There are numerous factors that can interfere with FRET-based detection. Here are a few items to consider:
a) Is there any component in the reaction solution that can quench green fluorescence?
b) Is there any component in the reaction solution that autofluoresces in the green or red emission range?
c) Too high of a substrate concentration may result in diffusional FRET: Lower the final working concentration of the substrate.
d) Ensure adequate dispersal of the substrate during the reaction. BODIPY dyes are very hydrophobic, and aggregates of the green product, BODIPY FL, can form an excimer and emit in the red range.
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