GUS assay with substrate MUG - is there oxidative degradation of MU?

Dirk Müller Kraweel at web.de
Thu Mar 18 11:31:16 EST 2004


I have a severe problem with my GUS assay. I measure proteinextract
from fungal cells, expressing b-glucuronidase with the substrate MUG.
The b-glucuronidase converts MUG to the fluorophor MU by removing the
glucuronide. The fluoreszence ist measured with a microtiter plate
reader at the appropiate wavelength. In nearly all samples I observe a
signal reduction from time point 0 min to 20 min and then an increase
for over night incubation. The curve looks like a "V". The microtiter
plate is not sealed between the first two time points; for over night
incubation we seal it with a special foil. Is it possible that oxygen
(or light) damages the MU in a way that it looses its ability to act
as a fluorophor at the normal wavelength? In the over night reaction
the plates are sealed from light and oxygen so that glucuronidase can
again accumulate MU from MUG without "destroying" the MU?

I hope someone can help me or has observed a similar effect - this is
really a pressing problem.

thank you, Dirk...



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