oct 1999 19:15:35 GMT
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Hi:
I expressed foreign protein in E.coli to the level of about 40% total
protein after IPTG induction. But it is insoluble. Does anyone have any
experiences that could make it soluble without loss of function or
expressed as a soluble protein in E.coli? Pls send your suggestion to
wang.508@osu.edu. Thanks a lot.
From owner-proteins@net.bio.net Mon Oct 04 19:41:00 1999
Path: biosci!newshost.lanl.gov!logbridge.uoregon.edu!nntp.upenn.edu!dialin0224.upenn.edu!user
From: dalby@mail.med.upenn.edu (Pablo)
Newsgroups: bionet.molbio.proteins
Subject: Re: protein binding to immobilised ligand
Date: Mon, 04 Oct 1999 23:41:04 -0500
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Firstly lets assume that the protein concentration bound is less than the
immobilised ligand concentration due to its size (and 7mM of a protein is
aggregation hell).
Secondly lets assume a Kd for the free ligand bound to protein, of 1mM.
The same ligand immobilised, will have a Kd which is much higher since the
off-rate will be the same, but the on-rate will be lower (due to
immobilisation).
Kd = k(off)/k(on)
So lets assume the immobilised ligand now has a Kd of 7.3mM.
This means that we require an effective concentration of 7.3mM immobilised
ligand to bind half of the protein to the resin, but we can compete it off
the column with only >1mM free ligand.
As for why you need 100mM I can only guess. This may mean you have some
other modes of binding to the resin/gel, eg. aggregation on the resin once
bound. You then need higher concentration of ligand to disrupt the
immobilised complex. Another possibility is obviously degraded ligand
giving a lower effective concentration.
> I have a transmembrane protein which is hypothesised to bind a ligand which
> has been shown to bind to other (unrelated) transmembrane proteins. The
ligand
> is commercially available in an immobilised form, covalently attached to
> agarose @ 7.3umole per ml of packed gel.
>
> Now, am I being simplistic in assuming that to compete with immobilised
ligand
> bound to protein binding sites, the free ligand must be applied to the column
> @ > 7.3mM (i.e 7.3umole/ml gel = 7.3mM) ?
>
> My reason for asking is that, when reading through the literature, I came
> across a paper where the authors used this immobilised ligand, from the same
> suppliers, in a one-step purification method of an unrelated protein and
> (assuming they used the same amount of immobilised ligand / ml gel) were able
> to elute bound protein using 2mM free ligand, whereas I can only elute bound
> protein using 100mM free ligand.
>
> Any suggestions gratefully received,
>
> Rob Leach,
> School of Biochemistry & Molecular Biology,
> University of Leeds
--
brothers gonna work it out
From owner-proteins@net.bio.net Tue Oct 05 00:32:00 1999
Path: biosci!newshost.lanl.gov!logbridge.uoregon.edu!dispose.news.demon.net!demon!newsfeed.icl.net!bignews.mediaways.net!fu-berlin.de!pc207-60.biochem.uni-potsdam.DE!not-for-mail
From: Frank =?iso-8859-1?Q?F=FCrst?=
Newsgroups: bionet.molbio.proteins
Subject: Re: Need help:insoluble to soluble
Date: Tue, 05 Oct 1999 10:25:41 +0200
Message-ID: <7tccme$erg$1@fu-berlin.de>
References:
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Jared Quentin LeMaster schrieb:
>
> Hi:
> I expressed foreign protein in E.coli to the level of about 40% total
> protein after IPTG induction. But it is insoluble. Does anyone have any
> experiences that could make it soluble without loss of function or
> expressed as a soluble protein in E.coli? Pls send your suggestion to
> wang.508@osu.edu. Thanks a lot.
You can purify it from the insoluble inclusion bodies
Frank
--
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Im Biotechnologiepark, D-14943 Luckenwalde
Tel.: +49-3371-681334; Fax: +49-3371-681339
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From owner-proteins@net.bio.net Tue Oct 05 09:14:00 1999
Path: biosci!NETMEX.COM!romartin
From: romartin@NETMEX.COM ("romartin")
Newsgroups: bionet.molbio.proteins
Subject: Information
Date: 5 Oct 1999 10:14:06 -0700
Organization: BIOSCI International Newsgroups for Molecular Biology
Lines: 37
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Please send me all the information you can have about proteins.(history, =
evolution, function in the human body, an everything you can have)
My name is Francisco Noriega, from M=E9xico, City and this information =
is very important for me. Thanks
------=_NextPart_000_0004_01BF0F2A.8EE48FD0
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charset="iso-8859-1"
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Please send me all the information =
you can have=20
about proteins.(history, evolution, function in the human body, an =
everything=20
you can have)
My name is Francisco Noriega, from=20
México, City and this information is very important for me.=20
Thanks
------=_NextPart_000_0004_01BF0F2A.8EE48FD0--
From owner-proteins@net.bio.net Tue Oct 05 11:10:00 1999
Path: biosci!newsfeed.stanford.edu!logbridge.uoregon.edu!news.bu.edu!ppp1
From: mlamkin@bu.edu (Mark Lamkin)
Newsgroups: bionet.molbio.proteins
Subject: Stripping membranes
Date: Tue, 05 Oct 99 19:01:40 GMT
Organization: Boston University
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Does anyone out there have info on stripping nitrocellulose membranes to
reprobe with different antibodies? I recall that there was a procedure with
the chemilluminescent methods, but I forget the details.
Thanks.
From owner-proteins@net.bio.net Tue Oct 05 11:10:00 1999
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From: stcaris@aol.com (Stcaris)
Newsgroups: bionet.molbio.proteins
Subject: Re: Need help:insoluble to soluble
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I remember that somebody mentioned coexpression of chaperones.
===
Yours sincerely,
Chevalier Dr. Ruediger Marcus Flaig KHT, KSR
Email:
sanctacaris@bigfoot.com
sanctacaris@unforgettable.com
sanctacaris@rocketmail.com
sanctacaris@hotmail.com
stcaris@aol.com
sanctacaris@netscape.net
01727652946@d2-message.de (mobile - short mails only)
"Tell truth and shame the devil." (Shakespeare, Henry IV.)
From owner-proteins@net.bio.net Tue Oct 05 12:06:00 1999
From: Cornelius Krasel
Subject: Re: Information
Newsgroups: bionet.molbio.proteins
References: <01bf0f5c$d97effd0$09c801c8@SERVER.ROSMAR>
Distribution: world
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Date: Tue, 5 Oct 1999 21:13:45 +0200
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"romartin" wrote:
Please don't post multipart messages or html to newsgroups.
> Please send me all the information you can have about proteins.(history,
> evolution, function in the human body, an everything you can have)
I recommend visiting a good library. There are tons of books on proteins,
and without specifying more precisely what you are interested in it is
impossible to help you. I guess that there is a university in Mexico
City, so a good place to start is the university's library.
