How to detect para-nitroaniline with fluorescence

Eric at
Thu Oct 23 00:07:23 EST 2003

John Hines wrote:
> Never tried this before, but I'm sure someone out there has done something
> similar.  We could use some advice to prevent us from wasting our $$$.
> We are using a substrate which contains a fluorogen to detect the presence
> of our favorite enzyme.  As a result of the enzymatic reaction,
> p-nitroaniline is produced and can be detected via fluorescence.  While we
> have an automated plate-reader that can do fluorescence readings, we need
> to buy the appropriate filter(s) (for the platereader) to get the proper
> wavelengths to work with p-nitroaniline.
> The literature is a bit confusing on this point.  Some sources cite an
> excitation wavelength of 340 nm; others 355 nm; and one even said 405 nm
> (although this last one might be to detect p-nitroaniline via simple
> absorbance).
> If anyone out there is well-versed in fluorescent detection of
> p-nitroaniline, can you suggest an optimal excitation wavelength (and the
> cognate emission wavelength)?

You could use a fluorescence spectrophotometer, e.g., Hitachi F-2000 or
equivalant, to empirically determine the best excitation/emission
wavelengths for your samples.


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