Questions on methylation interference assay
beatriz at pine.circa.ufl.edu
Sat Nov 14 21:01:00 EST 1992
While waiting to get a good exposure of my DNAse I footprinting gels
(3-4 weeks), I decided to try methylation interference. I basically
followed the protocol in Current Protocols in Mol. Biol. After
methylating my end-labelled 250 bp probe, I did as for the gel
mobility assays, and isolated the unbound and retarded complexes.
However, recovery was very low and I kenw I was going to have ot
expose it longer than my DNAse footp. gels. So I tried doing the
binding, and then methylating. Areas of the DNA protected by binding
proteins will not be methylated (I figured). For this I used enough nuclear
extract to shift all of my label. As a control, DNA w/o protein was
also methylated. Then I did as usual, piperidine cleavage, etc.
As a result I got a beatiful ladder, identical for both samples. A
friend in the lab says she's seen it done like this before, but has no
reference. I thought this way was faster than running a gel
retardation and exposing overnight, and so on.
Any comments on this will be appreciated. It is not "life or death",
but I hate trying a protocol that has worked for others, and then
finding I can't get it to work.
beatriz at ufpine beatriz at pine.circa.ufl.edu
Cuando suena el reloj despertador por la man~ana, recuerdo que "al que
madruga Dios lo ayuda", pero como "nunca es tarde si la dicha es buena"
y "no por mucho madrugar amanece mas temprano", sigo durmiendo.
More information about the Methods