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Figure 1 - Overlap (shaded area) between absorbance and emission spectra of Cy5 (left) and Allophycocyanin (right)

Figure 2 - Reabsorption of fluorescence in a dye solution

Figure 3 - Nonlinearity of Allophycocyanin (APC) fluorescence as a function of concentration for two different detection windows

Figure 4 - Fluorescence effect of serial additions of biotinylated Allophycocyanin (APC) to two different concentrations of Streptavidin-B-Phycoerythrin conjugate (SA-BPE)

B-Phycoerythrin (BPE) was paired with allophycocyanin (APC) to simulate a FRET assay.

At a low concentration of donor (B-PE/SA = 0.4 ug/mL), all available donor is used by the time about 1 ug/mL of acceptor (APC-biotin) is added, and subsequent additions of acceptor serve only to increase self-quenching.

A higher concentration of donor (B-PE/SA = 4 ug/mL) leads to significantly enhanced fluorescence.  But even in the presence of this tenfold higher concentration of donor, increasing acceptor concentration above  1 - 2 ug/mL is counterproductive due to self-quenching.

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