APS & TEMED in acid gels
Louis Hom
So I'm running these HOAc-urea polyacrylamide gels (15% resolving, 4%
stack) and each part takes >1hr to polymerize at the concentrations of APS
and TEMED that I currently use. Will I mess things up by increasing the
APS & TEMED concentrations so that polymerizatin occurs more on the order
of 30 minutes instead? I won't be transferring or eluting the proteins
from the gel for anything.
--
_______________________________________________________________________________
Lou Hom >K '93
lh...@nature.berkeley.edu
http://www.ocf.berkeley.edu/~lhom
Matthew Parker
Sure, dump some more in there, shouldn't hurt. How much do you use per
ml of gel?
Louis Hom
In article <32E3E5...@topaz.microbio.uab.edu>,
Matthew Parker <par...@topaz.microbio.uab.edu> wrote:
>Sure, dump some more in there, shouldn't hurt. How much do you use per
>ml of gel?
For the resolving gel (15%, 12.5ml) I use 125ul 10% APS and 33ul TEMED. For
the stack (4%, 5ml) I use 90ul APS, 25ul TEMED.
Bernard Murray
In article <1997Jan22....@news.iup.edu>, jf...@grove.iup.edu says...
>
>If you can the amounts of APS and TEMED, you will change the kinetics of
>gelation (great, that's what you want).
Agreed. You can also control kinetics by changing the temperature.
> You will also change the porosity of
>the gel - the relative amount of cross-linking.
Is this really true? I was under the impression that the porosity
was governed by the % acrylamide and by the ratio of acrylamide/bis
(or other cross-linker). The concentration of TEMED and/or persulphate
shouldn't have any effect unless they become limiting - so that the
gel fails to polymerise completely no matter how long it is left.
Since they only catalyse the polymerisation of the acrylamide monomer
they play no real role in the eventual structure of the gel.
Can someone correct or confirm my assumption?
Bernard
Bernard Murray, Ph.D.
ber...@elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
The Great American Gene Company
In <5c5r41$r...@light.nih.gov> ber...@elsie.nci.nih.gov (Bernard
Dear Bernard:
It is most definitely true. There is a literature (albeit somewhat
obscure) on the subject. I can give you one reference:
MacDonell, M.T. et al. 1993. Minimization of pore diameter in
polyacrylamide by crosslinker concentration is temperature dependent.
Mol. Biol. (Life Sci Adv.) 12:47-48
Send me a fax number and I will fax it to you!
-Mike
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| nothing about them." Isaac Asimov |
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Steven Cohen
This does not make sense to me either. Perhaps the kinetics of the
polymerization will be increased, but it is the amount of Bis-Acrylamide and
total Acrylamide concentration that controls the quantity of cross-linking
which controls the porosity of the gel. Just like other catalysts, APS and
TEMED do not effect the final product but the speed in which equalibrium is
reached.
J C Ford
If you can the amounts of APS and TEMED, you will change the kinetics of
the gel - the relative amount of cross-linking. You don't want that. If
you're doing highly precise work, you'll see a change in the migration
pattern.
Edmundo Castro
Steven Cohen wrote:
>
> This does not make sense to me either. Perhaps the kinetics of the
> polymerization will be increased, but it is the amount of Bis-Acrylamide and
> total Acrylamide concentration that controls the quantity of cross-linking
> which controls the porosity of the gel. Just like other catalysts, APS and
> TEMED do not effect the final product but the speed in which equalibrium is
> reached.
> >
> >Is this really true? I was under the impression that the porosity
> >was governed by the % acrylamide and by the ratio of acrylamide/bis
> >(or other cross-linker). The concentration of TEMED and/or persulphate
> >shouldn't have any effect unless they become limiting - so that the
> >gel fails to polymerise completely no matter how long it is left.
> >Since they only catalyse the polymerisation of the acrylamide monomer
> >they play no real role in the eventual structure of the gel.
