HDTenEightyP/NetNews huggingface dataset:
From: KBHATNAG@CCMAIL.SUNYSB.EDU
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: gentle raidioiodination protocols
Date: 13 Oct 92 16:49:10 GMT
Distribution: bionet
Organization: State University of New York, Stony Brook


>What is the best protocol around these days for radioiodinating a protein 
>_without_ disrupting its function too much (chloramine T, for instance, seems 
>too aggressive for this purpose)?

We use the Enzymobeads Reagent from Pierce extensively for radioidinating our
peptides and also making radioiodinated photoactivable analogs with much 
success and retention of activity. Loss of function can be attributed to "over"
radioiodination, which can be reduced by controlling the reaction time and 
molar excess of 125I. Also, you could label an aromatic residue or a 
photoactivable group during peptide synthesis or modification for better
selectivty in radioiodination. Other advantage is that the work up is easy. 

Another poster was curious about the Iodobeads reagent. Again, its a very clean
method for radioiodination. We have not used it for peptides but for 
photolabels.

Chloramine T method is very harsh indeed, and we have used it only in organic
synthesis.  
      
Kavish 

------------------------------------------------------------------------------
Kavish Bhatnagar
Dept. of Chemistry                           Fax: 516 632 7962
SUNY @ Stony Brook                           Phone: 516 632 7935
Stony Brook, NY 11794-3400                   Email:kbhatnag@ccmail.sunysb.edu   
------------------------------------------------------------------------------
From: scottk@nuclease.berkeley.edu ( Number Ten Ox)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: whence Coomassie?
Date: 16 Oct 92 02:33:09 GMT
Distribution: bionet
Organization: Biochemistry and Molecular Biology, U.C. Berkeley
NNTP-Posting-Host: nuclease.berkeley.edu

In article <92Oct06.063852.21808@acs.ucalgary.ca>,
 kfvanomm@acs.ucalgary.ca (Fintan VanOmmen Kloeke) writes:
|> I have a question that follows from the Coomassie one.
|> At the moment I am running protein gels on Pharmacia's PhastGel
|> system. It seems that high sensitivity silver stainning works
|> better if the gel is Coomassie dyed first. 
|> 
|> Does anyone know why this is so? The Pharmacia reps didn't have a
|> clue.
|> 
|> Thanks in advance.


Well, I can't say I know *why* this is true, but it is a fact that
double-staining (coomassie followed by silver) is better for many
applications than silver staining alone.  A paper in BioTechniques
a couple of years back (1990, vol 9 no 5, p 532) claimed a 40-fold
increase in silver stain sensitivity when gels were first stained
with a colloidal coomassie stain. In my hands, 2 to 5 fold is more
like it. Your mileage may vary. The double staining also 
evens out staining of some proteins, i.e. some proteins which are
stained poorly or not at all with silver are stained well by
the double stain protocol.

This brings up another little tidbit: using colloidal coomassie to
stain gels. The best (only?) reference for the technique is Neuhoff
et al (1988) Electrophoresis 9 pp 255-262. They report *coomassie*
stain sensitivities down to 1 ng and lower. Yes, nanogram. I've
easily detected 10 ng in 0.75 mm thick SDS minigels.

Basically, the protocol involves staining with coomassie in the 
presence of ammonium sulfate and methanol. In an ammonium
sulfate solution, Coomassie forms a colloid which is excluded from
the gel, while the methanol releases some of the dye into a molecularly 
dispersed form which can diffuse into the gel and bind the protein.
The result is staining with essentially no background; no background
means no destaining, which means no loss of stain from the
protein. Check it out.

Integrated Separations Systems sells a kit for those
who don't want to play with optimizing the system for themselves.