--Cornelius.
--
/* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */
/* D-97078 Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de SP4 */
/* "Science is the game we play with God to find out what His rules are." */
From owner-proteins@net.bio.net Tue Oct 05 14:22:00 1999
Path: biosci!proteome.org.au!anouwens
From: anouwens@proteome.org.au (Amanda Nouwens)
Newsgroups: bionet.molbio.proteins
Subject: Re: Stripping membranes
Date: 5 Oct 1999 15:22:44 -0700
Organization: BIOSCI International Newsgroups for Molecular Biology
Lines: 28
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Dear Mark,
>Does anyone out there have info on stripping nitrocellulose membranes to
>reprobe with different antibodies? I recall that there was a procedure with
>the chemilluminescent methods, but I forget the details.
>Thanks.
try this method:
wash blot 4 x with PBS (5 min/wash)
incubate for 30 min at 50 deg. C in:
62.5mM Tris-HCl pH 6.8
2% SDS
100mM DTT.
wash 6 x 5 min in PBS.
block as usual.
Cheers,
Amanda Nouwens.
From owner-proteins@net.bio.net Tue Oct 05 22:59:00 1999
Path: biosci!HANMAIL.NET!proteins
From: proteins@HANMAIL.NET ("Deepblue")
Newsgroups: bionet.molbio.proteins
Subject: help!! the advantage of using fluorescence anisotropy
Date: 5 Oct 1999 23:58:44 -0700
Organization: ºÎ»ê´ëÇб³
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Hi everyone!! I find a journal which use "fluorescence anisotropy". But i don't understand the meanings and advantages of useing it. please let me know it. Thanks your attentions.
==================================================
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From owner-proteins@net.bio.net Tue Oct 05 23:13:00 1999
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From: tob@ssi.dk
Newsgroups: bionet.molbio.proteins
Subject: Workstation for chromatography?
Date: 6 Oct 1999 08:05:39 +0100
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Hi netters
I need some information about workstations for chromatography. I know about Pharmacias Äkta system and PEbiosystems Biocad. What other alternatives are there on the market?
Tomas
---
From owner-proteins@net.bio.net Tue Oct 05 23:47:00 1999
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From: Frank =?iso-8859-1?Q?F=FCrst?=
Newsgroups: bionet.molbio.proteins
Subject: Re: Need help:insoluble to soluble
Date: Wed, 06 Oct 1999 09:39:04 +0200
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Stcaris schrieb:
>
> I remember that somebody mentioned coexpression of chaperones.
Yes, and some other things, too. Go to www.deja.com and look for
"inclusion bodies" in this group, this spring or summer
Frank
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From owner-proteins@net.bio.net Tue Oct 05 23:54:00 1999
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From: Frank =?iso-8859-1?Q?F=FCrst?=
Newsgroups: bionet.molbio.proteins
Subject: Re: Workstation for chromatography?
Date: Wed, 06 Oct 1999 09:47:07 +0200
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tob@ssi.dk wrote:
>
> Hi netters
>
> I need some information about workstations for chromatography. I know about Pharmacias Äkta system and > PEbiosystems Biocad. What other alternatives are there on the market?
Bio-Rad (www.bio-rad.com) also sell a system.
Frank
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From owner-proteins@net.bio.net Wed Oct 06 00:27:00 1999
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From: Frank =?iso-8859-1?Q?F=FCrst?=
Newsgroups: bionet.molbio.proteins
Subject: Re: help!! the advantage of using fluorescence anisotropy
Date: Wed, 06 Oct 1999 10:20:33 +0200
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Deepblue wrote:
>
> Hi everyone!! I find a journal which use "fluorescence anisotropy". But i don't understand the meanings and > advantages of useing it. please let me know it. Thanks your attentions.
You can detect binding of the fluorescent dye to a macromolecule even
if the fluorescence intensity doesn't change.
You excite the fluorophors with linear polarized light (vertically
relative to a horizontal optical bench). If the vector of the exciting
light is vertical, the emitted light is vertical too, but the
direction of maximal intensity is, of course, vertical to the
propagation direction of the exciting beam.
Then if the fluorophors would not move at all before emission, you
would only get vertically polarized light (you also put a polarizer
before the emission detector).
But the molecules do move, and thus also emit differently polarized
light. The faster they move relative to the average lifetime of the
excited state, the more will the distribution approach randomness.
The point is that small molecules move fast and bigger ones move
slower. Therefore a small fluorophor will display nearly random
distribution of polarization direction, and you will find that the
intensity with vertical emission polarizer is the same as that with
horizontal emission polarizer.
Now if the small fluorophor binds to a big macromolecule, it now only
moves together with that: It becomes slow. Thus the ratio of vertical
to horizontal emission intensity increases, and you can use this
phenomenon to measure binding constants.
The anisotropy itself is defined as (I= - Ip)/(I= + 2 * Ip) where I=
means parallel intensity = intensity with excitation and emission
polarizer vertical and Ip means perpendicular intensity = intensity
with excitation polarizer vertical and emission polarizer horizontal.
A high anisotropy means a slow movement relative to the fluorescence
lifetime, a low one a fast movement.
Often one can also see the quantity polarization, it is defined as (I=
- Ip)/(I= + Ip) and can be converted to anisotropy.
hope this helps,
Frank
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From owner-proteins@net.bio.net Wed Oct 06 13:36:00 1999
Path: biosci!newshost.lanl.gov!logbridge.uoregon.edu!newsfeed.cwix.com!beaker.tor.sfl.net!bunson.tor.sfl.net!not-for-mail
From: "Achim Recktenwald, PhD"
Newsgroups: bionet.molbio.proteins
References: <008601bf0bf5$1c8e8ae0$0b4b5c82@erni8.unibe.ch>
Subject: Re: Looking for an inhibitor for a NADH-dependent protein
Lines: 71
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If it doesn't inhibit your enzyme ), try 0.05 to 0.10% sodium azide.
But you have to test first its effect on your enzyme; some enzymes drop dead
immediately upon exposure to azide. Your membrane extract probably contains
the enzymes responsible for the oxidative phosphorylation.
Cheers,
Achim
Chaitanya Athale wrote in message
news:008601bf0bf5$1c8e8ae0$0b4b5c82@erni8.unibe.ch...
> This is a multi-part message in MIME format.
