From owner-proteins@hgmp.mrc.ac.uk  Wed Mar  1 10:45:06 2000
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From: "Martin Offterdinger" <martin.offterdinger@akh-wien.ac.at>
X-Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins
Subject: a-P-Y Western with PBST??
Date: Wed, 1 Mar 2000 11:51:05 +0100
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Hi
We are currently facing serious problems using a-PY Westerns; we cannot
detect our activated receptor-tyrosine kinase.
I have found a hint that only TBST must be used for P-Y.
Is there anyone who can confirm this statement?
A P-Y antibody should detect phosphotyrosine and not inorganic phosphate,
isn't it?
But I have tried both 4G10 and PY20 without success.
Is there a general recommendation which is the best available AB for
Westerns?
There is also a mixture of monoclonals available from Zymed.
Are there any experiences with it?
Thank you!
Martin




From owner-proteins@hgmp.mrc.ac.uk  Wed Mar  1 11:58:39 2000
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From: "ssultang" <ssultang@dreamx.net>
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Subject: Strong 5'AMP-sepharose 4B (?)
Date: Wed, 1 Mar 2000 21:03:26 +0900
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Hi, Everyone!

I am purifying some aldehyde dehydrogenases requiring NAD with
5'AMP-sepharose 4B.
But,I could not find the activity after 5'AMP-sepharose 4B affinity
chromatography

In column work,
    - starting and washing buffer : 10mM sodium phosphate buffer
    - eluting bnuffer : containing 0.5, 3, 7,10mM NAD
    - and at last eluting with 100mM sodium phosphate buffer.
Where is the enzyme?
How can I detach the guys from 5'AMP-sepharose 4B?

Please send me your idea.
With best regards.
Thank you in advance for your reply......




From owner-proteins@hgmp.mrc.ac.uk  Wed Mar  1 13:29:43 2000
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From: Oddmund =?iso-8859-1?Q?Nordg=E5rd?= <oddmundn@biokjemi.uio.no>
X-Newsgroups: bionet.molbio.proteins
Subject: 6xHIS tag and immunofluorescence
Date: Wed, 01 Mar 2000 14:29:35 +0100
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Hello!

I`m planning to make a 6xHis tagged fusion protein. Afterwards I want to use the

tag as an epitope for immunofluorescence. Has anybody tried this? Which antibody

do you suggest?

Have anybody tried the 6xHis antibody from Clontech? Or the one from Amersham

Pharmacia Biotech? Dianova? Qiagen?

What is your experience with expression of His-tagged proteins in mammalian cell

systems?

Thank you very much for any help!

Oddmund

--
*******************************************

  Oddmund Nordgård
  Ph.D. student
  Institute of Biochemistry
  Box 1041 Blindern
  0316 OSLO
  NORWAY
  Phone: 22 85 66 99
  Fax:   22 85 44 43
  Email: oddmundn@biokjemi.uio.no

  Private:
  Kalbakkv. 21
  0953 OSLO
  Phone: 22 25 23 93

********************************************
Powered by Linux! http://www.linuxnorge.com





From owner-proteins@hgmp.mrc.ac.uk  Thu Mar  2 00:58:58 2000
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From: roger.murphy@ludwig.edu.au
X-Newsgroups: bionet.molbio.proteins
Subject: Re: 6xHIS tag and immunofluorescence
Date: Thu, 02 Mar 00 01:58:55 GMT
Organization: The University of Melbourne	
Lines: 62
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References: <38BD1B3E.B34CDD5D@biokjemi.uio.no>
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Oddmund,

We've found that the 4-His antibody from Qiagen is very good for Western blot 
staining, better in fact than there other antibodies in our hands.

We've been expressing 6-his tagged proteins in CHO cells with mixed degree of 
success.  One protein we made in E.coli worked very well (~10% of total 
inclusion body protein) but was difficult to purify because it formed 
multimers.

Hope this helps.

Roger

In article <38BD1B3E.B34CDD5D@biokjemi.uio.no>, Oddmund 
=?iso-8859-1?Q?Nordg=E5rd?= <oddmundn@biokjemi.uio.no> wrote:
>Hello!
>
>I`m planning to make a 6xHis tagged fusion protein. Afterwards I want to use
> the
>
>tag as an epitope for immunofluorescence. Has anybody tried this? Which
> antibody
>
>do you suggest?
>
>Have anybody tried the 6xHis antibody from Clontech? Or the one from Amersham
>
>Pharmacia Biotech? Dianova? Qiagen?
>
>What is your experience with expression of His-tagged proteins in mammalian
> cell
>
>systems?
>
>Thank you very much for any help!
>
>Oddmund
>
>--
>*******************************************
>
>  Oddmund Nordgård
>  Ph.D. student
>  Institute of Biochemistry
>  Box 1041 Blindern
>  0316 OSLO
>  NORWAY
>  Phone: 22 85 66 99
>  Fax:   22 85 44 43
>  Email: oddmundn@biokjemi.uio.no
>
>  Private:
>  Kalbakkv. 21
>  0953 OSLO
>  Phone: 22 25 23 93
>
>********************************************
>Powered by Linux! http://www.linuxnorge.com
>
>
>


From owner-proteins@hgmp.mrc.ac.uk  Thu Mar  2 01:10:38 2000
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From: Fred <dynamin@hotmail.com>
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Subject: Freezing GSH-agarose for "pull-down fever (PDF)"
Date: Thu, 02 Mar 2000 12:10:04 +1100
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We all know that freezing agarose or sepharose beads in aqueous
solutions fractures them and ruins them for gel filtration.  I want know
if they actually break apart into small pieces.  In other words, can
they still be used for pull-downs.

We attach GST-pusion proteins to GSH-agarose, and need to keep the beads
for a long time for a frenzy of pull-downs we are doing.  We find they
get bacterial contamination or lose activity after a month or so at
4degC.  The purified protein off the beads are all stable when frozen
for a year.  So we are thinking of freezing the beads with the fusion
protein attached.  The beads are only there for the pull-down part of
the assay, so cracks and fractures are not a problem in theory.  (We
tried azide, to disastorous effect on our activity).  Anybody got any
comments on whether this might work?  It will save us expressing the
same proteins over and over...

A related question.  How does one dry these beads in a way that prevents
the cracking?  I presume we would add sucrose or trehalose? or some
such.  What? How much?  Is a lyophilyser ok?  This might also solve our
problem, if our protein reconstitutes functionally.

Phil R	Sydney


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From owner-proteins@hgmp.mrc.ac.uk  Thu Mar  2 03:11:48 2000
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From: "Sara Valafar" <svalafar@cns.bu.edu>
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Subject: METMBS 2000 DEADLINE EXTENSION (MARCH 6)
Date: Wed, 1 Mar 2000 22:16:39 -0500
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    The 2000 International Conference on Mathematics and Engineering
Techniques in Medicine and Biological Sciences
(METMBS'2000)
<http://www.cns.bu.edu/metmbs/>
(E-mail submissions are encouraged)

June 26 - 29, 2000
Monte Carlo Resort, Las Vegas, Nevada, USA
Call for Papers/Abstracts



Recent advances in computer technology have provided the tools and the
environment to study, analyze, and better understand complex systems. This
technological development has enabled researchers to collect and analyze
massive amounts of data to a scale previously not possible. The impact of
this technology is now being felt in the medical field and in the biological
sciences.   In recent years, research in interdisciplinary areas such as
Bioinformatics and computer assisted medical decision-making has
dramatically intensified.  METMBS'2000 aims to provide a platform for
researchers to present and discuss recent breakthroughs in this area.

The METMBS'2000 Conference will be held concurrently (i.e., same location
and dates) with the International Conference on Parallel and Distributed
Processing Techniques and Applications (PDPTA'2000), the International
Conference on Imaging Science, Systems, and Technology (CISST) and the
International Conference on Artificial Intelligence (IC-AI).

You are invited to submit a one-page abstract or a draft paper of about 4
pages, and/or a proposal to organize a technical session (see below for
submission information).  All accepted submissions will be published in the
conference proceedings.

