HDTenEightyP/NetNews huggingface dataset:
From: ED@micro.uct.ac.za (Ed Rybicki)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: whence Coomassie?
Date: 5 Oct 92 10:45:44 GMT
Reply-To: ed@micro.uct.ac.za
Distribution: bionet
Original-To: mills_j@edu.upenn.med.mscf, methods@uk.ac.daresbury
X-Mailer: Pegasus Mail v2.1c R5.


> BI1RGW@ibm.shef.ac.uk writes:
> > Hello Netland.
> >
> > Can anyone remind me of the details of what, how and when Coomassie R/G bind
> > to proteins?  As I recall, R binds slower but is more sensitive - but what do
> > they bind?  I have at the back of my mind some vague inkling about lysine
> > residues - but what about arginine, and what about the N-terminus?
> >
> > I'm sure this is a FAQ, but...all comments, recollections, anecdotes welcome.
>
>     You know, I spent a little bit of time researching just what exactly Coo-
> massie dye binds to.  The basic conclusion that I reached at the time was
> that either nobody knows, or I was looking in the wrong places.  There is a
> whole chapter on various anionic dyes in a 1985 book on electrophoresis, but
> as I remember it, other than a lot of interesting stuff about the history and
> some explanations of sensitivity and a good bit of detail about linearity of
> staining intensity to mass of protein, there wasn't much about binding.  All
> I remember was that Coomassie was supposed to bind free amino groups.  Of
> course, that would dictate lysine binding, but I got the impression the in-
> teraction between dye and protein wasn't that specific.
>
>     I'd be a little surprised if this were a FAQ, but if it is, or if there
> are any dye organic chemists out there chomping at the bit to enlighten this
> cell biologist who's out of his league, I'd be interested.

I think you may find it actually binds the peptide bond - there are a lot
of proteins with no free lysine which bind CBB just fine (like tobacco
mosaic virus coat protein).  I recall writing an essay on protein
quantitation some time in '77 where that was the received wisdom - but I'm
buggered if I can remember a reference, and the essay got thrown out a
while back....

 ____________________________________________________________________
| Ed Rybicki, PhD             |    "Now you've got the hang of it    |
| (ed@micro.uct.ac.za)        | There's nothing you can't do with it |
| Dept Microbiology           |        If you're very into it        |
| University of Cape Town     |         You can't go wrong...."      |
| Private Bag, Rondebosch     |                                      |
| 7700, South Africa          |               -Mad John              |
| fax: 27-21-650 4023         | (Ogden's Nut Gone Flake, Small Faces)|
 --------------------------------------------------------------------
From: djt2@po.CWRU.Edu (Dennis J. Templeton)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: (none)
Date: 2 Oct 92 21:31:55 GMT
Reply-To: djt2@po.CWRU.Edu (Dennis J. Templeton)
Organization: Case Western Reserve University, Cleveland, OH (USA)
NNTP-Posting-Host: thor.ins.cwru.edu


In a previous article, KNECHT@UCONNVM.UCONN.EDU () says:

>Hi Netters-  I remember reading somewhere that you can use ethidium bromide in
>restriction digestions to make partial digests.  Does anyone know a protocol or
> reference to this technique.  Thanks- Dave Knecht (KNecht@uconnvm.edu)
>
The earliest reference I have for this is Parker, Waton, and Vinograd, PNAS
74 (1977) pp851-855.  We have used it ir a variant several times.

Two comments:  It works well only for *some* enzymes.  They used HindIII
and HpaI, I have confirmed that it works very well for HindIII.  Other
enzymes are slowed, but not fully inhibited after the first cut.

Also, these workers (and I) used empirical tests to determine the optimal
concentrations of EtBr and enzyme.  A typical reaction is 1ug plasmid
+5-20ug EtBr + 2-10 U enzyme in 20 ul. The Parker paper also varied the
temp from 4 to 55 degrees, and said that higher was better.

