Autoradiography of PAGE gels - please help

Megan oxberry at numbat.murdoch.edu.au
Sun Jun 2 20:45:20 EST 1996


I am trying to identify the protein to which a drug is binding in a
unicellular organism using autoradiography and native PAGE gels.  I
have been adding tritium labelled drug to sonicated cells (with
protease inhibitor), incubating the lysate at 4¡C for 24h then running
it on a gel.  I then fix the gel in 2-propanol/water/acetic acid
(25:65:10) for 30min and soak it in Amlify (Amersham) for 30min in the
dark.  I then dry the gel at 80¡C for 2h on to filter paper and place
it against Hyperfilm MP (preflashed) in a cassette for 1-2 weeks at
-80¡C.

I have had no success at all with this technique even with the positive
control I have been using.  

I would be most grateful for any suggestions regarding this.

Thankyou,
Megan



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