THE NAMES OF TECHNICAL SESSION CHAIRS WILL APPEAR AS ASSOCIATE EDITORS ON
THE COVER OF THE CONFERENCE PROCEEDINGS.



SCOPE

Topics of interest include, but are not limited to, the following:

o Bioinformatics: This includes informatics techniques in genomics gene
sequencing, gene pattern discovery, gene pattern-function studies, and other
genomics related studies).

o Data mining in medicine and biological sciences.

o Pattern recognition in medicine and biological sciences.

o Signal processing in medicine and biological sciences (e.g. biomedical
signal processing, etc.)
o Image processing in medicine and biological sciences (e.g. biomedical
image processing, biomedical imaging, etc.)

o Medical decision-making.
o Medical Physics.
o Biomedical Engineering.
o Biomedical Electronics.
o Biosignal interpretation.
o Any application of computers in Medicine and biological sciences (protein
structure-function analysis, drug and protein design, molecular modeling and
simulation, etc.)
o Application of information technology in biomedicine (e.g. medical
database management, information retrieval and use of computers in
hospitals)
o Application of Computational Intelligence (artificial neural networks,
fuzzy logic, and evolutionary computing) in medicine and biological sciences
o Medical and bio-computing.
o Computer-based medical systems (automation in medicine, etc.)
o Recent history (1990-1999) of Mathematics and engineering techniques in
medicine and biological sciences, and what to expect during the next decade
(2000-2009); New horizons. Review articles)
o Other aspects and applications relating to technological advancements in
medicine and biological sciences.



SUBMISSION OF PAPERS

Prospective authors are invited to submit three copies of their one-page
abstract or draft paper (about 4 pages) to F. Valafar (address is given
below) by March 6, 2000.  E-mail and Fax submissions are also
acceptable.  The length of the Camera-Ready papers (if accepted) will be
limited to 7 pages.  Papers must not have been previously published or
currently submitted for publication elsewhere.

The abstract and the first page of the draft paper should include: title of
the paper, name, affiliation, postal address, E-mail address, telephone
number, and Fax number for each author.  The first page should also include
the name of the author who will be presenting the paper (if accepted) and a
maximum of 5 keywords.


PROPOSAL FOR ORGANIZING TECHNICAL SESSIONS

Each technical session will have at least 6 paper presentations.  The
session chairs will be responsible for all aspects of their sessions,
including soliciting papers, reviewing, selecting, ...

The names of session chairs will appear as Associate Editors in the
conference proceedings.  After the conference, some sessions will be
considered for publication in appropriate journals as Special Issues with
the session proposer as the Guest Editor of the journal.

Proposals to organize technical sessions should include the following
information: name and address (+ E-mail) of the proposer, title of session,
a 100-word description of the topic of the session, and a short description
on how the session will be advertised (in most cases, session proposers
solicit papers from colleagues and researchers whose work is known to the
session proposer).

Mail your proposal to F. Valafar (address is given below); E-mail
submissions are preferred.


EVALUATION PROCESS

Papers will be evaluated for originality, significance, clarity, and
soundness.  Two researchers in the topical area will referee each paper.
The Camera-Ready papers will be reviewed by one person.


PUBLICATION

The conference proceedings will be published by CSREA Press (ISBN).  The
proceedings will be available at the conference.  Some accepted papers will
also be considered for journal publication (soon after the conference).


ORGANIZERS/SPONSORS

A number of university faculty members and their staff, in cooperation with
the Monte Carlo Resort (Conference Division, Las Vegas), will be organizing
the conference.  The conference will be sponsored by Computer Science
Research, Education, & Applications Press (CSREA: USA Federal EIN #
58-2171953) in cooperation with research centers, international
associations, international research groups, and developers of
high-performance machines and systems.  The complete list of sponsors and
co-sponsors will be available at a later time.

The last conference's sponsors included: CSREA, the National Supercomputing
Center for Energy and the Environment - DOE, The International Association
for Mathematics and Computers in Simulation, The International Technology
Institute (ITI), The Java High Performance Computing research group, Korea
Information Processing Society, World Scientific and Engineering Society,
Sundance Digital Signal Processing Inc., the Computer Vision Research and
Applications Tech., and more.


LOCATION OF CONFERENCE

The conference will be held in the Monte Carlo Resort Hotel, Las Vegas,
Nevada, USA.  This is a mega hotel with excellent conference facilities and
over 3000 rooms.  The hotel is minutes from the Las Vegas airport with free
shuttles to and from the airport.  This hotel has many vacation and
recreational attractions, including: waterfalls, casino, spa, pools & kiddie
pools, sunning decks, Easy River water ride, wave pool with cascades,
lighted tennis courts, health spa (with workout equipment, whirlpool,
sauna,...), arcade virtual reality game rooms, nightly shows, snack bars, a
number of restaurants, shopping area, bars, ...  Many of these attractions
are open 24 hours a day and most are suitable for families and children.
The negotiated hotel's room rate for conference attendees is very reasonable
($79 + tax) per night (no extra charge for double occupancy) for the
duration of the conference.

The hotel is within walking distance from most other Las Vegas attractions
(major shopping areas, recreational destinations, fine dining and night
clubs, free street shows, and more).

For the benefit of our international colleagues: the state of Nevada
neighbors with the states of California, Oregon, Idaho, Utah, and Arizona.
Las Vegas is only a few driving hours away from other major cities and
attractions, including: Los Angeles, San Diego, Phoenix, the Grand Canyon,
and more.


EXHIBITION

An exhibit is planned for the duration of the conference.  We have reserved
20+ exhibit spaces.  Interested parties should contact F. Valafar (address
is given below).  All exhibitors will be considered to be the co-sponsors of
the conference.


IMPORTANT DATES

March 6, 2000 (Monday): One-page Abstracts or Draft papers (about 4
pages) due
April 3, 2000 (Monday):  Notification of acceptance
May 1, 2000 (Monday):  Camera-Ready papers & Preregistration due
June 26 - 29, 2000:  METMBS'2000 Conference

Proposals to organize technical sessions should be submitted as soon as
possible.  All accepted papers are expected to be presented at the
conference.


MEMBERS OF PROGRAM & ORGANIZING COMMITTEES

The Program Committee is currently being formed.  Those interested in
joining the Program Committee should e-mail F. Valafar (faramarz@cns.bu.edu
<mailto:faramarz@cns.bu.edu>) the following information: Name, affiliation
and position, complete mailing address, e-mail address, tel/fax numbers, a
short biography together with research interests.


OTHER INFORMATION

Last year PDPTA, CISST, and IC-AI had research contributions from over 44
countries (over 900 participants from all over the world.)  To make this a
more complete suite of conferences, we have added METMBS.  METMBS will also
have a strong international flavor and offers its participants an
introduction to a wide range of related interdisciplinary subjects through
the other three conferences.


CONFERENCE CONTACT:

          Faramarz Valafar
          Cognitive and Neural Systems
          Boston University
          677 Beacon Street
          Boston, MA 02215

          Tel: (617) 353-5134
          Fax: (617) 353-7755
          E-mail: Faramarz@cns.bu.edu <mailto:Faramarz@cns.bu.edu>















From owner-proteins@hgmp.mrc.ac.uk  Thu Mar  2 09:51:58 2000
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Dear Netters,

Invitek GmbH has changed the internet-address. You will find now
details of our products (MT1-MMP, MT2-MMP, MT3-MMP, MT4-MMP, 
MMP 2 [Progelatinase A], MMP 8 [Neutrophil Procollagenase], 
MMP 9 [Progelatinase B], MMP 13 [Procollagenase-3] and antibodies
against MT-MMP) at

     http://www.invitek.de/MMP.htm

Within the next weeks we will open an e-shop at www.invitek-shop.de

Yours sincerely

Dr. L. Essers




___________________________________________________________

Invitek GmbH                     
Robert-Rossle-Str. 10
Biomedizinischer Forschungscampus
13125 Berlin Buch

Tel. ++49 (0)30 / 9489 3796  Fax ++49 (0)30 / 9489 3795
___________________________________________________________



---


From owner-proteins@hgmp.mrc.ac.uk  Thu Mar  2 12:47:02 2000
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From: Peeter Toomik <peeter.toomik@ut.ee>
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Subject: Re: Freezing GSH-agarose for "pull-down fever (PDF)"
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Fred wrote:

> We all know that freezing agarose or sepharose beads in aqueous
> solutions fractures them and ruins them for gel filtration.  I want know
> if they actually break apart into small pieces.  In other words, can
> they still be used for pull-downs.