You *never* get exclusively full length linears, but EtBr does help getting
linears over just limiting enzyme.

For routine partials, we just do serial dilutions of enzyme into DNA
(plasmid is cheap) and run all the products out on a long gel with good
markers.  Usually somewhere between 0.5u/ug and 100x less gives reasonable 
partials.

By the way, it is MUCH easier to use partial digests if you cut fully with
one enzyme, then do partial digests (by dilution) with the second enzyme. 
That way (since you know the location of the first cut) you can identify
the appropriate partial with ease, AND since the two ends are different you
can force clone it into vector cut with two enzymes.  Fully cut
contaminating DNA won't interfere, since it won't (with two similar ends)
ligate into the twice cut vector.

good luck

dennis
From: bsh@MED.PITT.EDU (Basavaraju Shankarappa)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re:  Looking for a good oligo design program. Help!
Date: 28 Sep 92 13:59:33 GMT
Distribution: bionet

Here is some program that may be quite useful.  We have been using
OLIGO to look at our primers.  Everytime we use it, the inventor of tat
program goes to higher hell because of our curses.  I think it is one
program that people did not want anybody to make use of.  The manual is
so unfreindly you just donot feel like using it.

The PRIMER program looks like a very good program at least from their
descriptions.  The logic and methodology has been explained quite
decently and all of it does make sense.  Specially they have used 
a base stacking method to calculate Tm of the primers and it is supposed
to give a single value for the Tm which is much less pain (however accurate)
than the 3 outrageously different Tm provided by OLIGO.
	I have not been able to use the program yet because I have not been
able to compile the program with my knowledge and available facilities.
I believe they will supply a executable program if requested.  So I am
waiting.  If anyone else has had the opportunity to use it by now, I would
appreciate their input.  

	You can download the source codes and all the files by ftp as below.
You have to acknowledge the receipt of the program for some proprietory
considerations.  
To get the primer program

by anonymous.ftp

ftp 
to 

genome.wi.edu
and copy the programs from the folder

distribution/primer.0.4


or Contact Dr. Eric Lander 
Whitehead Institute for Biomedical Research
9 Cambridge center
Cambridge  MA 02142


Hope to see some discussion.
Raj Shankarappa
Univ of Pittsburgh
Newsgroups: bionet.molbio.methds-reagnts
From: txt15@po.CWRU.Edu (Tao Tao)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: cm amplification?
Date: 29 Sep 1992 14:29:35 GMT
Organization: Case Western Reserve University, Cleveland, Ohio (USA)
NNTP-Posting-Host: slc12.ins.cwru.edu


I really don't think that you need to mplify pUC based vector, sicne each
cell has about 200 copies.  What you need to do is to increase the final
cell density, which could be achieved by better nutrition.  See BRL's
focus or catalog about the TB (terrific broth) medium.

Tao
-- 
p
Newsgroups: bionet.molbio.methds-reagnts
From: endo@angis.su.OZ.AU (Prince of Wales Endocrinology Unit)
Newsgroups: bionet.molbio.methds-reagnts
Subject: LUCIFERASE EXPRESSION
Date: 30 Sep 92 01:31:19 GMT
Distribution: bionet


Hi I'm using luciferase as a reporter gene in primary cultures
of rat Leydig cells.  I posted a message previously asking if
anyone has encountered the problem that I'm having.  I know
that my cells are successfully transfected (the mRNA is present)
and the extraction and assay are working well in other cells yet
I get no detectible activity in my Leydig cell extracts.  

Haso anyone had a similar experience?

I would like to get my hands on a luciferase antibody next to
see if the protein is being translated or not.  Could someone
please tell me if the antibody is available?  and fromhom??