-- 
       ____         Scott Keeney, DNA repair-queer         ____
       \  /           scottk@mendel.berkeley.edu           \  /
        \/        Biochemistry and Molecular Biology        \/ 
                            U.C. Berkeley
Newsgroups: bionet.molbio.methds-reagnts
From: zxmkr08@mailserv.zdv.uni-tuebingen.de (Cornelius Krasel)
Subject: Re: RT-PCR
Organization: Comp. Center (ZDV) U of Tuebingen, FRG
Distribution: bionet
Date: Sun, 11 Oct 1992 17:35:49 GMT

In <9210090425.AA07044@cscgpo> Klaus.Matthaei@anu.edu.au writes:

>G'Day Netters

>I have some questions regarding PCR from RNA.  The standard method is to
>use Reverse Transcriptase and oligi-DT to synthesise cDNA from total RNA
>followed by standard PCR.  Another method is to use the dowmnstream primer
>for the cDNA synthesis followed by PCR.  More recently Amersham has brought

About a year ago, I worked as an undergrad student on a similar problem.
What worked fine for me, was:

1. Purification of total RNA by Guanidinium-SCN method (as described in
   Maniatis).
2. Preparation of polyA-RNA by using of oligo-dT columns.
3. Reverse translating of the RNA using the MMLV SuperScript from BRL and
   random hexamers.
4. Purifying the cDNA (with a Pharmacia Spun column, which is basically
   Sephacryl 2000, I think).
5. PCR using ordinary Taq polymerase.

We tried also PCR from cDNA prepared as above and transcribed with oligo dT
primers but got worse results.

Hope that helps, Cornelius.
--
/* Cornelius Krasel, Department of Physiological Chemistry, U Tuebingen    */ 
/* email: krasel@mailserv.zdv.uni-tuebingen.de (Internet)                  */
/*        krasel@chemie.uni-tuebingen.dbp.de (WIN/X400)                    */
/* "People are DNA's way of making more DNA." (anonymous)                  */
From: TAFFETS@SNYSYRV1.EARN
Newsgroups: bionet.molbio.methds-reagnts
Subject: Immunization and Adjuvants
Date: 11 Oct 92 23:52:00 GMT
Distribution: bionet
X-Original-To: methods@daresbury.ac.uk, TAFFETS
Original-To: methods@UK.AC.DARESBURY


 
 
     I'm wondering if anyone has any opinions on the new adjuvants that
have appeared in the past few years.
 
     In the past I have been successful at making good antisera using
complete Freund's adjuvant with my antigen.  In the last year I've tried
several of the new commercially available adjuvants (at the request of the
animal use committee).  I obtained antisera with a high titre by ELISA but
these sera were ineffective in some assays especially western blotting.
While it might be the antigens that I've used, I keep wondering about the
adjuvants.  I've noticed that every report describing these adjuvants only
has ELISA data and does not test the resulting serum in a variety of
assays.  Is it possible that these adjuvants induce a high titered serum
with low affinity or avidity?  Has anyone had great success (or failure)
with these adjuvants?  Are any of these adjuvants better than others?
 
     Thanks for your input.
 
        Steve
*******************************************************************************
Steven Taffet, Ph.D.
Associate Professor
Department of Microbiology and Immunology
S.U.N.Y. Health Science Center
750 E. Adams Avenue
Syracuse, New York 13210
 
Phone (315) 464-5419
FAX   (315) 464-4417
Bitnet  TAFFETS@snysyrv1
Internet TAFFETS@vax.cs.hscsyr.edu
*******************************************************************************
From: apk@phoenix (Ari-Pekka Kvist)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Automated sequencing: opinions please!
Date: 12 Oct 92 07:59:55 GMT
Organization: University of Oulu, Finland
X-Newsreader: TIN [version 1.1 PL6]

rybicki@uctvax.uct.ac.za wrote:
: Dear Netlanders / colleagues in cyberspace:
: 
: My turn to ask a FAQ - we would like to share the experiences of those of you
: who have tried out automated sequencing setups.  We are inclining towards the
: Pharmacia system; however, we would very much appreciate hearing from anyone
: who has any experience with this or anything else, or who can point us in the
: direction of info available on comparison of these systems.  Depending on what
How about Millipore's Basestation? Does anyone have any experiences?
I think that machine is quite new and it might not be as popular as
previous one's but anywaay I would like to hear opinions from that 
equipment too