> > Can someone correct or confirm my assumption?
If you have an excess of APS the ratio of radicals generated would be changed.
Instead of having an optimal number of acrylamide radical being generated (which will
react with the bis-acrylamide) you will have an excess of APS radicals which:
1- will react with each other thereby terminating the reaction (they can no longer
generate acrylamide radicals).
2- more importantly: will react with acrylamide radicals thereby preventing the
acrylamide radical reaction with other acrylamide radicals and bis-acrylamide.
But, I have increased the amounts of APS and TEMED in the past (usually APS by
50%) and have had no problems. Usually, I only use freshly made APS for
sequencing gels, when I use old APS (up to 4 weeks old) in gels for westerns I bump
up the amount of APS.
BTW, warming up the mix slightly can significantly speed up the polymerazation
process.
EC
Elizabeth Rogers
On 22 Jan 1997, Bernard Murray wrote:
> In article <1997Jan22....@news.iup.edu>, jf...@grove.iup.edu says...
> >
> >If you can the amounts of APS and TEMED, you will change the kinetics of
> >gelation (great, that's what you want).
>
> Agreed. You can also control kinetics by changing the temperature.
>
> > You will also change the porosity of
> >the gel - the relative amount of cross-linking.
>
> Is this really true? I was under the impression that the porosity
> was governed by the % acrylamide and by the ratio of acrylamide/bis
> (or other cross-linker). The concentration of TEMED and/or persulphate
> shouldn't have any effect unless they become limiting - so that the
> gel fails to polymerise completely no matter how long it is left.
> Since they only catalyse the polymerisation of the acrylamide monomer
> they play no real role in the eventual structure of the gel.
> Can someone correct or confirm my assumption?
> Bernard
>
> Bernard Murray, Ph.D.
> ber...@elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
>
>
>Yes, the amt of initiators DOES affect the gel. Increasing the amt of
TEMED and APS shortens the polymer chain length. In extreme cases excess
initiator can produce a gel solution which does not appear to polymerize
at all. This from Bio-Rad bulletin 1156. On the other hand, I routinely
add extra TEMED to speed up my sequencing gel polymerization without any
apparent effects. Depends on how analytical your gel has to be I guess.
Scott Bean
In article <5c0ch6$q...@agate.berkeley.edu>, lh...@nature.berkeley.edu says...
>
>So I'm running these HOAc-urea polyacrylamide gels (15% resolving, 4%
>stack) and each part takes >1hr to polymerize at the concentrations of APS
>and TEMED that I currently use. Will I mess things up by increasing the
>APS & TEMED concentrations so that polymerizatin occurs more on the order
>of 30 minutes instead? I won't be transferring or eluting the proteins
>from the gel for anything.
>--
>______________________________________________________________________________
_
Can you change catalysts? We use ascorbic acid and ferrous sulfate
(heptahydrate) for the polymerization of acid gels. This mix will polymerize
so fast we have to cool the mixture before pouring or it will polymerize before
you can get the gel poured!
Scott Bean
Grad. student
Kansas State Univ.
O.C. Meijer
In article <5c5r41$r...@light.nih.gov>, ber...@elsie.nci.nih.gov says...
>Since they only catalyse the polymerisation of the acrylamide monomer
>they play no real role in the eventual structure of the gel.
length of chain is governed by rate of polymerisation and that by the
conc of aps & temed
Dr S. Robertson
Hideyuki Kajiwara (kaji...@abr.affrc.go.jp) wrote:
: In article <5cdmu9$9...@highway.leidenuniv.nl>,
: o.me...@chem.leidenuniv.nl wrote:
: > In article <5c5r41$r...@light.nih.gov>, ber...@elsie.nci.nih.gov
: says...
: >
: > >Since they only catalyse the polymerisation of the acrylamide monomer
: > >they play no real role in the eventual structure of the gel.
: >
: polymerisation is by radical reaction.