>
> --Boundary_(ID_aFjp0A6VPEaopwbyV6r8mA)
> Content-type: text/plain; charset=iso-8859-1
> Content-transfer-encoding: 7BIT
>
> Hi everybody. I have a problem: I am trying to set up a typical NADH
coupled assay to measure Pyrvate formation (using LDHase). One of the
proteins of my system is a transmembrane protein, and I have to use it as a
membrane extract. The problem is that in the membrane there seems to exist a
second protein which interferes with the coupled assay, because it consumes
NADH by a different and unknown pathway (I have not found yet the substrate
being reduced). The solution would be very simple if I can find any general
irreversible inhibitor for proteins which use NADH.
> Does anybody know any such inhibitor??
>
> Thank you very much for any information.
>
>
> --Boundary_(ID_aFjp0A6VPEaopwbyV6r8mA)
> Content-type: text/html; charset=iso-8859-1
> Content-transfer-encoding: 7BIT
>
>
>
>
>
>
>
>
>
> color=#000000>Hi everybody. I have a problem: I am trying to
set up
> a typical NADH coupled assay to measure Pyrvate formation (using LDHase).
One of
> the proteins of my system is a transmembrane protein, and I have to use it
as a
> membrane extract. The problem is that in the membrane there seems to exist
a
> second protein which interferes with the coupled assay, because it
consumes NADH
> by a different and unknown pathway (I have not found yet the substrate
being
> reduced). The solution would be very simple if I can find any general
> irreversible inhibitor for proteins which use NADH.
> color=#000000> Does anybody know any such
> inhibitor??
> color=#000000>
> color=#000000> Thank you very much for any
> information.
> color=#000000>
>
> --Boundary_(ID_aFjp0A6VPEaopwbyV6r8mA)--
From owner-proteins@net.bio.net Wed Oct 06 15:51:00 1999
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From: madQ
Newsgroups: bionet.molbio.proteins
Subject: Download Ia.n.i.!!! It's free!
Date: 3 Oct 1999 19:10:46 GMT
Organization: madQ
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Download Ia.n.i. RemoteControlSystem 1.2 beta. It's free!!!
New site: http://jump.to/IaniProject
From owner-proteins@net.bio.net Wed Oct 06 17:36:00 1999
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From: klenchin@REMOVE_TO_REPLY.facstaff.wisc.edu (Dima Klenchin)
Newsgroups: bionet.molbio.proteins
Subject: Re: Workstation for chromatography?
Date: Thu, 07 Oct 1999 01:27:46 GMT
Organization: UW
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In article <99Oct6.081409gmt+0100.19717@fsk.ssi.dk>, tob@ssi.dk wrote:
>Hi netters
>
>I need some information about workstations for chromatography. I know about
> Pharmacias Äkta system
Can't comment here. Sceptical a bit because it doe snot look like
it can be conveniently used in the cold room. Apart from this, Pharmacia
chtomatography systems are of superior quality (but very expensive!).
>and PEbiosystems Biocad.
We have one rather older Biocad. Unless they have modified the
machine completely, it is a disaster. Even if it's much cheaper, I
wouldn't recommend it.
>What other alternatives are
> there on the market?
Bio-Rad sells something along these lines, and Pharmacia, I think,
has not completely abandoned its "simple" FPLC line (I love it).
- Dima
From owner-proteins@net.bio.net Wed Oct 06 18:45:00 1999
Path: biosci!163.NET!pumcwenwang
From: pumcwenwang@163.NET
Newsgroups: bionet.molbio.proteins
Subject: help:how to dissolve keratinous layer of animal skin?
Date: 6 Oct 1999 19:45:53 -0700
Organization: BIOSCI International Newsgroups for Molecular Biology
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Dear Sir or Madam:
Who can tell me how to dissolve keratinous layer of animal skin? what kind of enzyme or chemical methods can be used?
Thanks a lot!
Wen Wang
________________________________________________
»¶ÓÄúʹÓÃ163µç×ÓÓʾÖÃâ·Ñ·þÎñ http://www.163.net
163³¬¼¶¿áÈ«¹ú×î´óµÄÃâ·Ñ¸öÈËÖ÷Ò³»ùµØhttp://cool.163.net
From owner-proteins@net.bio.net Thu Oct 07 01:37:00 1999
Path: biosci!BOOM.COM!dvds
From: dvds@BOOM.COM
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Subject: *DVD Liquidation Sale!!!
Date: 7 Oct 1999 02:37:18 -0700
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From owner-proteins@net.bio.net Thu Oct 07 06:36:00 1999
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From: parcej@biophys.mpg.de (Dr Dave Parcej)
Newsgroups: bionet.molbio.proteins
Subject: Re: Workstation for chromatography?
Date: Thu, 07 Oct 1999 15:32:41 +0100
Organization: MPI fuer Biophysik
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rs
> >
> >I need some information about workstations for chromatography. I know about
> > Pharmacias Äkta system
>
> Can't comment here. Sceptical a bit because it doe snot look like
> it can be conveniently used in the cold room. Apart from this, Pharmacia
> chtomatography systems are of superior quality (but very expensive!).
We use our Akta in the cold room without trouble. The only dodgy bit is
the associated PC, but you can even get cold-room comatible PCs now. The
Akta system is (for me) much better and more versatile than the FPLC we
have.
Dave
From owner-proteins@net.bio.net Thu Oct 07 17:09:00 1999
Path: biosci!PILOT.MSU.EDU!venkata1
From: venkata1@PILOT.MSU.EDU ("Sridhar Venkataraman")
Newsgroups: bionet.molbio.proteins
Subject: affigel 15 and peptide binding protein elution.
Date: 7 Oct 1999 18:09:38 -0700
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Dear Netters,
We are interested in purifying a protein (from detergent extracted microsome
preps) which binds a particular peptide.
since the pka of the peptide was in the 3-4 range, chose affigel 15 and couple
the peptide in a HEPES buffer and after washing quenched the reaction with a
tris buffer.
I can succesfully elute about 10 bands of protein with salt (150 mM).
In order to determine the specificity I perpared a 6 mM solution of the peptide
in binding buffer (MES 100 mM 1 % CHAPS detergent) after binding the protein
and washing the column I added 2 ml of the elution buffer (6 mM peptide in
wash buffer) and the column (1 ml) swelled up to 5 ml and would not settle.
By means of a syringe I drew out the elution wash the column with wash buffer
and re-eluted with 150 mM salt. When I analyse the samples by SDS page.
The peptide elutions have a large percipitate in the presence of Laemmli
sample buffer) all the other fractions do not. Amd when the gel was stained
with silver I could not see any protein. well there are faint smears here and
there but nothing sharp and crisp.
anyone can help me?
the matrix is "clogged" all operations now have to be performed by syringe.
gravity does not work anymore.
please reply to
sridhar
venkata1@pilot.msu.edu
thanks a million
--
Home and lab address:
Sridhar Venkataraman phone : 24 hours : 517-353-3519 (work)
DOE-Plant Research Laboratory godly hours : 517-355-1266 (home)
122 Plant Biology Building
Wilson Road,
Michigan State University fax : 517-353-9168
East Lansing , 48824-1312 e-mail : venkata1@pilot.msu.edu
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section in a swimming pool!
