PE/Cy5 is a tandem conjugate. The PE is excited at 488nm and functions as an energy donor for the Cy5. The energy is transferred via FRET (Fluorescence Resonance Energy Transfer) to the Cy5, which emits then the energy received in the form of long wavelength light at 670nm.
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 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, in order that the PE-Cy5 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 usually gives optimal results. 50ug of antibody corresponds to an Ab:PE-Cy5 molar ratio of 1:1. The amount of antibody used for labeling should be the same as the pack size of LL-PE-Cy5. 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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