----

      Researcher           (Bitnet   : lbio-apk at finou.bitnet)
      Ari-Pekka Kvist      (IBM      : lbio-apk at finou.oulu.fi)
                           (UNIX     : apk@phoenix.oulu.fi or
      Oulu university                  kvist@convex.csc.fi)
      Department of Medical Biochemistry
      Kajaanintie 52 A
      SF-90220 Oulu
      Finland

      Tel. 358-(9)81-5375840
Newsgroups: bionet.molbio.methds-reagnts
From: kfvanomm@acs.ucalgary.ca (Fintan VanOmmen Kloeke)
Subject: Re: whence Coomassie?
Date: Tue, 06 Oct 92 06:38:52 GMT
Distribution: bionet
Organization: The University of Calgary, Alberta

I have a question that follows from the Coomassie one.
At the moment I am running protein gels on Pharmacia's PhastGel
system. It seems that high sensitivity silver stainning works
better if the gel is Coomassie dyed first. 

Does anyone know why this is so? The Pharmacia reps didn't have a
clue.

Thanks in advance.
Newsgroups: bionet.molbio.methds-reagnts
From: eboye@radium.uio.no (Erik Boye)
Subject: dam- e. coli
Organization: University of Oslo
Date: Thu, 8 Oct 1992 12:50:17 GMT


Re: problems in transforming dam- strains of e. coli. A methylated plasmid will 
    after one round of replication become hemimethylated in a dam- host.
    Several origins of replication (chromosomal and plasmid) are poorly 
    initiated when hemimethylated. Tranformation efficiency is both strain and
    vector (origin) dependent. Unmethylated plasmids usually transform
    dam- hosts with high efficiency. So, when in trouble, switch host cell or
    switch to another cloning vector (preferably high copy number).
From: eboye@radium.uio.no
From: rybicki@uctvax.uct.ac.za
Newsgroups: bionet.molbio.methds-reagnts
Subject: Automated sequencing: opinions please!
Date: 7 Oct 92 15:07:03 GMT
Organization: University of Cape Town

Dear Netlanders / colleagues in cyberspace:

My turn to ask a FAQ - we would like to share the experiences of those of you
who have tried out automated sequencing setups.  We are inclining towards the
Pharmacia system; however, we would very much appreciate hearing from anyone
who has any experience with this or anything else, or who can point us in the
direction of info available on comparison of these systems.  Depending on what
comes back, I will summarise and send on to Paul Hengen for his FAQ list.

Many thanks in advance,
 _________________________________________________________________________
|                                 |                                       |
|  Ed Rybicki                     |    Now you've got the hang of it      |
|  Dept Microbiology              | There's nothing you can't do with it  |
|  University of Cape Town        |         If you're very into it        |
|  PB Rondebosch 7700             |         You can't go wrong....        |
|  South Africa                   |                                       |
|  fax 27 21 650 4023             |              - Mad John               |
|  ed@micro.uct.ac.za             | (Ogden's Nut-Gone Flake, Small Faces) |
|_________________________________|_______________________________________|
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Tightest promoter repression ?
From: robison1@husc10.harvard.edu (Keith Robison)
Date: 13 Oct 92 03:33:48 GMT

jgraham@bronze.ucs.indiana.edu (the End) writes:

>I need to find a promoter which is very tigthly regulated for use in reducing
>the expression of downstream Lac sequence to a very low level.

I don't have personal experience with it, but you should check out
Studier's work with a T7 promoter -- T7 RNA polymerase -- T7 lysozyme
-- lac repressor system.  T7 lysozyme is an inhibitor of T7 RNA polymerase
(1).  The deluxe system is therefore:

	(a) T7 lysozyme & lac repressor under constituitive promoters
	(b) T7 RNAP under an inducible promoter
	(c) your gene driven by a T7 promoter, with a lac operator
	    between the two.