After freezing you will have a weak gel, but not a broken one. You can try
to freeze and thaw a piece of agarose (electrophoresis) gel and see what
happens.
Or you can try to freeze the gel from glycerol. I hope it will help.

>
> We attach GST-pusion proteins to GSH-agarose, and need to keep the beads
> for a long time for a frenzy of pull-downs we are doing.  We find they
> get bacterial contamination or lose activity after a month or so at
> 4degC.  The purified protein off the beads are all stable when frozen
> for a year.  So we are thinking of freezing the beads with the fusion
> protein attached.  The beads are only there for the pull-down part of
> the assay, so cracks and fractures are not a problem in theory.  (We
> tried azide, to disastorous effect on our activity).  Anybody got any
> comments on whether this might work?  It will save us expressing the
> same proteins over and over...
>
> A related question.  How does one dry these beads in a way that prevents
> the cracking?  I presume we would add sucrose or trehalose? or some
> such.  What? How much?  Is a lyophilyser ok?  This might also solve our
> problem, if our protein reconstitutes functionally.

> Phil R  Sydney

No problems with lyophilising, but you must add ca 30% of sucrose, dextran
or something like.

Peeter Toomik



From owner-proteins@hgmp.mrc.ac.uk  Thu Mar  2 12:47:19 2000
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Subject: Re: Freezing GSH-agarose for "pull-down fever (PDF)"
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This message has been posted by:  612 right <i.hinners@REMOVE-THIS-TO-SENDicrf.icnet.uk>

Why dont you freeze the protein and bind it to the beads when needed? Or
did I miss something out?

How about adding glycerol i fyou want to store the beads with bound
proteins at -20C?

Kind regards, Ina



Fred wrote:

> We all know that freezing agarose or sepharose beads in aqueous
> solutions fractures them and ruins them for gel filtration.  I want know
> if they actually break apart into small pieces.  In other words, can
> they still be used for pull-downs.
>
> We attach GST-pusion proteins to GSH-agarose, and need to keep the beads
> for a long time for a frenzy of pull-downs we are doing.  We find they
> get bacterial contamination or lose activity after a month or so at
> 4degC.  The purified protein off the beads are all stable when frozen
> for a year.  So we are thinking of freezing the beads with the fusion
> protein attached.  The beads are only there for the pull-down part of
> the assay, so cracks and fractures are not a problem in theory.  (We
> tried azide, to disastorous effect on our activity).  Anybody got any
> comments on whether this might work?  It will save us expressing the
> same proteins over and over...
>
> A related question.  How does one dry these beads in a way that prevents
> the cracking?  I presume we would add sucrose or trehalose? or some
> such.  What? How much?  Is a lyophilyser ok?  This might also solve our
> problem, if our protein reconstitutes functionally.
>
> Phil R  Sydney

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




From owner-proteins@hgmp.mrc.ac.uk  Thu Mar  2 17:17:04 2000
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From: Kai Schmengler <schmeng@uni-muenster.de>
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Subject: absorption coefficients
Date: Thu, 02 Mar 2000 18:17:31 +0100
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Dear all!

I determined Km-values of an enzyme with the substrates guaiacol and
potassium iodide. Unfortunately I chose wavelengths (near published
ones) I didn’t have the absorption coefficients (extinction
coefficients) for. Now I’m looking for a table or papers in which those
coefficients could be published. The wavelenghts were 405 nm for
potassium iodide and 450 nm for guaiacol.


Thank you very much.

          Kai




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Kai Schmengler schrieb:
> 
> Dear all!
> 
> I determined Km-values of an enzyme with the substrates guaiacol and
> potassium iodide. Unfortunately I chose wavelengths (near published
> ones) I didn’t have the absorption coefficients (extinction
> coefficients) for. Now I’m looking for a table or papers in which those
> coefficients could be published. The wavelenghts were 405 nm for
> potassium iodide and 450 nm for guaiacol.

If you know literature values at some wavelengths, why don't you just
measure accurate spectra and calculate the absorption coefficients at
the desired wavelengths?

Frank
-- 
Die Verwendung von mehreren Ausrufezeichen macht die Aussage nicht
ausrufender sondern ausufernder. [Michael Bauer in dnq]


From owner-proteins@hgmp.mrc.ac.uk  Fri Mar  3 00:53:33 2000
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From: Fred <dynamin@hotmail.com>
X-Newsgroups: bionet.molbio.proteins
Subject: Re: Freezing GSH-agarose for "pull-down fever (PDF)"
Date: Fri, 03 Mar 2000 11:52:58 +1100
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Thanks for the good suggestion Ina.  Freezing in glycerol is probably
one of the best ideas raised so far.  The beads can be quickly washed
after bringing to 4decC, to remove glycerol.

We don't freeze the protein and bind it to the beads later, because we
already have so much protein and bead available each time we do the
expression (we don't like to waste it).
- For free protein, we have to elute from the GSH-agarose with GSH, then
dialyse to remove GSH, then freeze, then re-apply to the column - works
ok, but we lose a number of the fusion proteins on dialysis (not all of
them, some behave well), and it takes a couple days.  
- We don't freeze the bacterial pellet very often, as this requires
homogenisation, centrifugation, binding the column, washing, then
mini-gels to verify the amount of protein bound to the column - again,
works ok, but takes a couple of days to be able to end up with exactly
the same amount of protein bound/ul bead.  We like to do all this work
*once* (or once-in-a-while), then use the beads for pull-downs.  Its
*far* easier to have a constant supply of beads that we have
characterised already, than to redo it for each experiment.

Phil R
CMRI, Sydney

nospam@our.site wrote:
> Why dont you freeze the protein and bind it to the beads when needed? Or
> did I miss something out?
> 
> How about adding glycerol i fyou want to store the beads with bound
> proteins at -20C?
> 
> Kind regards, Ina
> 
> Fred wrote:
> 
> > We all know that freezing agarose or sepharose beads in aqueous
> > solutions fractures them and ruins them for gel filtration.  I want know
> > if they actually break apart into small pieces.  In other words, can
> > they still be used for pull-downs.
> >
> > We attach GST-pusion proteins to GSH-agarose, and need to keep the beads
> > for a long time for a frenzy of pull-downs we are doing.  We find they
> > get bacterial contamination or lose activity after a month or so at
> > 4degC.  The purified protein off the beads are all stable when frozen
> > for a year.  So we are thinking of freezing the beads with the fusion
> > protein attached.  The beads are only there for the pull-down part of
> > the assay, so cracks and fractures are not a problem in theory.  (We
> > tried azide, to disastorous effect on our activity).  Anybody got any
> > comments on whether this might work?  It will save us expressing the
> > same proteins over and over...
> >
> > A related question.  How does one dry these beads in a way that prevents
> > the cracking?  I presume we would add sucrose or trehalose? or some
> > such.  What? How much?  Is a lyophilyser ok?  This might also solve our
> > problem, if our protein reconstitutes functionally.
> >
> > Phil R  Sydney
> 
> --
> Ina Hinners
> ICRF
> Secretory Pathways Laboratory
> 44 Lincolns Inn Fields
> WC2A 3PX London
> UK
> email: I.Hinners@icrf.icnet.uk


From owner-proteins@hgmp.mrc.ac.uk  Fri Mar  3 07:23:23 2000
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From: chenggy@iris.sipp.ac.cn (chenggy)
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Subject: grEST  purification
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Hi, Everyone! 

I am purifying two gravity related esterase, enzyme  are stable
at the present of low concentration of SDS and high resolution 
of (NH4)2SO4, but the activity lost at the present of SDS after
the phenol sepharose column.