Thanks in advance,
Tim Nason (I am not a computer geek)
From: awb@aber.ac.uk (Andrew William Berry)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Immunoprecipitation (again!)
Date: 28 Sep 92 11:11:08 GMT
Organization: University College of Wales, Aberystwyth

Dear all,
        Can someone suggest a supplier of anti-rabbit
IgG sepharose for immunoprecipitation? I have tried
our usual sources but with no success. Thanks in advance,
Andy
From: rpgrant@vax.path.ox.ac.uk
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Can anyone help with protein expression in pGEX vectors?
Date: 6 Oct 92 09:41:34 GMT
Organization: Oxford University Molecular Biology Data Centre
Nntp-Posting-User: rpgrant

In article <1992Oct6.003939.10137@uvm.edu>, lhanson@moose.uvm.edu (lhanson) writes:
> 
> 	I've been trying to express 2 cDNAs in pGEX-2T.  Both cDNAs are
> from a fungal library and encode proteins involved in mating reactions.
> The predicted fusion protein should be about 67 kdal; glutathione S-transferase
> (26 kdal) + cDNA encoded peptide (40 kdal).  I have followed the protocol
> given in "Current Protocols in Molecular Biology" and haven't been having
> much luck.  I can often see what appears to be an intensification of a
> protein band around 70 kdal in induced cultures (as compared to an uninduced
> control).  The fusion protein is expressed at significantly lower levels than
> the parental vector alone expresses GST (gluathione S-transferase).
> 
> 	Does anyone know of any "tricks" I might use to improve expression?
> I've tried inducing in early log phase (1.5 hrs after inoculation) as well as
> later (4-5 hrs post inoculation).

One thing you might try is to reduce the temperature of induction (and 
increase the time) with IPTG; try (say) 6h at 30C and/or overnight at 25C.  
This is reputed to help with insoluble fusion products.  Also try varying the 
final concentration of IPTG.

> cannot get affinity purification of the small quantity of fusion protein
> I can get expressed as I believe its in inclusion bodies!

See above.
Also, you might try the pMAL system from New England Biolabs.  This is 
conceptually the same, giving fusions with maltose-binding protein, so you can 
purify the products from an amylose column.  The cleavage from MBP is affected 
with Factor Xa (as with pGEX-3X).  The really neat thing is that they have a 
vector, pMALp2, which has the MAL signal sequence included, so you can purify 
products from the periplasm.  Again, this should help if you get inclusion 
bodies in the cytoplasm.  I did come across a protocol which purported to 
achieve recovery from inclusion bodies, but I can not remember where.

There is another system by Studier, and you can get a catalogue from the 
company which distributes them;

Novagen inc
565 science drive
Madison WI 53711 USA

fax 608-238-1388

phone  800-526-7319 or 608-238-6110.

(Thanks Rod!!!!)  The appropiate bits are the pET vectors.

Best of luck,

Richard Grant,
Sir William Dunn School of Pathology,
Oxford University

Email rpgrant@vax.path.ox.ac.uk
From: hsb@ucselx.sdsu.edu (Hirdeypal S. Bhathal)
Newsgroups: bionet.molbio.methds-reagnts
Subject: ***Chicken cell (second request)****
Date: 6 Oct 92 17:10:12 GMT
Organization: San Diego State University Computing Services




Hi netters

I am looking for an easy-to-grow, easy-to-maintain and easy-to-transfect
CHICKEN cell line. Just about any type of chicken cell line will meet our needs as long as it can be grown and transfected with relative ease. 
If anybody has such a chicken cell line I would appreciate hearing from you.
Any response is welcome. Thanks.


Hirdeypal Bhathal
Dept. of Chemistry
SDSU, San Diego 
CA-92182, USA
(619)594-3771
hsb@ucselx.sdsu.edu
hsb@chem.ucsd.edu
Newsgroups: bionet.molbio.methds-reagnts
From: rrumpf@magnus.acs.ohio-state.edu (Robert Rumpf)
Subject: Genebank on CDROM?
Organization: The Ohio State University
Date: Wed, 30 Sep 1992 14:48:40 GMT

I have heard that it is possible to get Genebank on a CDROM, and get updates 
every so often...who do we call/write/email, how much is it, and does it work 
on Macintosh CDROM readers?