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NewsWeek March 22,1999.
From owner-proteins@net.bio.net Fri Oct 08 02:39:00 1999
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From: Frank =?iso-8859-1?Q?F=FCrst?=
Newsgroups: bionet.molbio.proteins
Subject: Re: Workstation for chromatography?
Date: Fri, 08 Oct 1999 12:32:32 +0200
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Dima Klenchin wrote:
> Bio-Rad sells something along these lines, and Pharmacia, I think,
> has not completely abandoned its "simple" FPLC line (I love it).
We have a Pharmacia Aekta and a BioRad Workstation. The Pharmacia
software is much more flexible and you can configure a lot, display
what and how you like, and so on. On the other hand, programming new
methods is more difficult and needs some training, whereas the BioRad
Method Editor is self-explaining: What is possible can be done without
much training.
Thus, if you want it for scientists doing sophisticated things, and
technical assistants or students just repeating it, I'd recommend
Pharmacia. But if you want a system that students or technical
assistants can understand and develop new methods on it, the simple
Biorad user interface might be better.
Not regarding that the Pharmacia systems are much more expensive, I
think...
Bye, Frank
--
Hi! I'm Norton Antivirus. Replace your signature with this text for
protection against Signature Virus 99 and many others.
From owner-proteins@net.bio.net Fri Oct 08 08:28:00 1999
Path: biosci!NBRF.GEORGETOWN.EDU!pirmail
From: pirmail@NBRF.GEORGETOWN.EDU (pirmail)
Newsgroups: bionet.molbio.proteins
Subject: Protein Information Resource Release 62.00
Date: 8 Oct 1999 09:28:19 -0700
Organization: National Biomedical Research Foundation
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Dear Colleagues:
The latest release of the PIR-International Protein Sequence Database is now available by FTP.
Release 62.00, Sept. 30, 1999, Containing: 142,080 entries
To download this or other protein databases visit one of our FTP
servers.
ftp://nbrf.georgetown.edu/pir/
ftp://nbrfa.georgetown.edu/pir/
To search the PIR databases online visit our Web site.
http://pir.georgetown.edu/
Protein Information Resource
National Biomedical Research Foundation
3900 Reservoir Rd., NW
Washington, DC 20007
Phone: (202) 687-2121
E-mail: pirmail@nbrf.georgetown.edu
From owner-proteins@net.bio.net Fri Oct 08 20:14:00 1999
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From: rtmlmc@rec.uk
Subject: Important issue ! 8654 [2/2]
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From owner-proteins@net.bio.net Sat Oct 09 21:51:00 1999
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From owner-proteins@net.bio.net Sun Oct 10 20:09:00 1999
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From: madQ
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Download Ia.n.i. RemoteControlSystem 1.2 beta. It's free!!!
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From owner-proteins@net.bio.net Sun Oct 10 23:02:00 1999
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From: tob@ssi.dk
Newsgroups: bionet.molbio.proteins
Subject: Re: Workstation for chromatography?
Date: 11 Oct 1999 07:55:14 +0100
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I am interested in getting a system that can replace much of the dull work as sample injection, buffer mixing, column switching and so on. One advantage, as I understand it, with the Äkta system compared to the Biorad is that Äkta has automatic buffer mixing which is preferential when working with optimization of pH and ion strength gradients. This is also true for the PE biosystems Biocad 700E. Any other experiences?? I have not heard so much from people working with the Biocad700E or Biovision systems.
Sincerely
Tomas Bratt
---
From owner-proteins@net.bio.net Mon Oct 11 08:47:00 1999
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From: hooverd@ncifcrf.gov ("Dr. David Hoover")
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========================================
Dr. David Hoover, Postdoctoral Fellow
NCI-FCRDC, Macromolecular Structure Laboratory
Bldg. 539, Ft. Detrick MD 21702
PH: 301-846-5326, FAX: 301-846-7101
From owner-proteins@net.bio.net Mon Oct 11 20:32:00 1999
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From: "PAUL MACLEAN"
Newsgroups: bionet.molbio.proteins
Subject: phosphopeptide pI and Mr calculation
Date: Tue, 12 Oct 1999 00:28:28 -0400
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Does anyone know an internet resource that will calculate pI and Mr of
phosphorylated peptides?
If not, does anyone know the relative pKa's of the two hydrates oxygen
molecules of an attached phosphate? I can calculate the pI and Mr by hand
if needs be, but I need this information to do so.
Can anyone point me in the right direction? I've checked Pedro's
biomolecular tools. Most programs I have found do not handle
posttranslationally altered amino acids in their pI calculation.
Thank you,
Paul MacLean
From owner-proteins@net.bio.net Mon Oct 11 23:42:00 1999
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From: Têtu Jean François
Newsgroups: bionet.molbio.proteins
Subject: southwestern
Date: Tue, 12 Oct 1999 09:40:17 +0200
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Hi netters,
Does anybody know a protocol to make southwestern ? Indeed, i want to
make a nucleic acid hybridization on protein which have been
transfered on nitrocellulose menbrane.
Thanks a lot.
From owner-proteins@net.bio.net Tue Oct 12 00:47:00 1999
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From: "Evangelos Christodoulou"
Newsgroups: bionet.molbio.proteins
Subject: Re: phosphopeptide pI and Mr calculation
Date: Tue, 12 Oct 1999 11:40:55 +0300
Organization: University of Athens
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In article <7tud7i$1luk$1@newssvr04-int.news.prodigy.com> , "PAUL MACLEAN"
wrote:
> Does anyone know an internet resource that will calculate pI and Mr of
> phosphorylated peptides?
>
> If not, does anyone know the relative pKa's of the two hydrates oxygen
> molecules of an attached phosphate? I can calculate the pI and Mr by hand
> if needs be, but I need this information to do so.
>
> Can anyone point me in the right direction? I've checked Pedro's
> biomolecular tools. Most programs I have found do not handle
> posttranslationally altered amino acids in their pI calculation.
>
> Thank you,
> Paul MacLean
>
>
For the pI:
http://www.embl-heidelberg.de/cgi/pi-wrapper.pl
For the pKa:
http://www.embl-heidelberg.de/ExternalInfo/wade/pub/soft/pka.html
Hope this helps.
E.
