HDTenEightyP/NetNews huggingface dataset:
From: ED@micro.uct.ac.za (Ed Rybicki)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: hybridization problems
Date: 12 Oct 92 10:26:02 GMT
Reply-To: ed@micro.uct.ac.za
Distribution: bionet
Original-To: s882076@AU.OZ.rmit.cs.yallara, methods@uk.ac.daresbury
X-Mailer: Pegasus Mail v2.1c R5.

> From:       s882076@yallara.cs.rmit.OZ.AU (Sean Davidson)
> Subject:    Re: hybridization problems
> Date:       10 Oct 92 03:11:10 GMT

> Further to this discussion about different membranes, I recently began using
> the Digoxigenin-based system for Northern blotting. Initial attempts used
> Hybond N+, which is recommended for this procedure, but signal was always
> virtually undetectable amongst high background. When I began using
> Zetaprobe (Biorad) instead, results were (are) great.
>   So do you think this may have just been the batch of Hybond, or why are
> the results so much different?

I have seen a lab in the US to whom I had extolled the virtues of DIG,
fail miserably in many attempts to get Northerns to work in an environment
where 32P blots had been working well.  They also used to get "negative
bands" where the RNA should have been.  The local reps were of the
opinion that they should try the specialist Northern kit with RNAse-free
everything.  I then came home to find people gaily Northerning with plant
viral RNA and DNA probes with DIG, with excellent results - and they were
using Hybond-N, whereas my American colleagues were using AN other brand.
The answer is, who knows?  Probably one problem is the presence of RNAses
in the various blocking agents, etc, used with DIG - but then why does our
Genius kit work with our RNA?  Do we have a different batch of Boehringer
blocking agent?  Ours is still working, with chemiluminescence, and dye.
Maybe it'll stop, and we won't know why either...!  Will share protocols
if anyone is interested.

 ____________________________________________________________________
| 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: robert@arbo.microbiol.uwa.oz.au (Robert Coelen)
Newsgroups: bionet.molbio.methds-reagnts
Subject: BRL's cloneAmp system - any experience ?
Date: 13 Oct 92 01:53:51 GMT
Organization: University of Western Australia
NNTP-Posting-Host: mve.microbiol.uwa.edu.au


We are interested in using BRL's CloneAmp system, employing pfu
for accurate copying. Does anyone have any experience with this method ?
Whilst we're on the topic, what about their 3' and 5' RACE kits ?
Any comments gratefully received, acknowledged and summarised !


Robert Coelen
Dept of Microbiology
UWA
From: seh@bchm.biochem.duke.edu (Andrea Skantar)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Pierce Iodobeads
Date: 12 Oct 92 12:52:16 GMT
Distribution: bionet
Organization: Duke University Biochemistry Dept.

Speaking of iodination, does anyone have experience with Iodobeads from 
Pierce.  I've never done this sort of thing before and the thought of working
with free radioactive iodine creeps me out a bit.  I don't want to fry my
thyroid.  If anyone has any helpful hints or encouraging words, I'd love to 
hear them.  Thanks.

Andrea
From: Klaus.Matthaei@anu.edu.au
Newsgroups: bionet.molbio.methds-reagnts
Subject: Hybe conditions
Date: 15 Oct 92 23:40:11 GMT
Distribution: bionet

G'Day Netters

I have had a number of enquiries about the hybe conditions that I use
(which were worked out in the Reed Lab some years back) so I thought that I
would post them to avoid confusion. 

Note that these are for positvely charged membranes such as Hybond N+ after
alkaline transfer. 
 DO NOT USE THESE HIGH TEMPERATURES FOR NITROCELLULOSE.

DNA Probe DNA Target
2xPE, 7% SDS, 1% BSA hybe at 70 degrees or 
50% formamide, 7% SDS, 1% BSA at 43 degrees (To get the SDS into the hybe
dissolve the SDS in the formamide).
Wash down to 0.5x SSC, 1% SDS at 70 (or 0.2xSSC at 65).