Bob
From: hsb@ucselx.sdsu.edu (Hirdeypal S. Bhathal)
Newsgroups: bionet.molbio.methds-reagnts
Subject: ***Help CHICKEN cell line (urgent)******
Date: 6 Oct 92 01:50:22 GMT
Organization: San Diego State University Computing Services



Hi netters

I am looking for an easy-to-grow, easy-to-maintain and easy-to-transfect
CHICKEN cell line. Just about any type of chicken cell line will meet our needs as long as it can be grown and transfected with relative ease. 
If anybody has such a chicken cell line I would appreciate hearing from you.
Any response is welcome. Thanks.


Hirdeypal Bhathal
Dept. of Chemistry
SDSU, San Diego 
CA-92182, USA
(619)594-3771
hsb@ucselx.sdsu.edu
hsb@chem.ucsd.edu
From: wyan@CUMC.CORNELL.EDU
Newsgroups: bionet.molbio.methds-reagnts
Subject: protein biotinylation
Date: 1 Oct 92 05:11:54 GMT
Distribution: bionet



>
>Hi, everyone:
>
>    I am trying to screen a lamdaZAP library with a biotinylated GST-R ( R is 
>intracellular domain of a receptor) fusion protein, with the hope to find something downstream bind speceficly to the receptor. So far, all the "positive plaqes" I detected are also "positive " in the GST control.  Has anybody been lucky in the samilar approach ?  Any valuble suggestions on following points are greatly appreciated, e.g. protein biotinylation, buffer systerm used in protein-protein interaction.
>
>
>wei Yan
>
>Cornell University
>
>
From: asweatma@crc.ac.uk (A.K. Sweatman)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Xho I: A Bad Actor?
Date: 24 Sep 92 11:49:32 GMT
Organization: MRC Human Genome Resource Centre

In article <1992Sep23.160245.9136@ulrik.uio.no>, olam@radium.uio.no (Ola Myklebost) writes:
> Have you/they tried XhoI from another supplier? We had an XhoI-prep from one 
> supplier which seemed Ok when used analyically on plasmids, but when used on 
> genomic DNA in PFG electrophoresis gave a terrible smear. XhoI from 
> Pharmacia, on the other hand, was fine.
> 
> Ola Myklebost
> University of Oslo
> Norway


I've had the same problem with XhoI in PFGE. How did you solve it?
What buffer did you use?
How long did you digest for?
We you XhoI from Northumbria Biologicals.
Thanks
Angela
From: Klaus.Matthaei@anu.edu.au
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Genebank on CDROM?
Date: 30 Sep 92 23:11:21 GMT
Distribution: bionet

>I have heard that it is possible to get Genebank on a CDROM, and get updates 
>every so often...who do we call/write/email, how much is it, and does it work 
>on Macintosh CDROM readers?
>
>Bob


G'Day Bob

IMHO forget CDRom etc.   Your CD's are out of date before you get them.  It
is far better to log into available services which are updated daily such
as ANGIS here in Australia or better still the American hook up which
escapes me at the moment.  We also have a windows interface for my Mac so
you are not bogged down with UNIX if that is what bothers you.

Cheers, Klaus


--------------------------------------------------------------------------
Catherine Shang
Gene Targeting
The John Curtin School of Medical Research
The Australian National University
E-mail: Catherine.Shang@anu.edu.au


--------------------------------------------------------------------------
From: Klaus.Matthaei@anu.edu.au
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: hybridization problems
Date: 30 Sep 92 23:30:22 GMT
Distribution: bionet

>To molecular biologists:
>I am having a problem in probing Northern blot membranes ans slot blot 
>membranes (Hybond N+). Sllight hazy background is evident on the 
>membranes, suggesting that the probe is OK.  However, where the RNA is 
>applied in slot blots or where it has migrated to in Northerns appears 
>white.  The image looks like a negative.
>	If you have encountered this problem or if you know of a 
>possible solution, please leave a message here.
>Thanks. 
>Juan


G'Day Juan

You have a common problem of not blocking your membrane against
non-specific hybridisation.  Nylon membrane needs better blocking than
nitrocellulose.  The negative image is due to the probe not hybridising to
that part of the membrane and because you have no target = no signal.

