Gels keep cracking
S. McKay
I've been having some problems drying mini-gels. I'm using 14%
SDS-PAG to separate my protein (which is 35S-labelled). Following this I
have been fixing the gels in 7% Acetic Acid for 20', rinsing in dH2O and
then an amplification step using 0.5M Salicylic Acid for 20'. The gels are
then dried onto Whatmans 3mm Chromatography Paper under vaccum and heat on
a BioRad Gel Slab Dryer for 1.5hrs. Less time than this and the centre of
the gels do not dry. Unfortunately, I've been experiencing severe cracking
problems with the gels which is obviously unacceptable.
I am aware that the salicylic acid will remove much of the moisture
from the gel but 1.5hrs seems a long time.
I've also tried using the 0.5M Salicylic Acid combined with 2%
glycerol but this makes no difference and if anything the cracking is
worse.
This has become a very frustrating problem and any suggestions you
could make would be very much appreciated.
S.McKay
Richard V. Giles
I'd up the glycerol content to 10%.
In the past when we've dried SDS PA gels the final step was 5% acetic
acid, 10% glycerol. No cracks when dried as you have described. Don't
know how the 0.5M salicylic acid would interact with this though...
--
Richard V. Giles Ph.D.
School of Biological Sciences
University of Liverpool
Tel +44/151 794 4389
Fax +44/151 794 4349
http://www.liv.ac.uk/~giles/
kre...@my-dejanews.com
by using 10% V/V HAc, 10% V/V Glycerol without any success.
--
Kresten Lindorff Larsen
Dept. Yeast Genetics
Carlsberg Laboratory
Denmark
In article <365FD473...@liv.ac.uk>,
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Peter
samples use I125 as the label source. I do not fix the gels and I do not
use glycerol. But if you can get rid of the fixing step and dry the gel
immediately following the run you may have more success. The required
amplification may not be needed if you look for a "specialty film that is
more tuned toward S35.
I dry my gels with a uniform heting of the gels for 2 hrs @ 80 C under a
vacuum. Be careful not to strech the gel when laying it down on the filter
paper. I usually lay the gel on the filter paper from the bottom to the
top of the gel because the remenant stacker layer is really sticky and can
cause the gel to elongate at the corners.
Hope this helps
Peter
In article <v01510100630b6350aaa4@[132.234.31.46]>, S.M...@sct.gu.edu.au
Sam Michaelson
> I've been having some problems drying mini-gels. I'm using 14%
>
> SDS-PAG to separate my protein (which is 35S-labelled). Following
> this I
> have been fixing the gels in 7% Acetic Acid for 20', rinsing in dH2O
> and
> then an amplification step using 0.5M Salicylic Acid for 20'. The
> gels are
> then dried onto Whatmans 3mm Chromatography Paper under vaccum and
> heat on
> a BioRad Gel Slab Dryer for 1.5hrs. Less time than this and the
> centre of
> the gels do not dry. Unfortunately, I've been experiencing severe
> cracking
> problems with the gels which is obviously unacceptable.
I haven't run mini gels for a couple of years now, but I do remember
throwing the odd screaming fit trying to dry them down. I was drying
down 12% SDS-PAGE gels that were 0.75 or 1.5 mm thick (BioRad
Mini-Protean II apparatus). Through a series of trials and
observations, I found out that I got the best results by:
1) allowing gels to resaturate fully in water (tried various glycerol
concentrations up to 10% to no avail)
2) this seemed to do the trick: try not turning on the heater
immediately on the gel dryer. I found that by allowing the gel to
flatten and dehydrate a bit without the heater, then increasing the heat
gradually, I usually got quite a good result. Yes, it took a bit
longer, but for my gels, it seemed to stop them cracking.
This assumes, of course, that your gel dryer is fully functional. Does
the gel crack no matter where on the screen you place it? Do everyone
else's gels crack, too? (I ask because we had a certain amount of
trouble with a gel dryer that was not creating an even vacuum over the
whole surface - once we cleaned that up, it worked fine).
Good luck
Sam Michaelson
Jimmy Loh
Well, my colleague did something different. He was doing 1.5mm gels and
reckoned that it was due to the thickness of the gel that caused cracking
while drying. Our thin gels didn't crack...it was always the thick ones.
What he did was to do a final soak in (10%acetic acid,50% alcohol)
similar to destain solution overnight. This shrinks the gel n
reduces its thickness. THen he dried it down by placing on whatman n using
the gel dryer. Works for him. :)
Cheers
Jimmy Loh
Ian A. York
>
>the gels do not dry. Unfortunately, I've been experiencing severe cracking
>problems with the gels which is obviously unacceptable.
Make sure the gel dryer vaccuum is good. House vacuum and tap vaccumm
will often dry low-concentration gels but may not be not adequate for
high-concentration. If you use a vaccuum pump, be sure it's working
properly.
You might also, or instead, try a high-tensile-strength acrylamide. We
use Duracryl, which we started using when we had precisely the same
problem; we have only had one or two gels crack in the two-three years
since we started using Duracryl, and those were due to flagrant gel abuse
that would get you arrested in seventeen states.