DNA Probe RNA Target
50% formamide,2xPE, 7% SDS, 1% BSA at 50 degrees 
Was down to 0.2xSSC, 1%SDS at 70

Oligo probes
5xPE, 7% SDS, 1% BSA at the TH calculated from the oligo from
4x(G+C)+2x(A+T)-5 degrees C.
Wash down to 5xSSC, 1% SDS at the TH.

RNA Probe DNA Target.
50% formamide, 2xPE, 7% SDS, 1% BSA at 50 degrees.
Wash down to 0.2xSSc, 1% SDS at 70 degrees (if the background is high rinse
the membrane with 2xSSc and incubate in 1 ug/ml RNase A in 2xSSC for 15 min
and rewash in preheated 0.2xSSC 1% SDS etc).

RNA Probe RNA Target.
60% formamide, 2xPE, 7% SDS, 1% BSA at 60 degrees
Was as for RNA Probe DNA Target.

Washes are usually for 15 minutes only and a typical protocol is:
Rinse membrane after hybe briefly at RT in 2xSSc 0.1% SDS.
Wash 15 min RT 2xSSC 0.1% SDS with vigorous agitation.
Was in preheated hot wash as above and vigorous agitation.
Rinse in 2xSSC 0.1% SDS, Blot with 3mm, wrap in Saran wrap or similar film
(Caution some plastic films give you Saran-Zap from static electricity and
other background problems,  The Australian Glad-Wrap is also OK) and
expose.

Good Luck, 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: skspoidn@susssys1.reading.ac.uk
Newsgroups: bionet.molbio.methds-reagnts
Subject: RE: immune precipitation
Date: 7 Oct 92 08:29:20 GMT
Distribution: bionet
Original-To: methods@uk.ac.daresbury


(others have asked for this so...)
The only reply that I have got so far, which I have checked and unfortunately 
does not apply in my case, is that not all Abs bind protein A, and that there 
is a level of affinity at the Ig subclass level. It is best that the system
 be optomized empirically with the ab of interest.

The paper I originally got the protocol from (Borchelt et al., 1992: 
J Biol Chem v267 pp16188-16199) also suggests that the methanol 
precipitated proteins be resuspended in 3M guanidine thiocyanate,
 20mM Tris pH 7.5 for 10 min, followed by reprecipitation with 10
 vol methanol. This extra denaturation step apparently allows for 
a more quantitative dispersion of protein into DLPC. Note that this
 may only apply to the protein that they were working with (Prp).

This post was actually for a colleague, and he assures me
 that subclass affinity is not a problem in this case, so 
I repost my original question, being are there any (other)
 critical factors which influence immunoprecipitation?

Thanks in advance,

Mike Poidinger
Dept of Microbiology
University of Reading
From: mjones@crc.ac.uk (Dr. M.D. Jones)
Newsgroups: bionet.molbio.methds-reagnts
Subject: IN SITU PCR
Date: 14 Oct 92 13:13:52 GMT
Distribution: bionet
Original-To: methods@uk.ac.daresbury


Hi out there

I am posting this note for a colleague

He is having problems (numerous) on setting up in situ PCR.
If there is anybody out there with any experience, we would
love to hear from you.

:-) Should PCR In Situ be known as taking the P.I.S.

Correspondence to

Ian Teo
RPMS HIV Laboratory
Hammersmith Hospital
Du Cane Road
London,  W12 0NN, UK
Tel: 081-743-2030 extension 2305

or email to

mjones@uk.ac.rpms
mjones@rpms.ac.uk

Many thanks
Newsgroups: bionet.molbio.methds-reagnts
From: eboye@radium.uio.no (Erik Boye)
Subject: tightest promoter expression
Organization: University of Oslo
Date: Mon, 12 Oct 1992 13:52:29 GMT


It seems to me that you have three options:
1. The more easily tac-like promoters used in Herman Bujard's expression
vectors. I believe the promoter contains more operators.
2. The arabinose promoter, which has been used to clone genes for 
relatively toxic gene products.
3. The lambda pL promoter, which may be the best repressed promoter of 
the three (with excess cI857 repressor present). However, you will need
a temperature upshift to induce transcription.
From: beynonrj@uxb.liv.ac.uk (Dr. R.J. Beynon)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Protease inhibitors
Date: 16 Oct 92 07:44:52 GMT
Distribution: bionet
Organization: The University of Liverpool
X-Newsreader: Tin 1.1 PL5

NANTEL@UNCVX1.BITNET wrote:
: 
: 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
: 
this is curious - can't really explain that - antipain and leupeptin
are pretty benign

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

Hello Andre 

And I'm a biochemist playig at being a molecular biologist :-)

Preventing proteolysis during proten purification can be a pain. Some silly
questions first - 

Was the PMSF stock dissolved in water? - if so, it goes off with a half life
of minutes. Always make stock PMSF in dried solvents such as MeOH or PrOH.