For a homologous DNA probe try hybing in: 50% deionised formamide, 2 x PE,
7% SDS, 1% BSA (0.5mg/ml Salmon Sperm DNA optional) at 50 degrees.  Wash
down to 0.2x SSC 1% SDS at 70.

Good Luck..Klaus

--------------------------------------------------------------------------
Catherine Shang
Gene Targeting
The John Curtin School of Medical Research
The Australian National University
E-mail: Catherine.Shang@anu.edu.au


--------------------------------------------------------------------------
Organization: Queen's University at Kingston
Date: Wed, 30 Sep 1992 09:05:34 EDT
From: Dr. Peter T. Boag <BOAGP@QUCDN.QueensU.CA>
Newsgroups: bionet.molbio.methds-reagnts
Subject: Sequencing PCR Products - Best way?




                                We sequence mtDNA segments amplified from
total genomic DNA, using a DS amplification followed by asymetric
amplification and SS sequencing with Sequenase. We never have a problem with
DS amplification if the primers are good, but asymetric amplification
and subsequent sequencing have always been unreliable, with a high
failure rate. Quality and quantity of SS template seems the problem
but we have tried every purification technique in the book without
consistent results (precipitation, Millipore and AMicon spin dialysis,
gel slice excision, sepharose spin column, etc.).
 We have been considering alternatives such as
       cycle sequencing, or cloning
of DS PCR product, or lambda exonuclease production of single stranded
template, etc. Any experience from the net would be much appreciated.
We are leaning towards giving cycle sequencing a serious try and would
appreciate experience with various kits available that have been tried
with PCR products. Thanks in advance, I will summarize for NET if
response dictates. - Peter Boag, Biology, Queen's University, Kingston,
Ontario, Canada.
From: FGARBRECHT@mis.mcw.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: immune precipitation
Date: 4 Oct 92 02:55:43 GMT
Distribution: bionet
Organization: Medical College of Wisconsin (Milwaukee, WI)

If you stain your gel for protein before autoradiography,
and you see bands in the appropriate places for immunoglobulin,
that suggests that the monoclonal is indeed binding the SPA and is
being precipitated, and that you should look elsewhere for the
problem. One difficulty may be that the protein you are trying to
immunoprecipitate does not label efficiently under the conditions you
are employing.  For 35S-methionine labeling, make sure the protein
contains methionine!  You may also extend the labeling time overnight
to enhance labeling (at the expense of increased background).  Check
out "Current Protocols in Immunology" for detailed protocols and
troubleshooting tips.  Good luck.

Fred Garbrecht
Medical College of Wisconsin
From: mangalam@SALK-SC2.SDSC.EDU
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Primer compilation
Date: 28 Sep 92 22:54:44 GMT
Distribution: bionet

Richard J. Hughes writes:

>I have a copy of the source code for Lander's primer but I'm not a 
>programmer and I don't have a C compiler.  So far, at least, I haven't been 
>able to find anyone else who has either.
>
>So...could someone please mail me a copy of the executable files or post 
>these on an ftp site?  Why aren't the executables posted along with the 
>source code?

   The exe's aren't posted because there are a vast number of machines and
configurations and it's more efficient to let each person set the compiler
switches themselves to produce a version best suited to their machine/OS. 
However, for some machines/OSs (generic DOS, generic Mac), it would make
sense to post them.
   You didn't mention what your machine/OS is.  I have exe's for a number
of machines - what's your pleasure?

