GUS assay with substrate MUG - is there oxidative degradation
timm.maier at hispeed.ch
Thu Mar 18 17:53:43 EST 2004
at least my Methylumbelliferon standard in a ordinary lab bottle was
stable for a year or so at least ! I don't think you loose it that easily!
Dirk Müller wrote:
> 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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