FRET Conjugate Concentration Troubleshooting

I am using a cross-linked APC conjugate in a proximity assay (TR-FRET). When I increase the conjugate concentration, instead of getting a signal increase, I get a decrease!


The signal decrease seen with increased conjugate addition may come from self-quenching. This occurs with fluorescent dyes with small Stokes shifts due to overlap between their absorbance and emission spectra (Figure 1).

Because they absorb photons at some of the same wavelengths at which they emit, photons emitted by a given dye molecule may be absorbed by others in the reaction mixture before they reach the fluorescence detector (Figure 2).

At low dye concentrations reabsorption is minimal. As the dye concentration increases, it can lead to significant nonlinearity in the relationship between dye concentration and fluorescence (Figure 3).

In the extreme, measured fluorescence decreases with increases in dye concentrations.

The amount of self quenching observed will depend on the wavelength window used for detection (Figure 3), and can be minimized by the selection of an off-peak detection window, but the loss of signal that results can more than offset the benefits obtained.

Self-quenching is increased by any process that decreases the randomness of the distribution of molecules in solution. If the nature of the reagents (Table 1) or the assay system (Table 2) tends to bring dye molecules closer to one another than would occur if they were randomly distributed in solution, self quenching will be greater than predicted based on measured dye absorbance.

Experimental Design: Use of Excess Energy Donor.

Normally, it is better to have an excess of the energy donor dye (i.e. the dye that is initially excited by the source). Specifically, if one component of the assay system must be added in excess, design the system so that the donor is added in excess; minimize the amount of acceptor in the system. Figure 4 illustrates this principle.