Cheers,
Harry

Harry Mangalam                                   Vox:(619) 453-4100, x250
Dept of Biocomputing                                   Fax:(619) 552-1546
The Salk Institute                             mangalam@salk-sc2.sdsc.edu
10010 N Torrey Pines Rd                        mangalam@salk-sgi.sdsc.edu
La Jolla CA 92037                                    mangalam@salk.bitnet
Wed, 30 Sep 1992 14: 53:47 +0100
               Wed, 30 Sep 1992 14: 53:11 +0100
               Wed, 30 Sep 1992 16: 52:57 +0100
From: suter@VAX.MPIZ-KOELN.MPG.dbp.de
Newsgroups: bionet.molbio.methds-reagnts
Subject: re: cm amplification oof puc related plasmids
Date: 30 Sep 92 15:52:57 GMT
Distribution: bionet
Content-Identifier: 12
Original-To: methods@uk.ac.daresbury
Conversion: Prohibited

about cm amplification of puc plasmids:

+Subj:	Re: cm amplification?
+
+I really don't think that you need to mplify pUC based vector, sicne each
+cell has about 200 copies.  What you need to do is to increase the final
+cell density, which could be achieved by better nutrition.  See BRL's
+focus or catalog about the TB (terrific broth) medium.
+
+Tao
+--
+p

as should be clear from the discussion in the past few weeks, pUC based 
plasmids (are you sure about 200 copies per cell ??) can be amplified, 
enhancing the yield of a plasmidprep. 
TB has one disadvantage: the amount of contaminants (proteins or rna etc) 
also goes up, which may result in overloading of your purification system (be
it old fashioned CsCl or more modern methods).
thus, the ONLY advantage of tb is the lower volume to handle and (probably) 
the cost.
From: gessler@convex.HRZ.Uni-Marburg.DE (Gessler Manfred Dr.)
Newsgroups: bionet.molbio.methds-reagnts
Subject: cycle seq. of lambda
Date: 2 Oct 92 12:19:25 GMT
Distribution: bionet
Original-To: methods@uk.ac.daresbury

We would like to sequence lambda clones with cycle sequencing on an ABI
sequencer. Has anyone done this with dye-terminators, or do we need labelled
primers to get useful sequences? 
Any tips on protocols for DNA prep, oligo length etc. are welcome!

Thanks in advance

Manfred Gessler
Uni Marburg

gessler@convex.hrz.uni-marburg.de
FAX (49)6421-285630
From: ED@micro.uct.ac.za (Ed Rybicki)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Photographing Coomassie-stained Gels
Date: 30 Sep 92 10:06:50 GMT
Reply-To: ed@micro.uct.ac.za
Distribution: bionet
Original-To: muriana@edu.purdue.aclcb, methods@uk.ac.daresbury
X-Mailer: Pegasus Mail v2.1c R5.

> Dear Netters,
>
> does anyone have the appropriate photographic settings/conditions to get
> good quality black&white 35mm shots of Coomassie Blue-stained PAGE gels?
>
>   - filters for contrast?
>   - regular B&W film (100/125 ASA) vs. high contrast?
>   - backlighting (lightbox) preferred?
>   - instructions for photoprocessing prints (in regard to bracketed
>       exposure settings to see effect of lighter/darker exposure
>       since autoexposure print processing would make all of your
>       bracketed shots look the same)

1)  Kodak 1A red filter
2)  ordinary B&W film, 100/125 ASA
3)  on a diffuser light box (ie. backlit thru a diffuser screen)
4)  deliberately bracket exposures manually, or take 3 shots on
autoexposure but use the compensation dial (if you have one) to over- and
under-expose
5)  print on no. 3-5 to accentuate contrast, use hardest-looking neg with
lightest background.

Have fun!
Newsgroups: bionet.molbio.methds-reagnts