tritium-labelled proteins: best detection method

Jonathan B. Marder MARDER at agri.huji.ac.il
Sun Oct 2 01:29:23 EST 1994


In article <1994Sep25.144524.8738 at mbcf> heath at mbcf.stjude.org (Richard) writes:

>
>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....

Just to add, ... some years ago, I had success routinely using 1 molar sodium
salicylate (equilibrate gel, and dry directly) as a fluorographic enhancer.
While salicylate is noticeably inferior (e.g. to PPO) for S35 and
C14, for tritium the difference is virtually non-existant (probably due to the
shorter path of tritium beta emissions in the gel). I found it to give very
clean sharp bands. The main advantages of salicylate:-
1.  It is very cheap and easy to prepare (no solvents)
2.  Procedure is fast (adds 30-60 minutes to regular gel fixation + drying)
3.  Bands can be cut from dried gel and re-eluted with near 100% recovery (not
    true for other fluorography techniques)
__
Jonathan B. Marder                 '
Department of Agricultural Botany  |     Internet: MARDER at agri.huji.ac.il
The Hebrew University of Jerusalem | /\/ Bitnet:   MARDER at HUJIAGRI
Faculty of Agriculture             |/  \ Phone:    (08 or +9728) 481918



More information about the Methods mailing list