The basal lysozyme expression kills the basal T7 RNAP expression (2), 
and the lac repressor provides the insurance (3).  The system works well
enough to allow T7 RNAP to be cloned behind a T7 promoter --
a combination which is ordinarily lethal (4).

	1. Cell (1987) 49:221-227
	2. J.Mol.Bio. (1991) 219:37-44
	3. J.Mol.Bio. (1991) 219:45-59
	4. J.Mol.Bio. (1991) 219:61-68


Keith Robison
Harvard University
Department of Cellular & Developmental Biology
Department of Genetics / HHMI

robison@ribo.harvard.edu 



Newsgroups: bionet.molbio.methds-reagnts
From: jbh@anat.UMSMED.EDU (James B. Hutchins)
Subject: Re: Protease inhibitors
Summary: If you got it, use it 
Organization: Dept. of Anatomy, Univ. Mississippi Medical Center
Distribution: bionet
Date: Wed, 14 Oct 1992 01:26:26 GMT


Andre: Tried to respond by e-mail but it bounced; here's my answer.

   ----- Transcript of session follows -----
550 NANTEL@UNCVX1.BITNET... Host unknown


Andre,

It's OK to do biochemistry without a license...I'm a cell biologist without
a biochem license myself.

The best approach to protease inhibition was summarized by a friend of
mine: "If you've got it, use it".  Your problem with inhibition of column
binding is a refutation of this principle, I guess.

The two best sources (quick, too!) I know of are a recent Methods in
Enzymology entitled "Guide to Protein Purification" (Deutcher, M, ed.),
sorry I'm at home and don't have a more complete ref, and a *free* guide
put out by the Boehringer-Mannheim folks.

Basically, in my simplistic view, all protease inhibitors fall into groups:
serine protease inhibitors (PMSF, DFP), metalloprotease inhibitors (EDTA),
etc.

If you know what your protease activity is (ha!), use the drug specific to
your purpose.  If, like the rest of the world, you don't, then
trial-and-error is the rule...

We use aprotinin (serine protease inhibitor), leupeptin (serine/thiol
protease inhibitor), pepstatin (acid protease inhib), PMSF (serine again),
EDTA (metalloprotease inhib), not because we have any special knowledge,
but because that's what's evolved for us over the last 5 years and if it
ain't broke, don't fix it.

Hope this helps in some vague way.

Jim

-- 
Jim Hutchins                    []     E-Mail: jbh@anat.umsmed.edu
Dept of Anatomy                 []
Univ Mississippi Med Ctr        []

-- 
Jim Hutchins                    []     E-Mail: jbh@anat.umsmed.edu
Dept of Anatomy                 []
Univ Mississippi Med Ctr        []
From: ODILE@FRCGM51.EARN
Newsgroups: bionet.molbio.methds-reagnts
Subject: Cloned yeast GAL10 gene need
Date: 8 Oct 92 16:20:00 GMT
Distribution: bionet
X-Envelope-To: methods@daresbury.ac.uk
Original-To: methods@UK.AC.DARESBURY
X-Vms-To: IN%"methods@daresbury.ac.uk"

Bonjour !
 
We are looking for a plasmid bearing Saccharomyces cerevisiae GAL10 gene
to replace the gene of our reference strain which is unfortunally gal-...
and we don't want to do crosses to maintain the same genetic background.
 
Thank you for your help.
 
Odile Ozier-Kalogeropoulos (quiet difficult! :-).....)
CGM CNRS
91190 Gif sur Yvette
FRANCE
From: adavies@vax.oxford.ac.uk (Anthony Davies)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: ***Help CHICKEN cell line (urgent)******
Date: 7 Oct 92 07:50:08 GMT
Organization: Institute of Virology, Oxford, UK

In article <1992Oct6.201603.1@vax.path.ox.ac.uk>, jasper@vax.path.ox.ac.uk writes:
> The only lines that grow in culture other than primary ones are Marek's
> Lymphoma derived, there are (to the best of my knowledge) no others. I'm
> afraid I don't have a contact for these, though if pushed I *might* be able to
> contact a vetinarian who knows unless he has already gone to the Himilayas for
> 6 weeks vacation ! 
> 
> Anyone knowing of any others??? 

