We offer a variety of anti-fluorescent dye antibodies that recognize specific fluorophores and, in most cases, quench their fluorescence. These anti-dye antibodies, including our antibody to the BODIPYTM FL fluorophore, can serve as cell-impermeant probes for determining whether fluorescent dye-conjugated ligands, proteins, bacteria, or other biomolecules have been internalized by endocytic or pinocytic processes.
The anti-BODIPYTM FL antibody (A-5770) was raised against the BODIPY FL fluorophore and exhibits no appreciable cross-reaction with non-BODIPY dyes, although it will cross-react to varying extents with other BODIPY dyes. In solution assays, we have found that this antibody effectively quenches the fluorescence of the BODIPY FL fluorophore, but quenches the BODIPY TR dye to a lesser extent and does not significantly quench the BODIPYTM TMR dye.
Invitrogen uses a sensitive quenching assay to ensure that this antibody is provided at a consistently high titer value. As supplied, 20 µL of the antibody solution is certified to produce ≥50% of the maximal fluorescence quenching of 1 mL of a 50 nM solution of BODIPY FL dye, assayed in 100 mM sodium phosphate, pH 8.0. Maximal quenching for BODIPY FL dye is ~80% of the fluorescence of the free dye. Due to steric hindrance, maximal fluorescence quenching of the BODIPY FL fluorophore covalently bound to protein may be significantly less. It is a good practice to centrifuge protein conjugate solutions briefly in a microcentrifuge before use; only the supernatant should then be added to the experiment. This step will eliminate any protein aggregates that may have formed during storage, thereby reducing nonspecific background staining.
BODIPY™ FL dye is a bright, green-fluorescent dye with a high extinction coefficient and fluorescence quantum yield. In contrast to the highly water soluble fluorophores Alexa Fluor™ 488 dye and fluorescein (FITC), BODIPY FL dyes have unique hydrophobic properties ideal for staining lipids, membranes, and other lipophilic compounds.