In column work, 
   starting and washing buffer : 50 mM Tris-Cl buffer containing
                                    1.7 M (NH4)2SO4  
eluted with gradient (NH4)2SO4  from 1.7 M to 0 M. 

Where is the enzyme? How can I found the reson?


Please send me your idea. 
With best regards. 
Thanks a lot  for your reply...... 

~~~~~~
chenggy@iris.sipp.ac.cn

---


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In article <38C049FB.3207@iris.sipp.ac.cn>, chenggy
<chenggy@iris.sipp.ac.cn> writes
>In column work, 
>   starting and washing buffer : 50 mM Tris-Cl buffer containing
>                                    1.7 M (NH4)2SO4  
>eluted with gradient (NH4)2SO4  from 1.7 M to 0 M. 
>
>Where is the enzyme? How can I found the reson?

Still stuck on the column? Try adding in 20% ethylene glycol to your
buffers or drop down to zero salt and then elute with an increasing urea
gradient i.e. 0 - 4M in Tris buffer plus 20% ethylene glycol.

Duncan
-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382
http://www.dnamp.com
http://www.genesys.demon.co.uk


From owner-proteins@hgmp.mrc.ac.uk  Fri Mar  3 15:28:58 2000
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From: "Lars H. =?iso-8859-1?Q?=D8stergaard?=" <lho21@cam.ac.uk>
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Subject: Dimer->monomer->dimer
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Hi everybody

Any ideas on how to monomerize a dimer (hydrophobic interaction) - and
then afterwards
being able to form an active dimer again? Would a drop in
salt-concentration in the buffer
do the job? Or a mild detergent? Any suggestion would be gratefully
appriciated.

Thanks

Lars



From owner-proteins@hgmp.mrc.ac.uk  Fri Mar  3 16:52:02 2000
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Urbana, Illinois - The Theoretical Biophysics Group at the
University of Illinois is proud to announce the initial public release
of BioCoRE, a collaborative research environment.  BioCoRE software is
freely available for use at the Theoretical Biophysics Group website.
BioCoRE development is supported by the NIH National Center for
Research Resources.

Modern computational structural biology requires scientists to employ a
wide range of tools and techniques to solve complex problems while
keeping accurate and complete records of research activities.
Additional complications are introduced by the need to effectively
engage in interdisciplinary collaborations with geographically
dispersed colleagues. The software BioCoRE, a collaborative research
environment for molecular modeling and simulations, addresses these
challenges.

Initial design work has led to a web-based architecture focused on four
primary interface paradigms:

  + a WORKBENCH interface includes features for controlling molecular
modeling, simulation, and bioinformatics tools with convenient and
uniform access to collaboratory data.  Initial tools in this category
include the ability to start and monitor molecular dynamics simulations
via a web browser interface.

  + a NOTEBOOK interface automates recording of research activities.
Initial tools in this category include a notebook tool which can be used
by researchers to engage in offline discussions as well as review and
search the entire text of any prior chat session.

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across distances in real time or time-delayed sessions and will spawn
software for teleconferencing and synchronized visualization of shared
data at distant sites.  Initially, this category includes a text-based
chat mechanism where the researchers can choose topics and converse with
other researchers who are working on the same project.

  + a DOCUMENTS interface permits the preparation of multi-author
documents in a cross-platform revision control system.

BioCoRE consists of html and javascript and java where needed.

For additional information, please visit the BioCoRE website at
<http://www.ks.uiuc.edu/Research/collaboratory/>.

The Theoretical Biophysics group encourages BioCoRE users to be closely
involved in the development process through reporting bugs, contributing
fixes, periodical surveys and via other means.  Questions or comments
may be directed to collaboratory@ks.uiuc.edu.

We are eager to hear from you, and thank you for using our software!    





From owner-proteins@hgmp.mrc.ac.uk  Fri Mar  3 22:05:48 2000
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Use Beer's Law to calculate an average absorption coefficient from all of
the data you collected.  This should be accurate enough.  Here is Beer's
Law:

A=Ecl

A=Absorption (at 450 nm, in this case)
E=Absorption coefficient
c=Concentration of solute
l="length" (outer diameter of cuvette)

This is a pain in the ass compared to looking it up in a book, but it will
be good enough.

Justin Cobb
Sophomore, Biology-General
University of Illinois at Urbana-Champaign
School of Life Sciences
"Kai Schmengler" <schmeng@uni-muenster.de> wrote in message
news:38BEA22B.A78C7107@uni-muenster.de...
> Dear all!
>
> I determined Km-values of an enzyme with the substrates guaiacol and
> potassium iodide. Unfortunately I chose wavelengths (near published
> ones) I didn't have the absorption coefficients (extinction
> coefficients) for. Now I'm looking for a table or papers in which those
> coefficients could be published. The wavelenghts were 405 nm for
> potassium iodide and 450 nm for guaiacol.
>
>
> Thank you very much.
>
>           Kai
>
>




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On Thu, 2 Mar 2000 01:10:04, Fred <dynamin@hotmail.com> wrote:

ð We attach GST-pusion proteins to GSH-agarose, and need to keep the beads
ð for a long time for a frenzy of pull-downs we are doing.  We find they
ð get bacterial contamination or lose activity after a month or so at
ð 4degC.  

Some beads -- I think including agarose -- are shipped in 20% EtOH. Unless 
this would kill the glutathione, you could store the beads at 4deg in 20% 
EtOH. 


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/       |
| 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@hgmp.mrc.ac.uk  Sat Mar  4 10:20:15 2000
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From: jjmirujo@unav.es (" J. Martinez-Irujo")
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Subject: Re: Dimer->monomer->dimer
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"Lars H. Østergaard" wrote:

> Hi everybody
>
> Any ideas on how to monomerize a dimer (hydrophobic interaction) - and

> then afterwards
> being able to form an active dimer again? Would a drop in
> salt-concentration in the buffer
> do the job? Or a mild detergent? Any suggestion would be gratefully
> appriciated.
>
> Thanks
>
> Lars

In fact, it depends on the strength of the interaction. Try to  prepare
your protein in a solution containing different concentrations (5%, 10%,
15%, 20%...) of an organic solvent (methanol, ethanol, acetonitrile...)
in a suitable buffer at 0-4 oC (dilute 1:1 or  dialyze against this
buffer). Remember that the absolute concentration of the protein affects
to the monomer-dimer equilibrium, so test several concentrations of your
protein. Measure  the relative amount of monomer-dimer by gel filtration
chromatography (a high performance column  will be needed to separate
them). You can also follow the process by measuring enzyme activity (a
100-fold dilution may be necessary to avoid solvent effects). To get the
dimer again, dilute (or dialyze) your protein in a solvent-free buffer.
Adding a antichaotropic salt  (try  0,2-2 M NaCl or (NH2)2SO4) will
help. It is advisable not to dilute so much your protein in this step
since the dimer/monomer relation at the equilibrium will be decreased.
On the other hand, if excesive solvent remains after dilution you can
expect some monomer remaining at the end.

Good luck.


--
Juan J. Martinez Irujo

Departamento de Bioquimica
Universidad de Navarra
Pamplona, Spain


---


From owner-proteins@hgmp.mrc.ac.uk  Sat Mar  4 12:10:05 2000
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From: "ssultang" <ssultang@dreamx.net>
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Subject: HELP!!!! how to purify protein require for NAD
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Hi, Everyone!

I am purifying some aldehyde dehydrogenases requiring NAD with
5'AMP-sepharose 4B.
But,I could not find the activity after 5'AMP-sepharose 4B affinity
chromatography

In column work,
    - starting and washing buffer : 10mM sodium phosphate buffer
    - eluting bnuffer : containing 0.5, 3, 7,10mM NAD
    - and at last eluting with 100mM sodium phosphate buffer.
Where is the enzyme?
How can I detach the guys from 5'AMP-sepharose 4B?

Please send me your idea.
With best regards.
Thank you in advance for your reply......






From owner-proteins@hgmp.mrc.ac.uk  Sat Mar  4 23:37:46 2000
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From: Anton Tutter <atutter@aim.salk.edu>
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Subject: Re: Freezing GSH-agarose for "pull-down fever (PDF)"