Ian
--
Ian York (iay...@panix.com) <http://www.panix.com/~iayork/>
"-but as he was a York, I am rather inclined to suppose him a
very respectable Man." -Jane Austen, The History of England
theo...@medlib.georgetown.edu
pxp...@unixs.cis.pitt.edu (Peter) wrote:
> I routinely dry my 14% gels with about a 10% cracking frequency. My
> samples use I125 as the label source. I do not fix the gels and I do not
> use glycerol. But if you can get rid of the fixing step and dry the gel
> immediately following the run you may have more success. The required
> amplification may not be needed if you look for a "specialty film that is
> more tuned toward S35.
> I dry my gels with a uniform heting of the gels for 2 hrs @ 80 C under a
> vacuum. Be careful not to strech the gel when laying it down on the filter
> paper. I usually lay the gel on the filter paper from the bottom to the
> top of the gel because the remenant stacker layer is really sticky and can
> cause the gel to elongate at the corners.
> Hope this helps
> Peter
>
> In article <v01510100630b6350aaa4@[132.234.31.46]>, S.M...@sct.gu.edu.au
> (S. McKay) wrote:
>
> > Hi,
> >
> > SDS-PAG to separate my protein (which is 35S-labelled). Following this I
> > have been fixing the gels in 7% Acetic Acid for 20', rinsing in dH2O and
> > then an amplification step using 0.5M Salicylic Acid for 20'. The gels are
> > then dried onto Whatmans 3mm Chromatography Paper under vaccum and heat on
> > a BioRad Gel Slab Dryer for 1.5hrs. Less time than this and the centre of
> > problems with the gels which is obviously unacceptable.
> >
> > from the gel but 1.5hrs seems a long time.
> >
> > I've also tried using the 0.5M Salicylic Acid combined with 2%
> > glycerol but this makes no difference and if anything the cracking is
> > worse.
>
It's funny, but when I used to do a lot of metabolic labelling experiments, I
used good old PPO/DMSO, which seems to be out of fashion these days, but I
never had a gel break on the dryer (even 15% gels). They seem pretty tough
after the PPO is precipitated into the gel; you can pretty much throw them
around.
Nick Theodorakis
Peter
theo...@medlib.georgetown.edu wrote:
> It's funny, but when I used to do a lot of metabolic labelling experiments, I
> used good old PPO/DMSO, which seems to be out of fashion these days, but I
> never had a gel break on the dryer (even 15% gels). They seem pretty tough
> after the PPO is precipitated into the gel; you can pretty much throw them
> around.
But there is a good reason why this techinique has lost favor. The
chemicals are rather nasty.
Peter Pediaditakis
--
Peter
" Don't you eat that yellow snow
Watch out where the huskies go"
FZ
theo...@medlib.georgetown.edu
Well, I hate to start another thread like the one we had on the toxicity of
acrylamide, but... is it really true? I have often heard that PPO is supposed
to be nasty, but without any supportive evidence. OTOH, consider this MSDS
data..
(from http://www.packardinst.com/msds/msds139.htm)
OMNIFLUOR®
1. Identification of the Substance/Preparation and of the
Company
Company Name:
Packard BioScience B.V.
Chemical Operations
P.O. Box 9403
9703 LP Groningen
The Netherlands
Tel: (31) 50-5413360
Fax: (31) 50-5422292
Emergency Telephone: (31) 50-5413360
Chemical Family: Mixture of aryloxazole and aromatic
hydrocarbon
2. Composition/Information on Ingredients
Material CAS Number Concentration
2,5-Diphenyloxazole (PPO) 92-71-7 97-99%
p-Bis-(o-methylstyryl)-benzene (bis-MSB) 13280-61-0 1-3%
3. Hazards Identification
Symbol -
Special Risk for People and the Environment
None
^^^^
HMIS Hazard Code Ratings
Health 1
Flammability 0
Reactivity 1
Personal Protection B
<snip>
11. Toxicological Information
2,5- Diphenyloxazole
Eye: Irritating
Skin: Non-irritating
Toxicokinetics: Non-accumulative
Mutagenicity: Non-mutagenic
^^^^^^^^^^^^^
Carcinogenicity: Non-carcinogenic
^^^^^^^^^^^^^^^^
Teratogenicity: Non-teratogenic
^^^^^^^^^^^^^^^
(emphasis is mine)
-----------------------------------------------------------------------
Hung-yiu Ho
On 30 Nov 1998, Ian A. York wrote:
> In article <v01510100630b6350aaa4@[132.234.31.46]>,
> S. McKay <S.M...@sct.gu.edu.au> wrote:
> >
> >the gels do not dry. Unfortunately, I've been experiencing severe cracking
> >problems with the gels which is obviously unacceptable.
>
Peter
hy...@GATE.SINICA.EDU.TW (Hung-yiu Ho) wrote:
> where can I purchase Duracyl? Please respond.
Millipore