I would imagine you could stably transform Chicken PBL with Marek's virus using 
cyclosporin A, and that it would be pretty easy to do.  All you would need is 
an aliquot of Mareks, some CSA and a chicken!!  I could provide a contact for 
Mareks.
________________________________________________________________________________

Anthony Davies                Voice: 865-512361
Institute of Virology         Fax:   865-59962
Mansfield Road                Vax:   adavies@path.vax.ox.uk.ac (preferred)
Oxford OX1 3SR                       adavies@vax.ox.uk.ac (this account)
UK
________________________________________________________________________________
From: NANTEL@UNCVX1.BITNET
Newsgroups: bionet.molbio.methds-reagnts
Subject: Protease inhibitors
Date: 13 Oct 92 16:03:00 GMT
Distribution: bionet


Greetings Netters!!

I'm fishing for information concerning the use of protease inhibitors during
the purification of MBP-fusion proteins from E.coli. PMSF doesn't seem to
have any effects on the quality of my protein preps. Furthermore, when I
use antipain + leupeptin the efficiency of the purification step through
the amylose column is greatly reduced (7-10 fold lower yields). I feel
that protease inhibitors are necessary since one of my fusion protein is
chewed up from 95 to 80 kD during the purification.

I am faily new to the world of protein purification (yes, another molecular
biologist doing biochemistry without a licence) and any suggestions would
be most welcome.

Andre Nantel
Biology Dept.
University of North Carolina
From: BOTDJL@LURE.LATROBE.EDU.AU (LOWDEN,Darren)
Newsgroups: bionet.molbio.methds-reagnts
Subject: cross-linking
Date: 12 Oct 92 00:27:43 GMT
Distribution: bionet
Organization: La Trobe University
X-News-Reader: VMS NEWS 1.24

Dear fellow netters,

I am interested in trying to identify the cell surface receptor to which a
protein of interest binds to. I am using a chimaeric protein (fused with the
maltose binding protein) so I thought that one means of accomplishing this task
would be to cross-link the chimaeric protein in the presence of cells,
solubilise the cell membranes and pass the entire extract over an amylose
column in the hope that the maltose binding protein tag will bind. By washing
the column and then eluting bound material and reversing the cross-linking
process I hope to be able to resolve two bands on a SDS-PAGE gel, one band 
being the chimaeric protein and the other hopefully the receptor of interest.

Unfortunately I have no experience whatsoever with cross-linking reagents and
would welcome any advice with regards to reviews, references, protocols,
companies that supply reversible cross-linkers etc.

Thanks in advance for any help that you may be able to offer.

Yours sincerely,
Darren Lowen
botdjl@lure.latrobe.edu.au
From: Klaus.Matthaei@anu.edu.au
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: PCR max. lenght
Date: 7 Oct 92 23:18:33 GMT
Distribution: bionet

>Hi netters,
> 
>does anybody know the maximal lenght of the gene amplificable
>by PCR? I note, that we want to use it for the transfection
>into the eucaryotic cells.
> 
>Many thanks L.B.


G'Day

We have amplified 7kb and the literature suggests up to 15kb see:
 Kainz et al. Analytical Biochemistry 202,46-49 (1992) (but I haven't read
this paper closely).

