SDS-PAGE band dissolution for scintillation counting - (May/29/2006 )
does any of you have a good protocol to solubilize a SDS-PAGE protein band (12%).
I would like to analyze tritium incorporation by liquid scintillation counting.
Thanks in advance
here is the procedure used here:
60% HClO4 (perchloric acid)
30% H2O2 (hydrogen peroxide)
cut out your bands with a sharp blade and place into glass scintillation vials (20ml).
add 0.8 ml of peroxide in a slow, dropwise, fashion.
add 0.4 ml of perchloric acid in a similar fashion.
seal vials with teflon or polyethylene lined caps (not foil lined).
incubate at 70C (in oven) until gel slice is digested (usually about 3 hours).
adjust pH to 6-7 by the addition of 5N NaOH (about 0.4 ml), check with pH paper.
add scintillation fluid (with chemiluminescence quenchers, if available).
allow to sit, in dark, for about 1 hour (until quenched) then count.
hope this helps
Thanks a lot.
Just one more question : you cut the band after staining in coomassie blue, and maybe rinsed in water, that's right?
yes, you stain the gel. rinsing with water is not necessary but wouldn't hurt.
thank you mdfenko
you're welcome missele.