Date: Sat, 04 Mar 2000 15:40:20 -0800
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> ð We attach GST-pusion proteins to GSH-agarose, and need to keep the beads
> ð for a long time for a frenzy of pull-downs we are doing.  We find they
> ð get bacterial contamination or lose activity after a month or so at
> ð 4degC.

 
> Some beads -- I think including agarose -- are shipped in 20% EtOH. Unless
> this would kill the glutathione, you could store the beads at 4deg in 20%
> EtOH.
> 

i wouldn't do that if i were you!!!  i think peter misunderstood the
questions -- the beads are to be stored long-term *after* the GST-fusion
proteins are bound to the beads.  20% ethanol may denature the proteins.
what i would do is rapid-freeze the bead/protein complexes in liquid
nitrogen, making sure there was 20% glycerol in the final buffer before
storing. then store at -70 or colder.  the proteins should last a good long
time that way without losing any activity.  i regularly do pulldowns with
GST-fusions but i always bind my proteins fresh -- that is, i keep purified,
dialyzed stocks of my proteins at -80 prior to applying them to the beads,
and thaw them when it is time to bind them to the beads.  one hour of
binding shoud be more than adequate before you add whatever extracts you are
trying to pull down from.

what's the nature of your GST "bait" proteins?  i routinely use
transcription factor activation domains as bait and they generally are
resistant to at least two or three freeze/thaw cycles, provided there is 10%
glycerol or more (also slight detergent sometimes helps maintain solubility,
like 0.1% NP-40), and i freeze/thaw them as rapidly as possible (nitrogen
freeze, room temp water bath thaw).

--anton

tutter@salk.edu



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From: Anton Tutter <atutter@aim.salk.edu>
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Subject: Re: Freezing GSH-agarose for "pull-down fever (PDF)"
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> ð We attach GST-pusion proteins to GSH-agarose, and need to keep the beads
> ð for a long time for a frenzy of pull-downs we are doing.  We find they
> ð get bacterial contamination or lose activity after a month or so at
> ð 4degC.

 
> Some beads -- I think including agarose -- are shipped in 20% EtOH. Unless
> this would kill the glutathione, you could store the beads at 4deg in 20%
> EtOH.
> 

i wouldn't do that if i were you!!!  i think peter misunderstood the
questions -- the beads are to be stored long-term *after* the GST-fusion
proteins are bound to the beads.  20% ethanol may denature the proteins.
what i would do is rapid-freeze the bead/protein complexes in liquid
nitrogen, making sure there was 20% glycerol in the final buffer before
storing. then store at -70 or colder.  the proteins should last a good long
time that way without losing any activity.  i regularly do pulldowns with
GST-fusions but i always bind my proteins fresh -- that is, i keep purified,
dialyzed stocks of my proteins at -80 prior to applying them to the beads,
and thaw them when it is time to bind them to the beads.  one hour of
binding shoud be more than adequate before you add whatever extracts you are
trying to pull down from.

what's the nature of your GST "bait" proteins?  i routinely use
transcription factor activation domains as bait and they generally are
resistant to at least two or three freeze/thaw cycles, provided there is 10%
glycerol or more (also slight detergent sometimes helps maintain solubility,
like 0.1% NP-40), and i freeze/thaw them as rapidly as possible (nitrogen
freeze, room temp water bath thaw).

--anton

tutter@salk.edu



From owner-proteins@hgmp.mrc.ac.uk  Sun Mar  5 04:45:49 2000
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From: Fred <dynamin@hotmail.com>
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Subject: Re: Freezing GSH-agarose for "pull-down fever (PDF)"
Date: Sun, 05 Mar 2000 15:45:44 +1100
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Anton Tutter wrote:
> i wouldn't do that if i were you!!!  i think peter misunderstood the
> questions -- the beads are to be stored long-term *after* the GST-fusion
> proteins are bound to the beads.  20% ethanol may denature the proteins.

I completely agree. No ethanol for my beads (I, however, am a different
matter..).

> what i would do is rapid-freeze the bead/protein complexes in liquid
> nitrogen, making sure there was 20% glycerol in the final buffer before
> storing. then store at -70 or colder.  the proteins should last a good long
> time that way without losing any activity.  

Why -70?  We often find that 20% glycerol frezzes at that temp, vs -20? 
And why rapid freeze?  Does it offer some advantage? 

> i regularly do pulldowns with
> GST-fusions but i always bind my proteins fresh -- that is, i keep purified,
> dialyzed stocks of my proteins at -80 prior to applying them to the beads,
> and thaw them when it is time to bind them to the beads.  one hour of
> binding shoud be more than adequate before you add whatever extracts you are
> trying to pull down from.

I have thought of that too.  I am trying to eliminate the time involved
in eluting the fusion proteins and dialysing them, and then rebinding
them - roll it all into one - just freeze the initially washed beads. 
Also, some of our constructs don't survive dialysis!!
 
> what's the nature of your GST "bait" proteins?  i routinely use
> transcription factor activation domains as bait and they generally are
> resistant to at least two or three freeze/thaw cycles, provided there is 10%
> glycerol or more (also slight detergent sometimes helps maintain solubility,
> like 0.1% NP-40), and i freeze/thaw them as rapidly as possible (nitrogen
> freeze, room temp water bath thaw).

We use lost of GST bait constructs.
- Those that don't survive dialysis include a couple of protein kinase
catalytic subunits (although PKA is one example that is fine).  In this
case, we need to freeze the bacterial pellet and bind it to beads
whenever needed - works ok, just variable binding.
- Those that don't survive sodium azide include a couple of small G
proteins (ras-like).  In this case, we want to incubate the proteins
attached to the beads with GDP or GTP to "load" them.  The time involved
in characterising the beads after loading makes it *much* more
economical in time to make a big batch and keep the frozen.
- The rest are SH3 domains and a splicing factor we have.  These all
survive the above and work fine the way you suggest.  However, even
these have problems.  We, for example, need to use a battery of 4-10 SH3
domains to look at a specific protein binding.  I  takes a lot of time
to titrate the beads on mini-gels to equalise the protein content of
each lot.  It would be easier to do this only for each new bacterial
expression (ie once a year?).

Hence my original question.  Would it be feasible to simply bind our GST
fusion protein to the GSH beads, wash them, run a minigel to check
loading level, then freeze the lot in aliquots.  I am trying to find the
fastest, simplest approach that will be generally applicable to all our
protiens.

Freezing in glycerol seems to be the best bet so far.  I will post the
results when we have had a go at it.

Phil R,
CMRI, Sydney


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From: Antonin Tutter <tutter@salk.edu>
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Subject: Re: Freezing GSH-agarose for "pull-down fever (PDF)"
Date: Sat, 04 Mar 2000 23:05:26 -0800
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>
>
> Why -70?  We often find that 20% glycerol frezzes at that temp, vs -20?
> And why rapid freeze?  Does it offer some advantage?

i'm not a chemist or biophysicist, so i can't cite technical arguments for
freezing, but i was always taught to store proteins at -70, and the colder the
better.  as for glycerol, it somehow protects the proteins from the harsh effects
of water crystals - something to do with enhancing hydrophobic interactions.
rapid freezing also aids in protection from crystallization (disorderd
crystals?), but perhaps someone else in netland can supplement this claim with
better scientific explanation.

>
> > i regularly do pulldowns with
> > GST-fusions but i always bind my proteins fresh -- that is, i keep purified,
> > dialyzed stocks of my proteins at -80 prior to applying them to the beads,
> > and thaw them when it is time to bind them to the beads.  one hour of
> > binding shoud be more than adequate before you add whatever extracts you are
> > trying to pull down from.
>
> I have thought of that too.  I am trying to eliminate the time involved
> in eluting the fusion proteins and dialysing them, and then rebinding
> them - roll it all into one - just freeze the initially washed beads.
> Also, some of our constructs don't survive dialysis!!

some proteins are just so stubborn!

> Hence my original question.  Would it be feasible to simply bind our GST
> fusion protein to the GSH beads, wash them, run a minigel to check
