counting 3H bands

[Jonathan B. Marder]

In article <93317.123316XNRH at PURCCVM.BITNET> <XNRH at PURCCVM.BITNET> writes:
>Subject: counting 3H bands
>From: <XNRH at PURCCVM.BITNET>
>Date: Sat, 13 Nov 1993 17:33:16 GMT

>Hi folks:
>  I am trying to use a scintilation counter to measure the
>amount of tritium labeled product (proteins) in pieces of
>polyacrylomide gel. I expose the film and later on I slice
>the portion of the gel that contains the band, add water,
>incubate for a while, and finaly add enhancer fluid.
>  My problem is that the countings I am getting are much lower
>than expected. Could this be due to quenching?
>  Is there anyone who knows a technique for this procedure? Any
>way to destroy the gel slice (acidic or basic digestion). Since
>I could do this directly in the scintillation vial, I do not
>care about the proteins being also destroyed!

>                                  Thanks in advance.

>                                           Luiz
>                                     XNRH at PURCCVM.BITNET

I used to do this with Amersham's tissue-solubiliser NCS.  My recipe was
1.   30 microlitres NCS
2.  200 microlitres water
3.   4 ml Scintillation fluid (toluene based if I remember rightly)

This doesn't dissolve the polyacrylamide, but the gel piece swells enormously
and I guess most of the protein elutes.  It could be that there are now much
better cocktails/procedures available.

With tritium, you would in most cases expect to see significant quenching.

An alternative is to impregnate the intact gel with salicylate or PPO and do
quantitative fluorography + densitometry.
__
Jonathan B. Marder                 '
Department of Agricultural Botany  |     Internet: MARDER at AGRI.HUJI.AC.IL
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