Summary: Storage of SDS/PAGE Gels [LONG]
Samuel C. Blackman
blackman at uicvm.cc.uic.edu
Wed May 10 09:42:17 EST 1995
First off, thanks to all yea- and nay-sayers who offered tips and
suggestions with regard to my question, "Can you store SDS/PAGE gels
between running them and transfer (for Western blot)." I'll be testing
some of these methods, and will post any exciting results to the
newsgroup. Here, as promised, is a summary of replies:
Fergus R. McKenzie (MCKENZIE at naxos.unice.fr) of the Centre de
Biochimie, CNR, Universite de Nice, France offers:
Yes, you can store SDS-PAGE gels easily, we have two types of storage;
Short term: completely seal the gel in a plastic bag (to prevent
drying out) and keep the gel at 4¡C. This is good for two to three
no problem. In addition, the rate of protein diffusion in my
very slow, so the gel definition is maintained.
Longer storage: completely seal the gel in a plastic bag and freeze
at minus 20¡C. I've kept gels like this for a month and then used them
Western blots with no apparent degradation of either the signal
or the definition.
Of course if you are trying to maintain extremely high resolution
of two proteins which are at a similar molecular mass..... then I'm not
sure how this type of storage holds up.
Failing that, if the technician is hyper-lazy, then after gel migration
can leave the gel between the glass plates, with stacker, and put it in
cold room overnight. As before, diffusion will be very low.
Frank Meloni (Meloni1 at jeflin.tju.ede) wrote:
I really don't know if you can successfully store a SDS-PAGE gel
and then do a transfer. To me seems unlikely, but you can (and I
have) stored transfered proteins almost indefinitely at -70 C if
you block blots first and store damp in a seal-a-meal bag. You
could also block, wash once and store at 4C for a week or two.
So why not blot both instead? Actually it is more efficient
than "saving" a gel!
Sharon Michelhaugh (skmichel at tempest.rs.itd.umich.edu), a fine,
friendly, fellow pharmacologist told me:
It depends what you call significant diffusion. We've had gels, that
technical reasons, (buffer leaked out of upper chamber) that were *run*
for two days, and we noticed that the bands were 'fuzzier'. Depending
your protein, that could be a problem. If I was going to try storing a
gel, I'd soak it in Transfer buffer, and keep it at 4 degrees.
We've got a lazy tech, too, and what she sometimes does, is just
two sets of samples, runs one set and freezes the other. So she can
quickly set up and run another gel if she needs to. But I agree that
should just run another gel if needed. Probably the only way to know if
storing the gel will work, is to just try it, and either it'll work or
Ray Oomen (roomen at hookup.net) replied:
Hi: Something which has worked for us :
- put cling wrap on a small rigid support large enough to support your
gel ( a small block of lucite works well) ,
- place the gel directly onto cling wrap,
- lay a second sheet of cling wrap over the gel,
- wrap the cling wrap around the gel and support (snug & airtight)
- put into a -70 freezer.
The gel is fragile in this state, so before freezing, we put it in a
plastic box. You can thaw the next day (e.g. keep it wrapped and run
tap water over it) and then blot.
I don't know how long you can store a gel in this condition, but we use
this when our experiments put us late into the day.
I got this zinger from Tony Houthaeve
(Tony.Houthaeve at EMBL-Heidelberg.DE) at the EMBL in Heidelberg:
Sorry to tell you, but be a bit nicer to your technician. First she is
not to be called lazy. She is practical and intuitive. Because if you
store the gels at 4¡C, you can do this for some time. Only, why does
blotting have to take so long. Normally in 1 hour it can be done.
Detection means another half hour. Or are you performing Westerns ?
So even some hours later the other gel can be processed. If the
gel-pattern is not to complex, than diffusion is after some days not
dramatic. Virgin gels overall can be stored for several weeks at 4¡,
several companies sell them like that, even cheap.
So overall no problem.
By the way, motivate personal, by giving once cake,coffee, a beer or
flowers. You do not motivate people by calling them 'damn lazy', that
what I mean.
James Bassuk <bassuk at u.washington.edu> contributed this:
I store my gels in 40% MeOH/10% HOAc.....they keep very long..
months....then I can always blot them onto nitrocellulose...
Richard P. Grant (rpgrant at molbiol.ox.ac.uk) at Oxford wrote:
I've always thought that one has to either blot within 1 hour of
fix in MeOH/Acac.
However, a little thought will show that any fool can pour, run and
blot a gel in a morning. Once blocked, the filter can be stored at 4C
for up to two
weeks. The BioRad kit is designed to blot two minigels at the same
can do the same with Hoeffer.
Jim Warren (jwarren at zool.umd.edu), the osmoregulator at Maryland, said:
i read your post to b.m.proteins but for some reason my newsreader
could not rply to that newsgroup. We do single dimension westerns in
my lab and i have routinely stored gels to be tranferred for up to 4
weeks at -80oC.(i do this to have a back up replicate blots or to
stain for proteins without any noticable sample diffusion). i
the gels in 4oC towbins transfer buffer(0.025M tris base,0.192M
20% MetOH) for 15 min., pour off excess buffer and freeze them. thaw
room temp for 30 min.(i would not recommend agitation). this has been
with both biorad mini gels(7cmx10cm?) and 16cm x 18cm gels.
the subsequent blots with hrp and alk. phosphatase has worked
i did test this procedure with bsa standards and commercial anti-bsa
sigma i think)which might be a good idea to confirm that this procedure
compatable with your blotting protocal. however, i think you have lost
Brian Pak (3bp7 at qlink.queensu.ca) up at Queen's U. told me:
We routinely store our gels indefinitely at -70 degrees celsius. We
the semi-dry system to tranfer proteins onto membranes. So, the gels
placed in 'semi-dry blot buffer' (48 mM Tris, pH 9.2, 39 mM glycine,
mM SDS, 20% methanol) for a few minutes after electrophoresis and then
frozen at -70 degrees. The methanol in the solution prevents gel
cracking. We have had no problems with protein transfer after thawing
these gels. Also, there is no appreciable diffusion of the proteins.
Kazuki Sasaki, M.D. (ksasaki at ncc.go.jp), from the National Cancer
Center Research Institute, Tokyo, Japan added:
If you store gels in methanol/acetic acid solution, they are OK so long
they are not dried out.
I don't recommend it for blotting. In my hands, storage in a blotting
buffer at 4 degrees C overnight is the limit, though the buffer
contains 20% MtOH. I tried an old gel (2 days old) for blotting
before, without success.
I'm not sure how come it turned out to be, but some of the lab manuals
suggest immediate blotting following electrophoresis.
Again, thanks to all who replied!
Samuel C. Blackman ! InterNet : blackman at tigger.uic.edu
MD/PhD Candidate (2/7)! Disclaimer : Who cares what I say, I'm a
Dept. of Pharmacology ! Quote : I'm not a doctor, but I play one on TV.
UIC College of Med. ! Phone : 312/996-4983 (lab) 312/996-1225 (fax)
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