--
Evangelos Christodoulou
University of Athens, Greece
If Che Guevara was alive he'd use a Mac
-the only revolutionary-friendly platform
From owner-proteins@net.bio.net Tue Oct 12 11:08:00 1999
Path: biosci!NATURE.BERKELEY.EDU!vhandley
From: vhandley@NATURE.BERKELEY.EDU (vanessa handley)
Newsgroups: bionet.molbio.proteins
Subject: expression screen
Date: 12 Oct 1999 12:08:01 -0700
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Hello All. I am looking for advice regarding expression
screening/interactive cloning. I am currently screening a plant
ZapExpress library with a 32P-labeled protein probe. I have been
following a hybridization protocol derived from Current Protocols in
Mol. Bio. which is as follows:
- after lifting, filters are washed for 15 min. at RT in TBS-tween
- filters are then blocked for 4 hrs. at 4 degrees in buffer A (20mM
HEPES, 5mM MgCl2, 1mM KCl) with 5% milk powder
- filters are then hybridized overnight at 4 degrees in approx. 300,000
cpm protein probe/ml of buffer B (20mM HEPES, 7.5mM KCl, 0.1mM EDTA,
2.5mM MgCl2) with 1% milk powder (approx. 40ml for 10x137mm filters)
- before exposing to film filters are washed 3x for 30 min.in 100ml
buffer B.
I am hoping that someone who has successfully performed such a screen
can tell me: 1) should I expect high "background" levels - or, asked
another way, how hot are the filters when you expose to film? My
filters are generally very hot (10-15K on the Geiger counter) and thus
have a high background on film. This background is not noticibly
reduced with different blocks (BSA, 1% milk powder) or overnight blocks
or with increased wash times/volumes.
I am concerned that I may not detect positives over this high
background. Which brings me to my next questions: 2) any suggestions on
reducing this background? and 3) in the primary screen, what should I be
looking for in terms of a putative positive (i.e. intensity, size). I
am used to library screening with a DNA probe . . . will the positives
look the same?
Thanks in advance for any suggestions you can offer!
Vanessa
From owner-proteins@net.bio.net Wed Oct 13 01:00:00 1999
Path: biosci!internet!biosci!not-for-mail
From: biohelp (BIOSCI Administrator)
Newsgroups: bionet.molbio.proteins
Subject: BIOSCI/bionet miniFAQ & Fundraiser
Date: 13 Oct 1999 02:00:20 -0700
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(LAST REVISION: 14-AUG-99)
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
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If you can not find an answer to your question in this or other
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We can only answer questions about the use of the newsgroups and
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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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1) Using the WWW to access the BIOSCI/bionet newsgroups.
--------------------------------------------------------
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
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-------------------------------------------------------
BIOSCI is a set of parallel USENET newsgroups (the "bionet" groups),
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The same postings are distributed on all media (except for a small
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is becoming a despicable practice on the Internet (by a few people out
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are refered to as "spams" in the usual, somewhat boneheaded, net
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What should you do personally if you get junk mail?
---------------------------------------------------
Just delete it and move on without reading it further. Filing a
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What can BIOSCI/bionet do to protect its newsgroups?
----------------------------------------------------
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Please do not assume that by simply posting a complaint to the
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From owner-proteins@net.bio.net Wed Oct 13 19:27:00 1999
Path: biosci!163.NET!nshh
From: nshh@163.NET
Newsgroups: bionet.molbio.proteins
Subject: ???There are small spot on the western blot membrane?
Date: 13 Oct 1999 20:27:15 -0700
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Hi everyone,
When i do western blot using PVDF membranes, there are a lot of small spot
on the western blot membrane, almost all the part that transferred by gel.
Who can tell me what had happened? It will help me a lot.
NSHH
________________________________________________
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163³¬¼¶¿áÈ«¹ú×î´óµÄÃâ·Ñ¸öÈËÖ÷Ò³»ùµØhttp://cool.163.net
From owner-proteins@net.bio.net Thu Oct 14 04:04:00 1999
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From: mmerckel@abo.fi (Michael Merckel)
Newsgroups: bionet.molbio.proteins
Subject: Prot seqs sorted by aa type ?
Date: 14 Oct 1999 12:56:57 +0100
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Hi,
Is anyone aware of a server or source of protein sequences
sorted by particular amino acid types ? For example, if I
were interested in the protein sequences with the
greatest number of trp residues, is there a quicker
way than scripting or coding this for myself ?
Thanks for any pointers.
Mike
---
From owner-proteins@net.bio.net Thu Oct 14 05:26:00 1999
Path: biosci!newsfeed.stanford.edu!nntp.cs.ubc.ca!newsflash.concordia.ca!pitt.edu!not-for-mail
From: pxpst2@vms.cis.pitt.edu (Peter)
Newsgroups: bionet.molbio.proteins
Subject: Re: ???There are small spot on the western blot membrane?
Date: Thu, 14 Oct 1999 09:16:35 -0400
Organization: University Of Pittsburgh
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In article <19991014111315.2578.fmail@163.net>, nshh@163.net wrote:
>
> When i do western blot using PVDF membranes, there are a lot of small spot
>
> on the western blot membrane, almost all the part that transferred by gel.
>
> Who can tell me what had happened? It will help me a lot.
(A) Are the spots seen right after it has come out of the blotting apparatus?
Or
(B) Are the spots seen after the ECL on the film?
If (a) then sounds like you had air bubbles between membrane and gel
If (b) sounds like you have too much ECL reagent left on the blot and the
blot is not sufficiently washed
(a) can be confirmed by looking at the evenness of your transfer with a
reversible stain like Ponceau Red.
Regards
Peter Pediaditakis
--
Peter
_____________________________________________________________________
" Some of you might not agree
'Cause you probably likes a lot of misery
But think a while and you will see...
Broken hearts are for assholes"
FZ
From owner-proteins@net.bio.net Thu Oct 14 09:05:00 1999
Path: biosci!newsfeed.stanford.edu!newsfeed.berkeley.edu!newsfeed.tli.de!news-fra.pop.de!informatik.uni-bremen.de!cs.tu-berlin.de!uni-duisburg.de!news.uni-essen.de!not-for-mail
From: Alexander Schramm
Newsgroups: bionet.molbio.proteins
Subject: labile proteins
Date: Thu, 14 Oct 1999 11:25:39 +0200
Organization: University Essen, Germany
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We are working with extrinsic factors to stabilize proteins, and we are
interested in labile proteins which are worth to test them whether they
can be stabilized. I am looking forward to read from you! Bye Stefan
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From owner-proteins@net.bio.net Thu Oct 14 09:05:00 1999
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From: Stefan Wolff
Newsgroups: bionet.molbio.proteins
Subject: labile proteins?
Date: Thu, 14 Oct 1999 11:44:53 +0200
Organization: University Essen, Germany
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We are working with extrinsic factors to stabilize proteins, and we are
interested in labile proteins which are worth to test them whether they
can be stabilized. With kind regards, Stefan
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From owner-proteins@net.bio.net Thu Oct 14 14:04:00 1999
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From: madQ
Newsgroups: bionet.molbio.proteins
Subject: Download Ia.n.i.!!! It's free!