Cheers, Klaus
--------------------------------------------------------------------------
Klaus Matthaei
Gene Targeting
The John Curtin School of Medical Research
The Australian National University
E-mail: Klaus.Matthaei@anu.edu.au

"If all else fails : Read the instructions"
--------------------------------------------------------------------------
From: PHILROSS@BIOTECHNET.COM
Newsgroups: bionet.molbio.methds-reagnts
Subject: Method for Anchored PCR/RACE
Date: 15 Oct 92 14:36:58 GMT
Distribution: bionet

With regard to the recent message:

> From:	IN%"cytogen@chmeds.ac.nz" 15-OCT-1992 01:03:47.81
> To:	IN%"methods-and-reagents@net.bio.net"
> CC:	
> Subj:	Method for Anchored PCR/RACE

> The tailing buffer is a curse to prepare.  Check out your enzyme suppliers:  
> LifeTech/BRL and Promega both supply TdT with buffer.  Pharmacia (last time I 
> ordered), didn't supply buffer.  Pharmacia TdT is reputed to be the best.  I 

For anyone interested, Pharmacia now distributes the enzyme with buffer.
Oh, and no, this isn't a paid advertisement.

Phil Ross
Neurogen Corporation
From: coyne@uctvax.uct.ac.za
Newsgroups: bionet.molbio.methds-reagnts
Subject: subscribe
Date: 6 Oct 92 11:08:49 +0200
Organization: University of Cape Town

subscribe bionet.molbio.methds-reagnts

Newsgroups: bionet.molbio.methds-reagnts
From: bchs1b@Elroy.UH.EDU (Michael Benedik)
Subject: Re: Lambda libraries
Reply-To: bchs1b@Elroy.UH.EDU
Organization: University of Houston
Date: Thu, 15 Oct 1992 03:22:09 GMT

In article <1992Oct14.100936.121@chmeds.ac.nz>, cytogen@chmeds.ac.nz (Martin Kennedy) writes:
>In article <1992Oct13.100236.90319@vaxc.cc.monash.edu.au>, mic255z@vaxc.cc.monash.edu.au writes:
>> Dear Netters,
>> 
>> I am interested in using a lambda system to do some primary cloning.
>> Does anyone have any thoughts on what system is better ie., what are the
>> relative merits of lambda-ZAP and lambda-DASH compared with EMBL, GEM
>> or gt11 apart from obvious things like unique cloning sites.
>> 
>> 
>> Thanks in advance,
>> Helen Jost,
>> Department of Microbiology,
>> Monash University.
>
>
>I've used lambda ZAP several times, and it works beautifully; its main advantage
>is the "automatic excision" of inserts - ie, you don't have to make a lambda DNA
>prep, and subclone the insert, as addition of a helper phage causes copying of
>the phagemid portion of lambda ZAP which contains the multiple cloning site and
>your insert.  The phagemeid is then recovered as amp resistant colonies that
>contain a Bluescript "subclone"of your lambda insert.  Look at various volumes
>of Methods in Enzymology for good comparisons of lambda vectors.
>
>Cheers,
>
>Martin

Followup question for you lambda ZAP guru's out there. What is the efficiency
of excision from lambda zap to release the plasmids. In other words, we often
need to make small genomic libraries (bacteria) in plasmids. I was wondering
if it would make sense to make them in lambda zap (and use the high efficiency
of packaging) and then release the plasmid en masse to get a plasmid library.
I know making a plasmid library is not a big deal, but this would seem
easier. Am I missing something???

----------------------------------------------------------------------
 Michael Benedik				INTERNET: Benedik@uh.edu
 Dept. of Biochemical & Biophysical Sciences	
 University of Houston				BITNET: Benedik@uhou
-----------------------------------------------------------------------
Newsgroups: bionet.molbio.methds-reagnts
From: jgraham@bronze.ucs.indiana.edu (the End)
Subject: ung E.coli strains 
Organization: Indiana University
Date: Thu, 15 Oct 1992 15:18:53 GMT

In planning a chemical mutagenesis that will involve cytosine deamination,
I need an E. coli strain that will tolerate the presence of uridylate residues
in plasmid DNA, but will propagate them as A-T basepairs, rather than A-U pairs
We currenttly use CJ236 as a host which will introduce deoxyuridine. Are there
many ung only strains out there ? 

Any leads appreciated,


Jim
J. Graham
Biology and Chemistry Depts.
Indiana University -Bloomington