APS and TEMED are not strictly catalysts in this case they are initiators,
the greater the initiator concentration the more growing chains in
solution and the shorter the eventual chain length, therefore they do
play a role in the final stucture of the gel
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| University of Liverpool | Phone : +44 (0)151 794 4321 |
| School of Biological Science | Fax : +44 (0)151 794 4349 |
| Liverpool, L69 3BX. | |
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Hideyuki Kajiwara
Sonya Clark
I found using riboflavin is extremely useful for acid gels as the
APS/TEMED combination don't work well in acid conditions. Riboflavin acts
to accelerate polymerization.
To quote from Hames G., and Rickwood, D. Gel electrophoresis of proteins A
practical approach. 1990. - " In the APS-TEMED system TEMED catalyses the
formation of free radicals from persulphate and these in turn initiate
polymerisation. Since the free base of TEMED is required, polymerization
may be delayed or even prevented at low pH. increases in either the TEMED
or APS concentration increase the rate of polymerization. In contrast
.... the use of riboflavin/TEMED requires light to initiate polmerization.
This causes photodecomposition of riboflavin and production of the
necessary free radicals. Although gelation occurs when solutions
containing only acrylamide and riboflavin are irradiated, TEMED is usually
also included since under cetain conditions polymerization occurs more
reliably in its presence."
Stock solution: 4 mg riboflavin in 100 ml of water. Store at 4oC in dark.
I add half as much vol of this stock as I use of TEMED in stacking gels
for SDS-PAGE to get guaranteed perfect wells. Sorry, can't find how much
exactly you use for acid gels - I think it might be about the same ratio..
I have that info at home and will look it up if someone asks me! Ciao
Sonya Clark
PGEC-USDA
Albany CA
sac...@nature.berkeley.edu
Leonard Ornstein
Straight from (one of) the horses' mouths:
Louis' question has generated quite a thread; with a mixture of truth,
half-truth and error.
I'm one of the two (or four, depending how you look at it) inventors of
polyacrylamide electrophoresis; and it was B.J. Davis and I who first
introduced the APS/TEMED, TEMED/Riboflavin and APS/TEMED/Riboflavin
polymerization systems. Most of the details are available in Adobe
Acrobat versions of our two original manuscripts, which can be
downloaded from:
<http://www.pipeline.com/~lenornst/DiscElectrophoresis.html> .
My paper also discusses the effects of degree of cross-linking on
separation performance, Since, as already noted in this thread, the rate
of initiation is dependent upon the free radical concentration, and this
determines the number of chains that start to propagate, you ultimately
get many short chains, at high rates of polymerization, and fewer very
longer chains at lower rates. With high concentrations of BIS, this
hardly changes pore size. But with low concentrations of BIS, the
effective pore size can end up larger, and, as noted, in some cases the
solution, though viscous, might not even gel.
In addition, with a poorly optimized catalyts/initiator system, the
system may not consume all the monomer, and so the effective pore size
can end up larger than with an optimized system. (Incompletely
polymerized gels end up far-UV opaque, whereas completely polymerized
gels are usually far-UV transparent.)
Len Ornstein
J C Ford
>>
>>Is this really true? I was under the impression that the porosity
>>was governed by the % acrylamide and by the ratio of acrylamide/bis
>>(or other cross-linker). The concentration of TEMED and/or
>persulphate
>>shouldn't have any effect unless they become limiting - so that the
>>gel fails to polymerise completely no matter how long it is left.
>>they play no real role in the eventual structure of the gel.
>> Bernard
You're assuming that the cross-linker reacts with the same kinetic parameters
as the monomer. T'aint so. Thus, changing APS or TEMED concentrations will
change the relative reactivities of the cross-linker and the monomer, and
consequently, pore size.
Now, whether the change significantly alters your gel ... that's another
question. That's why I suggested, in my original response, that you try it
with several samples which would also be run under the original conditions.
Chances are that a modest change will have little visible effect (famous last
words).
JCF