PE/Cy5.5 is a tandem conjugate. In our Lightning-Link PE/Cy5.5 the best result is obtained when the tandem is excited at 488nm, wavelength at which the PE has its maximum absorption. The excited PE functions as an energy donor, where the energy is transferred via FRET (Fluorescence Resonance Energy Transfer) to the Cy5.5. The Cy5.5 emits then the energy received from the PE in the form of long wavelength light at 694nm.
The antibody is covalently bonded to the label in a directional and controlled process at near-neutral pH.In view of the large size of the PE-Cy5.5 label, the amount of antibody used in a labeling reaction with the tandem must always be less than the pack size of LL PE-Cy5.5, in order that the PE-Cy5.5 is in a slight molar excess. The best ratio for any new antibody reagent must be determined by experimentation but 50-60ug of IgG antibody for every 100ug of LL PE-Cy5.5 usually gives optimal results. 50ug of antibody corresponds to an Ab:PE-Cy5.5 molar ratio of 1:1. The amount of antibody used for labeling should be the same as the pack size of LL-PE-Cy5.5. For any new antibody, optimization of the ratio of antibody to dye is often worthwhile. If your antibody needs to be concentrated, purified, or cleaned up, consider purchasing an antibody clean up kit.
Glass vial(s) of Lightning-Link mix (1 or 3 vials, depending on pack size), 1 vial of LL Modifier reagent, 1 vial of LL Quencher FD reagent
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