PMSF only inhibits some serine proteases - there are another 3/4 classes
and some unknown class proteases lurking about. If you really want to eliminate
proteolysis, you have to be systematic, and start by making it happen (finding
conditions) and then trying to shut it down. There are dozens of inhibitors
that you could try.

You may find it helpful to have a look at the following:

Beynon, R.J. & Bond, J.S. Proteolytic Enzymes - A Practical Approach
Oxford University Press, (1989)

and 

Harris, E.L.V. & Angal, S. Protein Purification - A Practical Approach
same (1989) - I wrte a section for that on preventing proteolysis
pp40-50

and Walker, J. Methods in Molecular Biology, Vol 3 (1988) I wrote a similar
chapter for that book p1-23.

This may seem like a bit of drum-beating, and it's not meant to be - :-)
I just happen to have worked with thesedelightful enzyme classes for
quite a while. Can't understand why everyone else hates them ;-)
Hell - why not dump your protein and work on the protease instead :-)

Lastly, we discovered some years back that sample preparation for SDS-PAGE can
 cause some fragmentation too. Some of the degradation you see might be due to
gel analysis.

There are two reasons for this  

SDS unfolds substrates faster than proteases, so in a moment
of fleeting glory, the proteases go out with all guns blazing
and wipe out the substrate!

Excessive heating in SDS/reducing agent might be able to break up proteins
slightly as well.

We always TCA ppt proteins before dissolving in sample buffer.

Hope this helps
From: knoblauch@rz-berlin.mpg.de (Klaus v. Knoblauch)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: gentle radioiodination protocols
Date: 16 Oct 92 18:59:31 +0100
Organization: MPI f. molekulare Genetik, Berlin

In article <1992Oct12.090307.9414@vax.oxford.ac.uk>, adavies@vax.oxford.ac.uk (Anthony Davies) writes:
> 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)?
> 
Well, not quite from these days, but some years ago I used Bolton-Hunters-
Reagent (N-Succinimidyl 3-(4-hydroxy-5[125I]iodophenyl)propionate) to iodinate
Protein A which was subsequently used as a secondary antibody in immuno-
detection assays (western blots). The method was quite ok (as far as one can
say this about working with [125]I at all) and funktionally - well, it
funktioned in an immunological sense. But you may want to consider subsituting 
it with [35]S, if it is possible for you. BTW: The [125]I-labelling agent
as well as a [35]S labelling agent are both available from Amersham, so check
their catalog for ordering info (it might be available from others as well, 
but i at that time used theirs). 
finally, just to once again clear it up, I have worked only with the iodine  
reagent at that time only.

			have fun with it,
					Klaus
-- 
InterNet:  Knoblauch@rz-berlin.mpg.de
EAN     :  Knoblauch@mpimg-berlin.mpg.dbp.de
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
> Half a bee, philosophically, must ipse facto half not be. But can it be an >
> entire bee, if half the bee is not a bee, due to some ancient injury?      >
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>Monthy Phyton>>>>>>>>>>
Disclaimer: The above stated opinions are not necessarily shared by my
            employers. Some times not even by me.
From: jdboer@SOL.UVIC.CA (Johan deBoer)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Fluorescent guanine?
Date: 7 Oct 92 18:15:24 GMT
Distribution: bionet

Hello,

Maybe this is not the proper forum, but I will give it a try.
Does anybody know if guanine or some of its analogues, such as 
6-thioguanine or azaguanine fluoresces?
I would like to try to use this fluorescence as a probe for binding 
of guanine to an enzyme.

Any suggestions?

