tritium-labelled proteins: best detection method

Richard heath at mbcf.stjude.org
Sun Sep 25 15:45:24 EST 1994


In article <35s2km$bsm at mserv1.dl.ac.uk>, <dacoj3 at wptemp.kvl.dk> writes:
> We are looking at a putative methyltransferase from barley and have
> expressed the plant gene in coli as a fusion protein.
> 
> We have UV cross-linked tritiated SAM  to our fusion protein, as
> measured by scintillation counting nitrocellulose-bound material, and we
> are now ready to confirm whether the label is actually bound to our
> protein, as indeed all our controls suggest.
> 
> What is the current best method for detecting this after SDS-PAGE: we
> are talking about approx. 700 cpm. What about after a western blot? Is
> that sensitive enough?
> 
> all comments gratefully received
> 
> david collinge dacoj3 at wptemp.kvl.dk
> anders christensen andersc at biobase.aau.dk
> 
I routinely examine tritium labeled proteins by PAGE - after running the gel, I
soak in fixer, then in En3Hance (available from DuPont NEN), wash in water, dry
and slap a film on...  My autoradiograms are generally visible in 1-3 days, but
then I have much more than 700 cpm/sample!  (I uniformly label the prosthetic
group of ACP in E. coli by growing on labeled precursor).  I am awaiting
Molecular Dynamics to send me a Tritium screen for use with our PhosphImager
... this may well turn out to be more sensitive, but may well be out of some
budgets!!!  Unless you have a machine present, I suggest you try the En3Hance,
and expose the film at -80 for several weeks....

Good luck,

Richard Heath, Ph.D.
St Jude Children's Research Hospital
Department of Biochemistry
Memphis, TN 38101
heath at mbcf.stjude.org



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