FRET Reagent and System Tables

CHARACTERISTICS OF REAGENTS THAT AFFECT SELF-QUENCHING:

Table 1

  • Extent of overlap between excitation and emission curves - broad emission curves and small Stokes shifts lead to the greatest amount of self-quenching.
  • Quantum efficiency - dyes with high quantum efficiency are less subject to self-quenching because they can be used at lower concentrations to achieve a desired level of fluorescence, thus minimizing the absorbance of the solution.
  • Formation of molecular associations - many dyes (including phycobiliproteins and cyanine dyes) occur in solution in equilibrium between single molecules and associations of two or more molecules. Since these associations reduce the randomness of the molecules in solution, they increase self-quenching.

Table 2

CHARACTERISTICS OF ASSAY SYSTEMS THAT AFFECT SELF-QUENCHING:

  • Cell-based systems - where multiple dye molecules are attached to the surface of a cell, the average distance between molecules is reduced relative to molecules in solution and self-quenching increases.
  • Immobilized reactants - when the dye is attached to a surface (bead, plate, fiber, etc) randomness is reduced and self-quenching increases.
  • Incomplete mixing - if the reaction mixture is not well mixed, dye molecules will be localized and self-quenching increases.
  • Multiple tagging - when more than one dye molecule is attached to a reactant, self-quenching increases. NOTE: while this reduces fluorescence yield from the labeled reagent somewhat, it will not in itself lead to nonlinearity of the analyte-fluorescence relationship, provided the reagent is properly standardized.