Johan de Boer
jdboer@sol.uvic.ca

Newsgroups: bionet.molbio.methds-reagnts
From: msw1633@zeus.tamu.edu (Mark S. Whitsitt, N5RJF)
Subject: Re: TA Cloning kits
News-Software: VAX/VMS VNEWS 1.41    
Reply-To: MSW1633@SUMMA.TAMU.EDU
Organization: Texas A&M University, Biochemistry Department
Date: Wed, 7 Oct 1992 14:47:00 GMT

In article <92274.190051ETWLC@CUNYVM.BITNET>, Elli Wurtzel <ETWLC@CUNYVM.BITNET> writes...
>Any recommendations?

Make your own T-vector...  

see: NAR vol.19 no.5 pp.1154 (1991)

It works great for me!

Mark S. Whitsitt, N5RJF        Texas A&M University, Dept of Biochemistry
Internet:  msw1633@summa.tamu.edu 	  College Station, Tx. 77843-2128
AMPRnet:   n5rjf@n5rjf.ampr.org 			   (409) 845-0832
Packet/APLINK:  N5RJF @ KE5HE.TX.USA.NA
   "You can't throw darts when you're empty, man." - another Shadelism
From: jdboer@SOL.UVIC.CA (Johan deBoer)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Protein expression systems
Date: 8 Oct 92 05:29:03 GMT
Distribution: bionet

Forwarded message:
> 
> We have been using the Maltose-Binding System over the last year
> with success except in one area which I think is worth mentioning.
> One can cleave the MBP from the protein of interest with factor Xa.
> One is then supposed to be able to to separate the cleaved
> MBP from the target protein by passing the cleaved mix over the amylose affinity
> column.  The protein of interest should pass through and the MBP should be retained.
> We find that after cleavage, the MBP fails to bind to the column.  I have talked
> 
We have been using this system with pretty good succes. The first problem we
had was that with the original pMAL vector both cleavage and binding to amylose
was not very good. This was solved by using instead the pIH902 vector which
puts a short amino acid linker between the MBP and your protein. This 
apparently makes it better accessible to Xa and to the amylose column. It worked
a lot better. 
The problem with binding of the MBP to amylose after cleavage (we had that 
problem too) was because we could not really get rid of the maltose. What
we eventually discovered was that simple passing the cleaved mixture over
an hydroxy apatite ultrogel column separated the MBP and our protein 
beautifully. The MBP sticks to the column and our protein (APRT) runs
through. this may not work for all proteins and you may have to fiddle 
with the salt concentrations of the HA column and the sample, but in our
case it worked very well.

Johan de Boer
jdboer@sol.uvic.ca
From: jmv@canctr.mc.duke.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: 33P: results!
Date: 9 Oct 92 19:16:23 GMT
Reply-To: jmv@canctr.mc.duke.edu
Organization: Duke U. Med. Ctr., Durham, NC

Here, finally, is what I've found out about 33P. It works terrifically
with regular (2 primer) "hot" PCR, using the same parameters as with
32P. Now we must try to titer reactions to see whether the 33P can be
diluted by a factor large enough to offset the price vs. 32P.

For cycle sequencing (gamma 33P end-labelling the primer), 33P used
in the same amount & same conditions as 32P yields much sharper results,
though so light that exposure time is at least doubled. The amount ued
according to Promega fmol (tm) kit methods is so small that, since 33P
1/2-life is ~4x as long as 32P's, we get a better deal paying 2x as much.

Q: Has anyone used 33P for probe labelling (for Southerns, eg)?
Q2: Has anyone had success with 33P and the CircumVent cycle sequencing
kit? There's a NEB promo for the latter offering free 33P, & I'm wondering
if it's worth anything.

czes'c'(-:

DB Loeb
-Neurology Div., Duke U. Med. Ctr.
--Durham, NC  27710
From: Klaus.Matthaei@anu.edu.au
Newsgroups: bionet.molbio.methds-reagnts
Subject: RT-PCR
Date: 9 Oct 92 04:33:44 GMT
Distribution: bionet

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
out a taq polymerase called Tet-Z which has both RT and polymerase activity
(indeed Amplitaq from cetus does this also).  The reverse transcription and
the PCR is therefore done in the same tube in the same set of cycling
reactions, the first one being a 15-30 minute RT step followed by standard
PCR.  We have also tried these methods (when I say we, read 1 honours
student and a postdoc with little molecular experience, and I have limited
experience with RNA) and get extremely variable results.  Sometime we get
good results and othertimes no more than a smear.  Before I personally
start running some experiments to work this out I thought that I would ask
all of you out there in Netland for your opinion.

