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"I have used a wide variety of secondaries and Jackson ImmunoResearch has consistently been the best. The fluorophores are bright and stable and their selective (x reactivity removed) secondaries have always shown species specificity in multiple labeling."
Janet Duerr, Ohio University
FabuLight antibodies are Fab fragment secondary antibodies specific to the Fc region of IgG or IgM primary antibodies. They are available conjugated with 9 different fluorophores and biotin, and enable labeling of primary antibodies prior to incubation with cells or tissue. This provides a time saving alternative to sequential incubation flow cytometry and immunohistochemistry procedures, without compromising the active site of the primary antibody.
Possible uses of FabuLights also include labeling cell surface immunoglobulins without cross-linking and activating B cells, and labeling Fc chimeras (fusion proteins).
FabuLights are not provided cross-adsorbed against other species, so blocking steps may be required to avoid labeling endogenous immunoglobulins. For advice on developing protocols, refer to the example below, or the FabuLight white paper.
Incubation with FabuLight-labeled primary antibodies requires fewer washes than sequential incubation with primary antibodies and labeled secondary antibodies, thereby reducing damage to cells in flow cytometry protocols. Incubation steps in protocols requiring multiple primary antibodies from the same host animal are also reduced.
FabuLight binds to the Fc portion of the primary antibody (either anti IgG, Fcγ specific or anti-IgM, μ chain specific), leaving the antigen-binding region active.
The resulting complex of primary antibody with FabuLight does not precipitate or aggregate, because the dye-conjugated Fab anti-Fc secondary antibody fragments are monovalent. FabuLight-primary antibody complexes offer good tissue penetration and low non-specific binding.
Optimal protocols for each application must be established empirically. Label the less abundant target antigen first for optimal results. Complexing at a 3:1 molar ratio of FabuLight:Primary Antibody (equal weight ratios) provides a good degree of labeling of the primary antibody without excessive amounts of unbound Fab. Titrating Fab-labeled complexes vs. their target antigens will minimize the amount of free Fab anti-Fc, thereby minimizing potential cross-talk in a multiple labeling application.