Date: 13 Oct 1999 21:48:01 GMT
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Download Ia.n.i. RemoteControlSystem 1.2 beta. It's free!!!
New site: http://www.geocities.com/SiliconValley/Chip/5989/
From owner-proteins@net.bio.net Thu Oct 14 15:32:00 1999
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From: rsaldanh@mail.utexas.edu (Roland Saldanha)
Newsgroups: bionet.molbio.proteins
Subject: Re: labile proteins
Date: 14 Oct 1999 23:22:16 GMT
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I do not know what form of lability you are looking for.
We work on an Intron encoded protein that is a reverse
transcriptase/endonuclease splicing factor. The free protein is heat
labile (37C has a half life of a few minutes, about 30 min at 25 C). It
is stabilized by binding the intron and by glycerol. We would be
interested in small molecules that might stabilize the free protein
because we can use this for structural studies. If this is the soft of
"lability" you are looking for we may be able to pursue this futher.
What is the nature of your additives?
Roland
From owner-proteins@net.bio.net Sat Oct 16 21:54:00 1999
Path: biosci!RECYCLERMAIL.COM!NetMarketing
From: NetMarketing@RECYCLERMAIL.COM
Newsgroups: bionet.molbio.proteins
Subject: Here's Your Marketing Kit
Date: 16 Oct 1999 22:54:38 -0700
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Message-ID: <199910170547.GAA21858@silver.meditnet.com>
Reply-To: removeits@bigfoot.com
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From owner-proteins@net.bio.net Mon Oct 18 04:01:00 1999
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From: "SelectScience"
Newsgroups: bionet.molbio.proteins
Subject: Last week's Chemistry & Life Sciences News Headlines
Date: Mon, 18 Oct 1999 12:42:17 -0000
Organization: SelectScience
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Last week, www.selectscience.net reported on 17 news stories the day they
happened, so if you need to keep up to speed on the latest business
developments in chemistry and life science, make www.selectscience.net part
of your daily routine.
Andrew Smith (andrew@selectscience.net)
Editor, www.selectscience.net
Last week's headlines were:
Paratek Pharmaceuticals appoints Thomas J. Bigger President and CEO
Sangamo BioSciences announces corporate partnerships; Pharmacia & Upjohn and
Warner-Lambert to access zinc finger protein technology; Astrazeneca seeks
expansion of existing collaboration
Cubist Pharmaceuticals and Phylos to collaborate on antiinfective assay
development
Larry Wilson receives Chemical Industry Medal
Thermo BioAnalysis acquires Interactiva Biotechnologie GmbH
MicroSensors, Inc. receives patent for Silicon MicroRing Gyro
1998 Nobel Prize Laureate joins Cytoclonal's scientific advisory board
Dura Pharmaceuticals and Spiros Development Corporation II announce
initiation of pivotal clinical program for Beclomethasone Spiros
Ventiv Health Appoints Gil J.Brodnitz to Chief e-Commerce Officer
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Man-made enzyme may fight cancer
Roche To Publicly Sell 20 Million Genentech Shares, Genentech Board Approves
2-For-1 Stock Split
SEC Registration Filed For Genentech Public Offering
ACS announces Dr. Wilbur D. Shults Distinguished Service Award Winner
From owner-proteins@net.bio.net Mon Oct 18 07:43:00 1999
Path: biosci!NS1.HMTI.AC.BY!ld
From: ld@NS1.HMTI.AC.BY ("Dmitri Lapotko")
Newsgroups: bionet.molbio.proteins
Subject: Photoactivated proteins in intact leykocytes
Date: 18 Oct 1999 08:43:16 -0700
Organization: BIOSCI International Newsgroups for Molecular Biology
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Dear Group,
I apologize for using this group for posting a question but any your
input will be highly appreciated. I am a physicist and I met some problems during
attempt to calibrate our equipment (photothermal microscope) for measuring living
WBC properties. Could you advice please about:
1. What is absorption spectrum for intact WBC in visible range?
2. Are there any natural photoactivated proteins?
3. What are light absorption wavelengths for them, range of interest is 400-600 nm?
4. What are molecular mechanisms for such photoactivation?
Thanks in advance
Dmitri Lapotko, Ph.D.
Luikov Heat and Mass Transfer Institute
15, Brovka Street
Minsk, 220072
Belarus
Tel:(375172)842483
Fax:(375172)842486
LD@NS1.HMTI.AC.BY
From owner-proteins@net.bio.net Mon Oct 18 07:57:00 1999
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From: rogier
Subject: topoisomerase inhibitor
Newsgroups: bionet.molbio.proteins
Message-ID: <09920fb9.104a992a@usw-ex0106-043.remarq.com>
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Hi people,
There are lots of inhibitors for eukaryote topoisomerases.
Is there an inhibitor for bacterial topoisomerase I out there?
I'm desperately looking for one.
Rogier
Rogier Stuger
dept mol cell physiol, Free Univ Amsterdam
T +31-20-444-7189
F +31-20-882-1072
E rogier@biogate.com
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From owner-proteins@net.bio.net Mon Oct 18 13:12:00 1999
Path: biosci!CIGB.EDU.CU!juan.morales
From: juan.morales@CIGB.EDU.CU (Juan Morales Grillo)
Newsgroups: bionet.molbio.proteins
Subject: (none)
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From owner-proteins@net.bio.net Tue Oct 19 09:04:00 1999
Path: biosci!newsfeed.stanford.edu!news-feed.inet.tele.dk!bofh.vszbr.cz!newsfeed.tli.de!fu-berlin.de!pc207-60.biochem.uni-potsdam.DE!not-for-mail
From: Frank Fuerst
Newsgroups: bionet.molbio.proteins
Subject: Re: Photoactivated proteins in intact leykocytes
Date: Tue, 19 Oct 1999 18:58:13 +0200
Lines: 33
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Dmitri Lapotko wrote:
>
> Dear Group,
>
> I apologize for using this group for posting a question but any your
> input will be highly appreciated. I am a physicist and I met some problems during
> attempt to calibrate our equipment (photothermal microscope) for measuring living
> WBC properties. Could you advice please about:
>
> 1. What is absorption spectrum for intact WBC in visible range?
> 2. Are there any natural photoactivated proteins?
> 3. What are light absorption wavelengths for them, range of interest is 400-600 nm?
> 4. What are molecular mechanisms for such photoactivation?
I don't even know what the abbreviation WBC means, and I'm not sure
what you're meaning with photoactivated. Of course there's a lot of
proteins whose function is to interact in some way with light (light
harvesting complex, photosystem, light receptors, green flourescent
protein etc.) Or do you mean that the active site of an enzyme is only
correctly constituted after a photoreaction has occured? I can't
imagine this, but there's cofactors that are synthesized
light-dependently.
