counting 3H bands
Jonathan B. Marder
MARDER at agri.huji.ac.il
Mon Nov 15 11:19:23 EST 1993
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
> 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.
> 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
The Hebrew University of Jerusalem | /\/ Bitnet: MARDER at HUJIAGRI
Faculty of Agriculture |/ \ Phone: (08 or +9728) 481918
More information about the Methods