Any help will be appreciated.

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: rpgrant@vax.path.ox.ac.uk
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Protease inhibitors
Date: 14 Oct 92 12:20:21 GMT
Distribution: bionet
Organization: Oxford University Molecular Biology Data Centre
Nntp-Posting-User: liz

In article <9210131601.AA11710@net.bio.net>, NANTEL@UNCVX1.BITNET writes:
> 
> 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.
A couple of suggestions;

i)  During the lysis procedure, watch the sonication!!!  This has been a 
source of grief to others.  Excessive sonication does appear to result in 
degradation.  I find 4 x 15 second blasts, with 30s intervals, works well for 
25ml resuspended bugs.  A colleague uses 3 x 30s, with 30s intervals, for 40ml 
solutions.  Keep it on ice ALL the time.

ii) Do you know at what stage degradation occurs?  The relevant chapter in 
"Current Protocols in Molecular Biology" (the Red Book) suggests use of 
* protease-deficient *strains of E.coli, such as lon, htpR, DnaJ for pMALc2 
fusions, depP and tsp for pMALp2 fusions (and probably c2, as well), 
and ompT for both.  

> 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.

So who needs a licence? 

> Andre Nantel
> Biology Dept.
> University of North Carolina


Richard Grant
Sir William Dunn School of Pathology
Oxford.
From: BOTDJL@LURE.LATROBE.EDU.AU (LOWDEN,Darren)
Newsgroups: bionet.molbio.methds-reagnts
Subject: secretion vectors
Date: 13 Oct 92 07:12:39 GMT
Distribution: bionet
Organization: La Trobe University
X-News-Reader: VMS NEWS 1.24

Some time ago I posted a message to this news service asking for any help
whatsoever with regard to a secretion vector. As I have received no mail I can
only assume that one of two events happened. Either no-one out there was able
to help me with my problem (highly unlikely) or the message was not posted
correctly (highly probable).

I would therefore like to rule out one of these possibilities by posting this
message out differently to the way it was previously posted. If the former
possibility was correct ie no-one was able to help with this particular problem,
then I apologise in advance to everyone who has already read this message.

The message that I posted was as follows:

Dear Networkers,

I would be very grateful if anyone could offer me advice on the following
problem. I am interested in obtaining a vector (either commercially or
directly from a lab), that is able to be propogated in both prokaryotic and
eukaryotic cells. However the vector has to be a secretion vector in the sense
that it makes the protein of interest which is then exported out of the cell.

There is the vector pMAMneo-s available from Clontech, in which the gene of
interest is inserted downstream from the signal peptide of the tissue-type
plasminogen activator (t-PA), thus resulting in the secretion of the protein
out of mammalian cells. However, expression of the inserted gene is under the
control of an inducible promoter. For my work, I require constitutive
expression of the gene thus leading to the continued presence of protein in the
absence of any inducers.

Thanks for your help.

Darren Lowen
botdjl@lure.latrobe.edu.au
From: story@binah.cc.brandeis.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Prime-it Kit
Date: 13 Oct 92 19:19:04 GMT
Reply-To: story@binah.cc.brandeis.edu
Organization: Brandeis University

Hello,
Have you or someone you love used the Prime-It (TM) kit from Promega
to random-prime label?  The company description sounds wonderful:

-probes in 2 minutes, >10^9 cpm/ug, etc.

Before dropping the $130, I would like some personal testimonies, especially
since random priming the "old fasioned" way is not difficult.

Please RSVP direct, to: story@binah.cc.brandeis.edu.
It may be of general enough interest for the net, so why not send it out as
well?  Thanks a million.

Craig Story
Brandeis University
Waltham, MA 02254-9110