Detection of 3H-Leu by Autoradiography ?

Samuel P. Lee samuel.lee at utoronto.ca
Mon May 26 20:39:12 EST 1997


In article <EAAJA4.BHn at midway.uchicago.edu>,
   scb1 at midway.uchicago.edu (Samuel C. Blackman) wrote:

>My questions are:
>
> a.) Does any one do this and have protocol for it?
> b.) Does anyone have any idea about what brand/type of spray to use?
> c.) Can anyone provide any technical information (such as the minimum
>     specific activity of my 3H-Leu needed, exposure time, conditions,
>     etc.)?
>
>Any and all help would be, of course, greatly appreciated!
>
>Thanks!
>
>- Sam


I'm not sure about 3H-Leu, but there is extensive literature on 
autoradiography of fixed SDS PAGE gels after incorporation of 3H palmitate. In 
short, one electrophoreses the protein and then soaks the gel in a 
commercially available enhancing agent such as "Enlightening" from NEN or 
"Amplify" (I can't remember the manufacturer). The gel is then fixed in MeOH 
and Acetic acid and then dried. You can then expose the gel to film at -70 
degrees C and obtain an autoradiograph. Exposure time is unfortunately quite 
long usually (in the order of 3 - 6 weeks).

For details, I would suggest looking up a paper that looks at the 
incorporation of palmitic acid into proteins. I believe there was a section in 
 the "Methods in Enzymology" series in 1996 dedicated to palmitoylation of G 
proetins and G protein coupled receptors.

-------------------
Samuel P. Lee
samuel.lee at utoronto.ca
Department of Pharmacology
University of Toronto



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