Help with activity test

Cornelius Krasel krasel at wpxx02.toxi.uni-wuerzburg.de
Wed Jul 8 11:52:54 EST 1998


Heiko Koch <Heiko.Koch at ruhr-uni-bochum.de> wrote:
> The radioactive label is transferred to the target and the amount of
> labeled target is determine in a scintilator. 
> The reaction is stopped by adding 25 µl 50% TCA to the 100 µl
> incubation mixture.
> The mixture should be transfered to an Nitrocellulose filter
> and the filter must be washed to remove the unbound labeled NAD.
> 
> And this is the problem. Controls with no ribosyltransferase have as
> much cpm´s as the tests with ribosyltransferase
> 
> The problem is how to remove the non bound radioactive NAD.

How high is your background (i.e. buffer without protein on the
filter) ? What amount of incorporated radioactivity do you expect?

It is generally important to prewet the filters. If that doesn't
help, try separating the protein from nonincorporated NAD by
SDS gel electrophoresis. This method is obviously not suitable for
large numbers of samples but it should help you to pinpoint the
problem.

Good luck,
--Cornelius.

-- 
/* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */
/* D-97078 Wuerzburg, Germany   email: phak004 at rzbox.uni-wuerzburg.de  SP4 */
/* "Science is the game we play with God to find out what His rules are."  */



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