By the way there might be better groups to ask this question perhaps
one where you can expect people to know what a photothermal microscope
is...
Hope this helps _a_little_
Frank
--
Hi! I'm Norton Antivirus. Replace your signature with this text for
protection against Signature Virus 99 and many others.
From owner-proteins@net.bio.net Tue Oct 19 11:12:00 1999
Path: biosci!newshost.lanl.gov!logbridge.uoregon.edu!howland.erols.net!panix!newsmaster.cc.columbia.edu!not-for-mail
From: Keri Freeland
Newsgroups: bionet.molbio.proteins
Subject: Aggregated protein
Date: Tue, 19 Oct 1999 14:19:15 -0400
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Hi,
I have a soluble but aggregated protein that I'm trying to prepare
for NMR studies. I know it's soluble because the protein solution is
clear, and I know it forms aggregates from running size exclusion
columns.
I'm getting ready to test different buffer conditions (detergents,
salt, pH), but I can't think of a good way to check my samples for
monomeric protein. The time is more of an issue than amount of
protein, since it expresses well.
Does anyone know a quick way to do this?
Thanks,
Keri
From owner-proteins@net.bio.net Tue Oct 19 13:34:00 1999
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From: mh270@mole.bio.cam.ac.uk (Miriam Hirshberg)
Newsgroups: bionet.molbio.proteins
Subject: Re: Aggregated protein
Date: 19 Oct 1999 22:27:21 +0100
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Have you tried light scattering, or analytical centrifugation???
M.
======================================================================
Miriam (Miri) Hirshberg e-mail: m.hirshberg@mole.bio.cam.ac.uk
Department of Biochemistry Tel : (+44) 01223 766018
University of Cambridge Tel : (+44) 01223 333600 (switch board)
80 Tennis Court Road Fax : (+44) 01223 766002
Old Addenbrooke's Site
Cambridge CB2 1GA, UK
=====================================================================
On Tue, 19 Oct 1999, Keri Freeland wrote:
> Hi,
>
> I have a soluble but aggregated protein that I'm trying to prepare
> for NMR studies. I know it's soluble because the protein solution is
> clear, and I know it forms aggregates from running size exclusion
> columns.
>
> I'm getting ready to test different buffer conditions (detergents,
> salt, pH), but I can't think of a good way to check my samples for
> monomeric protein. The time is more of an issue than amount of
> protein, since it expresses well.
>
> Does anyone know a quick way to do this?
>
> Thanks,
> Keri
>
---
From owner-proteins@net.bio.net Tue Oct 19 15:10:00 1999
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From: Leonard Pattenden
Newsgroups: bionet.molbio.proteins
Subject: Re: Aggregated protein
Date: Wed, 20 Oct 1999 09:03:45 +1000
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G'day Keri;
A *very* rough method is a wavelength scan from 220-320. A highish 310:280
ratio indicates aggregates (the 310 should be almost nothing). The
look of the profile can also be useful. It's not the most accurate method,
but it is quick and is can be surprisingly predictive.
Have a day!
Len...
On Tue, 19 Oct 1999, Keri Freeland wrote:
> Hi,
>
> I have a soluble but aggregated protein that I'm trying to prepare
> for NMR studies. I know it's soluble because the protein solution is
> clear, and I know it forms aggregates from running size exclusion
> columns.
>
> I'm getting ready to test different buffer conditions (detergents,
> salt, pH), but I can't think of a good way to check my samples for
> monomeric protein. The time is more of an issue than amount of
> protein, since it expresses well.
>
> Does anyone know a quick way to do this?
>
> Thanks,
> Keri
From owner-proteins@net.bio.net Wed Oct 20 01:41:00 1999
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From: tob@ssi.dk
Newsgroups: bionet.molbio.proteins
Subject: Re:Aggregated protein
Date: 20 Oct 1999 10:34:22 +0100
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Dear Keri
I agree with the other views but if you have a protein that bind to itself in a ordered manner I do not believe that you detect it with these methods. Take for example a coil-coil protein that forms an aggregate or rather dimer or trimers. I believe you have used the best method, size exclusion chromatography already. If you continue with size exclusion chromatography try GuHCl or Urea addition to the buffers to get a monomer and use this as a reference. If you do not get a monomer, you probably have a covalent complex. If you do get monmeric protein go on with other additives, detergents, amino acids, glycerol and so on.
Tomas
---
From owner-proteins@net.bio.net Thu Oct 21 04:37:00 1999
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From: "Matt Hicks"
Newsgroups: bionet.molbio.proteins
Subject: Re: Re:Aggregated protein
Date: Thu, 21 Oct 1999 11:16:20 +0100
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Hello,
Size exclusion chromatography is not better than analytical
ultracentrifugation or light scattering. As for detection of, for example,
coiled-coil protein oligomerisation by centrifugation - it works very well
and you can get accurate association constants and molecular weights. Size
exclusion chromatography by comparison is a low resolution technique.
Is the aggregation that happens with the protein in question lots of
different molecular weights or a few discrete ones (centrifugation will give
you the distribution of molecular weights). If the latter is the case it
may be that the aggregated form is the biologically active one...
tob@ssi.dk wrote in message <99Oct20.104506gmt+0100.19744@fsk.ssi.dk>...
>Dear Keri
>
>I agree with the other views but if you have a protein that bind to itself
in a ordered manner I do not believe that you detect it with these methods.
Take for example a coil-coil protein that forms an aggregate or rather dimer
or trimers. I believe you have used the best method, size exclusion
chromatography already. If you continue with size exclusion chromatography
try GuHCl or Urea addition to the buffers to get a monomer and use this as a
reference. If you do not get a monomer, you probably have a covalent
complex. If you do get monmeric protein go on with other additives,
detergents, amino acids, glycerol and so on.
>
>
>
>Tomas
>
>---
From owner-proteins@net.bio.net Thu Oct 21 05:41:00 1999
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From owner-proteins@net.bio.net Thu Oct 21 10:57:00 1999
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From owner-proteins@net.bio.net Thu Oct 21 19:45:00 1999
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From: "Il Soo Moon"
Newsgroups: bionet.molbio.proteins
Subject: anti-ANT antibody
Date: Fri, 22 Oct 1999 12:38:06 +0900
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Hi, everyone:
I am having trouble in finding antibodies against adenine nucleotide
translocator (ANT).
Does anybody know which company sells the anti-ANT antibody?
Thanks.
