pgegen at rnaworld.bio.ukans.edu wrote:
>Well, here I am with a question instead of an answer...
>We need methods for solubilizing 10% acrylamide/7M urea gels so that [3-H]radiolabeled nucleic acids can be quantified. I used to use perchlorate.H2O2 for 3% gels, but that doesn't work with these gels. We typically have 10 to 30 bands from a slab gel that must be quantified.
>Please bear in mind that polymerized acrylamide quenches 3-H, so solubilization is probably the best way to go.
>Any help at all would be greatly appreciated, and will be summarized here. Thanks!
I have in the past used hydrogen peroxide at concentrations as low as 6%
to solubilise 10% polyacrylamide gels. The presence of urea does not
seem to make much, if any difference, to the solubilisation. Like Dima
Klenchin, I used to solubilise overnight at 60C. Counting for 3H after
solubilisation can still be a problem as the solubilised acrylamide (and
perhaps the peroxide, as mentioned by Dima) quench 3H. Some people dry
down the gel before adding peroxide, and some add ammonium hydroxide.
You might also consider using one of the more easily broken crosslinkers
instead of bis-acrylamide: diallyltartardiamide is the one which springs
to mind, which is solubilised with low concentrations (less than 0.1M) of
periodate at room temperature. For an equivalent gel, the concentration
of diallyltartardiamide has to be greater than that of bis.
There are other cleavable crosslinkers, ethylene diacrylate crosslinked
gels dissolve in strong mineral bases.
I also remember a paper on acrylamide-agarose gels with no cross-linking
which had similar sieving properties to conventional acrylamide-bis gels,
which had been developed to avoid the problems associated with chemical
cleavage of crosslinkers.
Mick.Partis at BBSRC.AC.UK Horticulture Research International