> loading level, then freeze the lot in aliquots.  I am trying to find the
> fastest, simplest approach that will be generally applicable to all our
> protiens.

that should be fine--especially if you are doing your pulldowns to probe with
antibodies, and you are not too concerned about trace contamination on the beads
from loading the original bacterial lysates.  i was silver staining the gels
after washing the beads and loading them on SDS-PAGE.  it's amazing how much
contamination the silver staining picks up from the bacterial lysate sticking
nonspecifically to the beads during the initial protein loading. re-loading the
dialysed proteins eliminated that problem for me.  but it was a problem only
because i was silver staining, otherwise i would have done it the way you
propose.

--anton



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From: "Marcel Dinger" <marcel@ihug.co.nz>
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Subject: Genamics Expression Sequence Analysis Software
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" J. Martinez-Irujo" wrote:
> 
> In fact, it depends on the strength of the interaction. Try to  prepare
> your protein in a solution containing different concentrations (5%, 10%,
> 15%, 20%...) of an organic solvent (methanol, ethanol, acetonitrile...)
> in a suitable buffer at 0-4 oC (dilute 1:1 or  dialyze against this
> buffer). 

You can also try small concentrations of denaturant (urea or guanidin
hydrochloride); a plateau in a denaturation transition is sometimes a
sign for folded monomers.

> Remember that the absolute concentration of the protein affects
> to the monomer-dimer equilibrium, so test several concentrations of your
> protein. 

And remember that some proteins won't form stable monomers! So perhaps
you'll just get aggregated protein, or unfolded monomeric chains.

> You can also follow the process by measuring enzyme activity (a
> 100-fold dilution may be necessary to avoid solvent effects). 

Sometimes even monomers are enzymatically active.

> Good luck.

>From me too, because it is not always possible.

Bye, Frank
-- 
Die Verwendung von mehreren Ausrufezeichen macht die Aussage nicht
ausrufender sondern ausufernder. [Michael Bauer in dnq]


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ssultang wrote:

> Hi, Everyone!
>
> I am purifying some aldehyde dehydrogenases requiring NAD with
> 5'AMP-sepharose 4B.
> But,I could not find the activity after 5'AMP-sepharose 4B affinity
> chromatography
>
> In column work,
>     - starting and washing buffer : 10mM sodium phosphate buffer
>     - eluting bnuffer : containing 0.5, 3, 7,10mM NAD
>     - and at last eluting with 100mM sodium phosphate buffer.
> Where is the enzyme?
> How can I detach the guys from 5'AMP-sepharose 4B?
>
> Please send me your idea.
> With best regards.
> Thank you in advance for your reply......

Have you checked the flow through to make sure it even bound to the
comlumn?

Colin




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From: PGegen@UKans.nolospamare.edu (Dr. Peter Gegenheimer)
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Subject: Re: Freezing GSH-agarose for "pull-down fever (PDF)"
Date: 6 Mar 2000 23:13:18 GMT
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On Sat, 4 Mar 2000 23:40:20, Anton Tutter <atutter@aim.salk.edu> wrote:

ð 
ð > Ð We attach GST-pusion proteins to GSH-agarose, and need to keep the beads
ð > Ð for a long time for a frenzy of pull-downs we are doing.  We find they
ð > Ð get bacterial contamination or lose activity after a month or so at
ð > Ð 4degC.
ð 
ð  
ð > Some beads -- I think including agarose -- are shipped in 20% EtOH. Unless
ð > this would kill the glutathione, you could store the beads at 4deg in 20%
ð > EtOH.
ð > 
ð 
ð i wouldn't do that if i were you!!!  i think peter misunderstood the
ð questions -- the beads are to be stored long-term *after* the GST-fusion
ð proteins are bound to the beads.  

Oops! I did misunderstand! Anton is quite right. In response to other 
questions, the presence of glycerol helps prevent ice crystal formation 
(that's why it's used for freezing bacterial cells). 

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/       |
| 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



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From: "elgansNet" <elegansnet@hotmail.com>
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hello,

I 'd like to purify a protein from saliva, but there were  many problems
for it's viscosity.

I used whole saliva and removed cell and tissue debris by
centrifugation. But the supernatant still had strong viscosity.
Is it normal procedure to purify a protein from saliva with this strong
viscosity?
or are there any methods to eliminate viscosity without any harmful
effects on saliva protein?

If you have good references about protein purification in saliva, please
send me the information about that paper and so forth.

thanks in advance



---


From owner-proteins@hgmp.mrc.ac.uk  Wed Mar  8 16:14:05 2000
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From: Paul <pd1@mole.bio.cam.ac.uk>
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Fred wrote:

> We all know that freezing agarose or sepharose beads in aqueous
> solutions fractures them and ruins them for gel filtration.  I want know
> if they actually break apart into small pieces.  In other words, can
> they still be used for pull-downs.
>
>

We store our favourite proteins for pull-down assays on glutathione agarose
or
amylose resin at -20 °C in 20 - 50% glycerol (depending on who made the
prep).
Even at the lower glycerol concs. where the mixture obviously freezes the
beads
still work fine for pulldowns and don't seem to behave any differently to
"fresh"
stuff.  I don't think we've kept anything as long as a year (yet), but this
approach
has worked well for periods of months at least.

Paul Digard




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From: jlb@ncgr.org ("Jeff L. Blanchard")
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This summer the BioPharmaceutical Technology Center Institute in Madison, WI
will be offering the following two bioinformatics-related courses.  For more
information see http://www.btci.org/courses/CourseList/courses.htm or
contact Jeff Blanchard at the address below.

--------------------------------------------------------------------------------
Techniques in Bioinformatics and Comparative Genomics June 18 - June
23, 2000

Objectives and Goals:  This five-day intensive computer laboratory
course is designed to help participants construct a working library of
bioinformatic tools and resources.  The format will combine lectures
and interactive problem-solving sessions with each participant working
individually at a computer.  An emphasis will be placed on analysis of
practical problems posed by the participants.  The flow of the course
will move from traditional sequence analysis techniques to the
opportunities afforded by the imminent flood of genomic information.
Afternoon research seminars by the instructors will sample academic
and private sector visions of bioinformatics and highlight creative
approaches to utilizing genomic data.  (Please note:  this course is
designed to provide an overview of the field; detailed training in
specific packages will not be provided.)

Instructors:

Jeff Blanchard, Ph.D., Research Scientist, National Center for Genome Resources
Tim Burland, Ph.D., Vice President and General Manager, DNASTAR 
Barbara Butler, Ph.D., Bioinformatics Training and Education Group Leader,
	Genetics Computer Group 
Ross Overbeek, Ph.D., Senior Computer Scientist, Integrated Genomics 
Ann Palmenberg, Ph.D., Professor, Institute for Molecular Virology
	Department of Biochemistry,University of Wisconsin 
Michael Slater, Ph.D., Senior Scientist, Promega Corporation 
Jeff Thorne, Ph.D., Assistant Professor, Department of Statistics, 
	North Carolina State University
Jennifer Weller, Ph.D., Senior Scientist, National Center for Genome Resources 
----------------------------------------------------------------------
Database Design and Development for Genomics Research June 29 - July
1, 2000

Course Overview:  Databases have been the quiet intermediary of
molecular biology research and in their current state offer a
wonderful example of using the web to share experimental results and
knowledge.  In recent years there has been a proliferation of
individual and community oriented databases that contain DNA sequence,
expression, structural, enzymatic, phenotypic, organismal and other
types of data.  Many scientists and private companies now face the
challenge of integrating their data into these heterogeneous databases
and/or synthesizing databases before they can finally get to the heart
of a research question.  This three-day "hands on" workshop will start
with a session on "What are databases?"  and move onto to database
issues in molecular biology related to storing, integrating,
