Countng prot bands after SDS-PAGE
mbrgw at s-crim1.dl.ac.uk
Wed Jan 26 06:39:43 EST 1994
In article <1994Jan25.212125.3206 at alw.nih.gov> djpf at helix.nih.gov (d fitzgerald) writes:
>I am cleaving a radioactive protein of 67 kD into two large fragments. I
>then separate the fragments from the parent protein by SDS-PAGE. I would like
>to get an accurate count of the radioactive fragments from slices that are
>excised from the gel. Is there a way to dissolve the gel and make the
>counts fully available to a liquid scintillation cocktail?
>Or is there some other trick that can be used to get the same result?
I asked about this a couple of weeks ago (THANKS to all those that replied -
I hadn't got round to reporting SUCCESS yet).
Having played with it a little bit, the best method (if you don't want to
spend money on special gel solubilisers) is that of Goodman (Anal. Biochem
42, 481 (1973)). It works even if you have dried the gel down onto paper!
Cut the band(s) of interest out, and put them in a vial, and add approx 0.25ml
(the less, the better) of a 99:1 mix of 30% H2O2:conc ammonium hydroxide (i.e.
ammonia in water) and incubate at 37C overnight (don't put the lids on too
tight, and watch out for nasty smelling stuff).
Then add scintillant (depending on the type, you may have to add somw water
Robin Walters. Robert Hill Institute, Sheffield UK.
A fact is an opinion that everyone agrees with.
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