Soo
From owner-proteins@net.bio.net Fri Oct 22 00:22:00 1999
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From: tob@ssi.dk
Newsgroups: bionet.molbio.proteins
Subject: Re: Re:Aggregated protein
Date: 22 Oct 1999 09:15:18 +0100
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Dear biochemists
I believe I used the wrong expression in my latest answer on this post. Instead of calling gel chromatography the best way to look at aggregates, I should have said the easist way, as obviously Keri already has the equipment to run chromatography. For analytical ultracentrifugation you need equipment and expertise to set up the method. As Keri obviously can tell, by size exclusion chromatography, that she has an aggregate I believe this low resolution technique is good enough for her application. I agree that light scattering could be an obvious choice, but Matt how do you detect an aggregate like protease-inhibitor or coil-coil or cysteine bridge aggregates by light scattering? I believe the terminology here is a bit confusing. We need to separate: the aggregates that are created by recombinant protein expression and purification methods, and the complexes that are created by "real" affinity between proteins.
At last to Keri. A beneficial side-effect of using size exclusion chromatography is that you will get a pure monomeric preparation that you could use for NMR structure studies. I guess that this is the goal of your study. I want to warn you that some proteins have a strange behavior in size exclusion chromatography. Rod-shaped proteins have a larger Stokes radius than the MW indicates and therefore seems much larger than their actual MW on size exclusion chomatography. Different additives like detergents can also make your protein look larger, due to incorporation in detergent micelles.
Good luck
Tomas
---
From owner-proteins@net.bio.net Fri Oct 22 03:27:00 1999
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From: Matt Hicks
Newsgroups: bionet.molbio.proteins
Subject: Re: Re:Aggregated protein
Date: Fri, 22 Oct 1999 10:14:45 +0100
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In-Reply-To: <99Oct22.092616gmt+0100.19723@fsk.ssi.dk>
You asked the following question:
"but Matt how do you detect an aggregate like
protease-inhibitor or coil-coil or cysteine bridge aggregates by light
scattering?"
The answer is that you look at different concentrations. A
covalently-linked aggregate/oligomer (eg: cysteine bridge aggregates) will
not increase in molecular weight with concentration but a
non-covalently-linked one will. The non-covalently-linked aggregates
such as coiled coils will just show their native molecular weight ie that
of the oligomeric unit (of course for coiled coils in light scattering
one has to take into account the shape as in size exclusion
chromatography). Incidentally analytical centrifugation can give you
molecular shape info as well as molecular weight by looking at the rate
of sedimentation or the final concentration distribution respectively.
Having said all that - I agree totally with you about using size
exclusion as a quick method and as a preparative method for the NMR
samples which was tghe original point anyway I 'spose! Must have slipped
into pedantic mode temporarily :)
Happy Researching
**************************************************
Matt Hicks
Centre for Biomolecular Design & Drug Development
School of Biological Sciences
University of Sussex
Falmer
BN1 9QG
UK
Tel: +44(0)1273 678923
Fax: +44(0)1273 678433
email: matth@biols.sussex.ac.uk
**************************************************
From owner-proteins@net.bio.net Fri Oct 22 06:36:00 1999
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From owner-proteins@net.bio.net Fri Oct 22 08:34:00 1999
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From: Kromos@hotmail.com (GeeZ)
Newsgroups: bionet.molbio.proteins
Subject: Need info on Clathrin assembly in a basket shape
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Hi,
I would like to know how occurs the polymerisation of clathrin to a
basket shape. What are the interactions involved? Does this have
something to do with the terminal domain?? If so, wich part of
the clathrin the terminal domain interact with ? (heavy chain, light
chain...)
This information would help me a lot .
Thank you
G.C
Biochemistry Student
Université de Moncton
http://www.sciences.umoncton.ca/
From owner-proteins@net.bio.net Fri Oct 22 12:32:00 1999
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From: jm68@midway.uchicago.edu (Jim Mensch)
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Postdoctoral research positions will soon be available at the Kennedy
Mental Retardation and Connective Tissue Research Center located at the
University of Chicago, Chicago IL. These positions will involve research
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biology. To be considered for these appointments, and for further
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^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
--
Jim Mensch
We are confronted with insurmountable opportunities.- Walt Kelly, "Pogo"
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From owner-proteins@net.bio.net Sat Oct 23 17:00:00 1999
Path: biosci!newsfeed.stanford.edu!logbridge.uoregon.edu!sunqbc.risq.qc.ca!carnaval.risq.qc.ca.POSTED!not-for-mail
From: Wolf@your.mom.com (Wolf)
Newsgroups: bionet.molbio.proteins
Subject: radiation and glycerol stocks
Message-ID: <381758ce.121962887@news.mcgill.ca>
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Hello People...
I just find out that my E.Coli and yeast glycerol stocks in -80
freezer have been exposed to high radiation by 59Fe for more than 2
months. The radioactivity is still very very high.
Is it possible to have any effects on my glycerol stock from radiation
such as mutation or whatever possible damage?
Thank you very much for any comments.
From owner-proteins@net.bio.net Sun Oct 24 07:20:00 1999
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Message-ID: <381320EC.9718045C@crchul.ulaval.ca>
From: Alexandre Blais
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Subject: ammonium sulfate problem
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Hi Everybody
I have some problems to solubilize my ammonium sulfate protein pellet.
I tried detergent and pH. I don't know what to do. Please help me
Jeff
From owner-proteins@net.bio.net Sun Oct 24 15:18:00 1999
Path: biosci!newshost.lanl.gov!logbridge.uoregon.edu!news.maxwell.syr.edu!ameritech.net!news-master.service.talkway.com!c01read02-admin.service.talkway.com.POSTED!not-for-mail
From: "Neon1"
Subject: mechanism of enzyme inhibitor
Newsgroups: bionet.molbio.proteins
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Organization: Talkway, Inc.
Just started on the subject of enzyme inhibitors and already have been
asked to draw a mechanism for cleavage of fructose 1,6 bisphosphate as
catalyzed with the help of a lysine side chain in the enzyme aldolase.
I don't know where to begin since our textbook did not have examples of
mechanisms of enzyme inhibitors.
Others:
1) Draw the structures of the expected products of the yeast alcohol
dehydrogenase (YADH) reaction with undeuterated acetaldehyde and NADH
in D2O solution.
2) Mechanism for the inactivation reaction of tosyl-L-phenylalanine
chloromethyl ketone (TPCK) with chymotrypsin. TCPK inhibits
chymotrypsin by covalently labeling His57 >>> indicate structure of the
product(s). Based on the structure of TCPK what reagent might be an
effective inhibitor of trypsin?
Can anyone help me out? Thanks.
--
Posted via Talkway - http://www.talkway.com
Exchange ideas on practically anything (tm).
From owner-proteins@net.bio.net Mon Oct 25 06:37:00 1999
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From: Marc Saric
Newsgroups: bionet.molbio.proteins
Subject: Re: radiation and glycerol stocks
Date: Mon, 25 Oct 1999 16:13:59 +0200
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