visualizing, analyzing, synthesizing and presenting data.  The
workshop is geared for people in molecular biology lab groups and
computer scientists getting into bioinformatics.  The format will
combine discussions with problem-solving sessions and each participant
will work individually at a computer.

Instructors:

Jeff Blanchard, Ph.D., Research Scientist, National Center for Genome Resources 
Carol Bult, Ph.D., Research Scientist, Mouse Genome Informatics Group, 
	The Jackson Laboratory
Elizabeth Shoop, Ph.D., Research Associate, Computational Biology Center, 
	University of Minnesota 
Lincoln Stein, Ph.D., Assistant Professor, Cold Spring Harbor Laboratory 
Matteo di Tommaso, Ph.D., Manager, Database Product Development, 
	Genetics Computer Group

----------------------------------------------------------------------



The BioPharmaceutical Technology Center Institute (BTCI) is a
not-for-profit organization operated exclusively for educational,
scientific and cultural purposes.  One goal of BTCI is to promote the
exchange of scientific, educational and cultural information between
industry, educators and the general public by providing facilities and
resources to support conferences, seminars, classes, and electronic
distribution of programs.





_____________________________________

Jeffrey L. Blanchard
Research Scientist
National Center for Genome Resources
2935 Rodeo Park Drive East
Santa Fe, NM 87505
tel: 505-995-4405
fax: 505-995-4432
http://www.ncgr.org/about/staff/jlb

---


From owner-proteins@hgmp.mrc.ac.uk  Wed Mar  8 19:06:44 2000
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From: "Antonin Tutter" <atutter@aim.salk.edu>
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Subject: Re: GST/His Tag expression woes
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> I have not varied the length of induction, nor the concentration of
> IPTG. Can anyone
> out there offer any suggestions as to which conditions to vary. Is this
> doomed to fail?
> Should I start looking at baculovirus or gateway technology?

you just suggested the right things to do.  it sounds like the fusion is
toxic to the cells at the current induction conditions, which explains why
you see a faint band around the size of gst-- it is expressing weakly and
getting degraded back down to gst--this is common. try milder induction
conditions, like 0.1-0.2mM IPTG instead of 1mM.  I routinely use 0.1mM for
all my gst fusions.  3 hour induction is a good starting point, i doubt
varying that much will help, but you may try one and two hour inductions.  i
have never used the strains you indicated, so i can't comment on them.  i
always use BL21 DE3.  if you are using a codon-optimized strain, then you
maight be over-expressing.  again, lower IPTG may help this.

--anton

_______________________________________
Antonin Tutter
Salk Institute for Biological Studies
RBIO-J
10010 N. Torrey Pines Rd.
La Jolla, CA  92037
email:  tutter@salk.edu


From owner-proteins@hgmp.mrc.ac.uk  Wed Mar  8 19:06:51 2000
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The following message includes the correct URL for the BTCI courses.

This summer the BioPharmaceutical Technology Center Institute in Madison, WI
will be offering the following two bioinformatics-related courses.  For more
information see http://www.btci.org/courses/CoursesList/courses.htm or
contact Jeff Blanchard at the address below.

--------------------------------------------------------------------------------
Techniques in Bioinformatics and Comparative Genomics June 18 - June
23, 2000

Objectives and Goals:  This five-day intensive computer laboratory
course is designed to help participants construct a working library of
bioinformatic tools and resources.  The format will combine lectures
and interactive problem-solving sessions with each participant working
individually at a computer.  An emphasis will be placed on analysis of
practical problems posed by the participants.  The flow of the course
will move from traditional sequence analysis techniques to the
opportunities afforded by the imminent flood of genomic information.
Afternoon research seminars by the instructors will sample academic
and private sector visions of bioinformatics and highlight creative
approaches to utilizing genomic data.  (Please note:  this course is
designed to provide an overview of the field; detailed training in
specific packages will not be provided.)

Instructors:

Jeff Blanchard, Ph.D., Research Scientist, National Center for Genome Resources
Tim Burland, Ph.D., Vice President and General Manager, DNASTAR 
Barbara Butler, Ph.D., Bioinformatics Training and Education Group Leader,
	Genetics Computer Group 
Ross Overbeek, Ph.D., Senior Computer Scientist, Integrated Genomics 
Ann Palmenberg, Ph.D., Professor, Institute for Molecular Virology
	Department of Biochemistry,University of Wisconsin 
Michael Slater, Ph.D., Senior Scientist, Promega Corporation 
Jeff Thorne, Ph.D., Assistant Professor, Department of Statistics, 
	North Carolina State University
Jennifer Weller, Ph.D., Senior Scientist, National Center for Genome Resources 
----------------------------------------------------------------------
Database Design and Development for Genomics Research June 29 - July
1, 2000

Course Overview:  Databases have been the quiet intermediary of
molecular biology research and in their current state offer a
wonderful example of using the web to share experimental results and
knowledge.  In recent years there has been a proliferation of
individual and community oriented databases that contain DNA sequence,
expression, structural, enzymatic, phenotypic, organismal and other
types of data.  Many scientists and private companies now face the
challenge of integrating their data into these heterogeneous databases
and/or synthesizing databases before they can finally get to the heart
of a research question.  This three-day "hands on" workshop will start
with a session on "What are databases?"  and move onto to database
issues in molecular biology related to storing, integrating,
visualizing, analyzing, synthesizing and presenting data.  The
workshop is geared for people in molecular biology lab groups and
computer scientists getting into bioinformatics.  The format will
combine discussions with problem-solving sessions and each participant
will work individually at a computer.

Instructors:

Jeff Blanchard, Ph.D., Research Scientist, National Center for Genome Resources 
Carol Bult, Ph.D., Research Scientist, Mouse Genome Informatics Group, 
	The Jackson Laboratory
Elizabeth Shoop, Ph.D., Research Associate, Computational Biology Center, 
	University of Minnesota 
Lincoln Stein, Ph.D., Assistant Professor, Cold Spring Harbor Laboratory 
Matteo di Tommaso, Ph.D., Manager, Database Product Development, 
	Genetics Computer Group

----------------------------------------------------------------------



The BioPharmaceutical Technology Center Institute (BTCI) is a
not-for-profit organization operated exclusively for educational,
scientific and cultural purposes.  One goal of BTCI is to promote the
exchange of scientific, educational and cultural information between
industry, educators and the general public by providing facilities and
resources to support conferences, seminars, classes, and electronic
distribution of programs.

_____________________________________

Jeffrey L. Blanchard
Research Scientist
National Center for Genome Resources
2935 Rodeo Park Drive East
Santa Fe, NM 87505
tel: 505-995-4405
fax: 505-995-4432
http://www.ncgr.org/about/staff/jlb

---


From owner-proteins@hgmp.mrc.ac.uk  Thu Mar  9 14:00:28 2000
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Subject: Re: GST/His Tag expression woes
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In article <8a670b$sb8$1@phobos.scripps.edu>, "Antonin Tutter" 
<atutter@aim.salk.edu> wrote:

> > I have not varied the length of induction, nor the concentration of
> > IPTG. Can anyone
> > out there offer any suggestions as to which conditions to vary. Is this
> > doomed to fail?
> > Should I start looking at baculovirus or gateway technology?
> 
> you just suggested the right things to do.  it sounds like the fusion is
> toxic to the cells at the current induction conditions,

I can't see the original poster's post, but with a toxic construct it's 
a good idea to use a strongly repressing strain (e.g. pLysS) so that you 
get good growth (i.e. you're preventing the bad effects from a leaky 
repressor), and then induce as normal.

R

-- 
Richard P. Grant MA DPhil          
Structural Studies Group, MRC-LMB
http://www2.mrc-lmb.cam.ac.uk/personal/rpg/index.html
Please reply to rpg 'at' mrc-lmb.cam.ac.uk


From owner-proteins@hgmp.mrc.ac.uk  Fri Mar 10 03:28:41 2000
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Message-ID: <38C86B9B.4949@antibodyresource.com>
From: The Antibody Resource Page <antibody@antibodyresource.com>
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The Antibody Resource Page (http://www.antibodyresource.com/) is your
