Creating Complex Fluorophore Spectra on Antibodies Through Combinatorial Labeling

Fluorescently-labeled antibodies are central to many biochemical assays, but they are not easy to multiplex beyond 3-4 colors. A long-term hypothesis of ours is that labeling antibodies with multiple fluorophores, in a way such that fluorescence resonance energy transfer (FRET) occurs, may provide a way to increase fluorescence multiplexing ability by creating a rich variety of complex emission spectra that could be deconvolved via spectral methods. However, it is not yet clear how one can effectively label antibodies with multiple fluorophores that exhibit FRET. Here, we show how to use Mix-n-Stain antibody labeling kits from Biotium to label antibodies with multiple fluorophores that exhibit FRET. Key to our approach is the use of Fab fragments, as opposed to full IgG molecules, since the full IgG molecules are generally too large to allow the fluorophore proximity necessary for observable FRET. We show that our approach works with two different sets of FRET-capable fluorophore combinations: CF405M/CF488A and CF568/CF640R. These results form the basis for continued development of approaches for increased multiplexing of fluorescent antibody measurements.