Compound Interference

Compound Interference

FLEXYTE™ Assays Correcting for Interference Effects

Protease cleaves substrate removing dynamic quenching of 9AA by an aromatic moiety (Ar) leading to an increase in FLT.

 

Caspase-3 Standard Curveinterference

  • The red line represents the correlation between average FLT and product formation for a Caspase-3 assay. Data is taken from triplicates of mixed samples of substrate and cleaved product peptides (1 mM)
  • The green line is from an identical experiment but now including the interfering dye, 7-diethylaminocoumarin-3-carboxylic acid (DEAC) (10 mM). This highly fluorescent dye has a similar excitation and emission wavelength to 9AA but with a much shorter FLT (< 1 ns)
  • On initial inspection, the effect of a 10-fold excess of DEAC is to reduce the observed average lifetime of the 9AA-labelled peptide, which could lead to false positives in screening situations
  • However, unlike other fluorescence based assay techniques, the information rich FLT output permits analysis and correction of data using Fluorescence Analysis Software Technology (FAST) (Edinburgh Instruments Ltd.).3 As the FLTs of the substrate and product peptides are accurately known, the influence of an interfering short lifetime component can be easily identified and corrected for, returning the data shown in blue above
  • Hence, FLT technology has the capacity to not only flag up fluorescent interfering compounds but to also correct for their effect, leading to a reduction in the number of false positives and negatives
Flash replacement image