number one source for information on antibodies.  Here is just some of
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See it all at: 

http://www.antibodyresource.com/


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Research Assistant/Associate in Protein Chemistry / Biochemistry

Looking for a Biochemist / Protein Chemist to work in the Mass Spectrometry 
facility at the National Institute on Aging in Baltimore. The ideal candidate
has a B.S. 
or M.S. degree, is experienced in working with protein and is computer 
knowledgeable. Experience in protein microchemistry, HPLC or mass spectrometry 
is desirable.  We are seeking a highly motivated individual that is meticulous
and 
reliable in his/her work.
We offer an exciting environment and the possibility of getting trained in the
field of 
protein mass spectrometry.   Salary is based on degree and experience.
For consideration send curriculum vitae and three Letters of Reference to:

Salvatore Sechi
Laboratory of Genetics
National Institute on Aging, NIH
TRIAD Building, Suite 4000
333 Cassell Drive
Baltimore, MD 21224-6825
---


From owner-proteins@hgmp.mrc.ac.uk  Fri Mar 10 18:20:10 2000
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From: kurbanic@uoguelph.ca (Kevin Urbanic)
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Subject: phosphohistidine
Date: 10 Mar 2000 18:16:00 GMT
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Can anybody tell me if you can purchase, or know of a lab that has, an 
anti-phosphohistidine antibody?  I am looking at non-radioactive methods 
of identify bacterial two component histidine kinase molecules that 
become phosphorylated.  

Can anti-phosphotyrosine antibodies be used to screen for 
phosphohistidine molecules?


Your help is much appreciated!

Kevin



From owner-proteins@hgmp.mrc.ac.uk  Fri Mar 10 19:21:49 2000
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From: Ellis Golub <ellis@biochem.dental.upenn.edu>
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Purifying a protein from whole saliva is not a good idea, as the bacteria
in the mouth are likely to alter the structure.  You should look into
methods for collecting duct saliva.  For example, parotid saliva can be
easily collectd with a suction cup apparatus.  Parotid saliva has a
viscosity similar to that of water.

It is also possible to collect submadibular saliva, but this requires the
fabrication of  specific collectors for each donor.  This material is also
quite viscous.

Ellis Golub

KimChaiWan wrote:

> hello,
>
> I 'd like to purify a protein from saliva, but there were  many problems
> for it's viscosity.
>
> I used whole saliva and removed cell and tissue debris by
> centrifugation. But the supernatant still had strong viscosity.
> Is it normal procedure to purify a protein from saliva with this strong
> viscosity?
> or are there any methods to eliminate viscosity without any harmful
> effects on saliva protein?
>
> If you have good references about protein purification in saliva, please
> send me the information about that paper and so forth.
>
> thanks in advance
>
> ---

--
=============================================================
Ellis Golub                  Phone: (215) 898-4629
Biochemistry Department      FAX:   (215) 898-3695
University of Pennsylvania   ellis@biochem.dental.upenn.edu
School of Dental Medicine
4001 Spruce Street
Philadelphia, PA 19104-6003
==============================================================





From owner-proteins@hgmp.mrc.ac.uk  Sun Mar 12 04:44:08 2000
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From: Tilman Lamparter <lamparte@zedat.fu-berlin.de>
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Subject: Re: phosphohistidine
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I have screened several calatogues for anti-phosphohistidine antibody and
havent found it. There a method from Pierce where the
phosphate group is detected by an Fe-containing compound. This is however 
not specific for phophohistidine, so you can use it only for a purified
protein to check whether it is phosphorylated or not. AntiPhosphoTyrosine
antibodies give a weak signal (to phosphotyrosine according to my
experience) and you should not wash the blot in the cold. However, even if
there is a slight affinity for phosphohistidine, it is very hard to tell
whether it s specific. 
 
On 10 Mar 2000, Kevin Urbanic wrote:

> Can anybody tell me if you can purchase, or know of a lab that has, an 
> anti-phosphohistidine antibody?  I am looking at non-radioactive methods 
> of identify bacterial two component histidine kinase molecules that 
> become phosphorylated.  
> 
> Can anti-phosphotyrosine antibodies be used to screen for 
> phosphohistidine molecules?
> 

=============================================
Dr. Tilman Lamparter
Freie Universitaet Berlin
Pflanzenphysiologie
Koenigin Luise Str. 12-16
D-14195 Berlin

Tel. +49 (0)30 838 54918
FAX  +49 (0)30 838 54357
e-mail lamparte@zedat.fu-berlin.de
URL www.biologie.fu-berlin.de



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Subject: Re: gradigels pre-cast PAGE gels- can anyone recommend?
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I have never come across the gels which you refer to, but I
can recommend gels which we have been using in our lab for
the last year or so.

They are made by a German company called NOVEX.

NOVEX ELECTROPHORESIS
11040 Roselle St.
San Diego
CA 92121
Tel: 1-800-456-6634
Fax: 1-619-542-6635
E-mail: nvxtech@novex.com
Website: www.novex.com

We have not had any problems with these gels, they run very
well and are reproducable.

They might be worth a look


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From owner-proteins@hgmp.mrc.ac.uk  Mon Mar 13 12:03:37 2000
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Sorry Keith

I forgot that Novex had merged with INVITROGEN

If the last US address if of no use you should maybe try:

1600 Faraday Ave
Carlsbad, CA 92008 USA
Phone: 1 (800) 955-6288
Fax: 1 760-603-7201


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From owner-proteins@hgmp.mrc.ac.uk  Mon Mar 13 12:07:49 2000
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From: David Coulson <d.coulsonNOd.SPAM@qub.ac.uk.invalid>
Subject: Colloidal Blue
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I was wondering if anybody could help me with a "recipe" for
making up Collodial Blue (Comassie G-250) solution for
staining SDS-PAGE gels.  Do you just make it up in water?,
and if so what sort of concentration?

Any help would be appreciated


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From owner-proteins@hgmp.mrc.ac.uk  Mon Mar 13 12:19:00 2000
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From: "Richard P. Grant" <rgrant@netscape.net>
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Subject: Re: Colloidal Blue
Date: Mon, 13 Mar 2000 12:18:56 +0000
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In article <109ec386.d1a7c612@usw-ex0109-066.remarq.com>, David Coulson 
<d.coulsonNOd.SPAM@qub.ac.uk.invalid> wrote:

> I was wondering if anybody could help me with a "recipe" for
> making up Collodial Blue (Comassie G-250) solution for
> staining SDS-PAGE gels.  Do you just make it up in water?,
> and if so what sort of concentration?

Coomassie brilliant blue R250?  There is something which sounds similar 
but doesn't work.  Can't remember what it is, exactly.

Anyhoo:  0.1 % coomassie in 40% methanol, 10 % glacial acetic acid in 
water (e.g. 2g dye, 800 ml MeOH, 200 ml GAA, make up to 2 l).  Store at 
4C and can be reused several times.  Stain for about 15 - 20 mins at RT.  
For destaining, I pour off the dye, then wash in dH20 (we have that on 
tap) twice, then wash in destain (same as above, minus the dye) with 
foam bungs to soak up the blue.  This can also be re-used if you lid the 
container to reduce evaporation.

HTH, email me if you're stuck.

R

-- 
Richard P. Grant MA DPhil          
Structural Studies Group, MRC-LMB
http://www2.mrc-lmb.cam.ac.uk/personal/rpg/index.html
Please reply to rpg 'at' mrc-lmb.cam.ac.uk


From owner-proteins@hgmp.mrc.ac.uk  Mon Mar 13 16:31:03 2000
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This message has been posted by:  612 right <i.hinners@REMOVE-THIS-TO-SENDicrf.icnet.uk>

Hi!
For collodial Blue use Coomassie G 250 and follow: Neuhoff et al,
Electrophoresis, 1988, 9, 255-262
They claim, you dont need to destain and its more sensitive, although I
never found that and returned to good old conventional
coomassie stain as Richard describes it.

Hope this helps, Ina


"Richard P. Grant" wrote:

> In article <109ec386.d1a7c612@usw-ex0109-066.remarq.com>, David Coulson
> <d.coulsonNOd.SPAM@qub.ac.uk.invalid> wrote:
>
> > I was wondering if anybody could help me with a "recipe" for
> > making up Collodial Blue (Comassie G-250) solution for
> > staining SDS-PAGE gels.  Do you just make it up in water?,
> > and if so what sort of concentration?
>
> Coomassie brilliant blue R250?  There is something which sou