Conjugation of Fluorochromes to Monoclonal Antibodies

Detection of cell surface molecules labeled by monoclonal or polyclonal antibodies conjugated to a fluorochrome is the most widely used application of flow cytometry. Here, we present protocols for tagging monoclonal antibodies with fluorescein, biotin, Texas Red, and phycobiliproteins. In addition, we provide a procedure for preparing a PE-Texas Red tandem conjugate dye that can then be used for antibody conjugation. These protocols enable investigators to label antibodies of their choice with multiple fluorochromes and permit more combinations of antibodies for multicolor flow applications. © 2023 Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol 1: Labeling an antibody with fluorescein isothiocyanate (FITC) Basic Protocol 2: Labeling an antibody with long-armed biotin Basic Protocol 3: Labeling an antibody with Texas Red-X Basic Protocol 4: Labeling an antibody with a synthetic organic fluor kit Basic Protocol 5: Labeling an antibody with phycobiliproteins Basic Protocol 6: Conjugation of Texas Red to R-phycoerythrin to produce an energy transfer fluorochrome.