Hi
I'm trying to set up an assay to detect the spatial location of laccase (enzyme oxidizing pnelocs under O2 consumption) acitivity in the stems of grass.
Assays are made by immersing cross section of stems in a substrate on microscopy slides.
When applying DAF (2,7-diaminofluorene) as a substrate I get good color development when dilluted in 0.25 M BisTris buffer (pH 5.9) but no development in a dilluted BisTris buffer (20mM, pH 5.9). 20 mM Tris (pH 8.0) also gives no color development. Color development in concentrated BisTris coincides with an assay using syringaldazine in ethanol as a substrate. A negative sample consisting of boiled material do not show color development for any of the substrates. Using ABTS as a substrate does not give any color development.
Does anybody have an idea why I do not get color development in dilluted buffer but do see a reaction when using the stock buffer.
Other ideas for making such assays are very welcome.
Cheers
Anders
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