BODIPY™ 650/665-X NHS Ester (Succinimidyl Ester)

BODIPY™ 650/665-X dye is bright, far-red fluorescent dye with similar excitation and emission to Cy5 or Alexa Fluor™ 647. It has a high extinction coefficient and fluorescence quantum yield and is relatively insensitive to solvent polarity and pH change. In contrast to the highly water soluble fluorophores Alexa Fluor™ 488 dye and fluorescein (FITC), BODIPY™ dyes have unique hydrophobic properties ideal for staining lipids, membranes, and other lipophilic compounds. BODIPY™ 650/665-X dye has a relatively long excited-state lifetime (typically 5 nanoseconds or longer), which is useful for fluorescence polarization-based assays and a large two-photon cross-section for multiphoton excitation. In addition to reactive dye formulations, we offer BODIPY™ 650/665-X dye conjugated to a variety of antibodies, peptides, proteins, tracers, and amplification substrates optimized for cellular labeling and detection.

The NHS ester (or succinimidyl ester) of BODIPY™ 650/665-X is the most popular tool for conjugating the dye to a protein or antibody. NHS esters can be used to label the primary amines (R-NH₂) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The resulting BODIPY™ 650/665-X conjugates exhibit bright fluorescence, narrow emission bandwidths, and relatively long excited-state lifetimes, which can be useful for fluorescence polarization assays and two-photon excitation (TPE) microscopy.

This reactive dye contains a seven-atom aminohexanoyl ('X') spacer between the fluorophore and the NHS ester group. This spacer helps to separate the fluorophore from its point of attachment, potentially reducing the interaction of the fluorophore with the biomolecule to which it is conjugated.

Detailed information about this BODIPY™ 650/665-X NHS ester:

Fluorophore label: BODIPY™ 650/665-X dye
Reactive group: NHS ester (succinimidyl ester)
Reactivity: Primary amines on proteins and ligands, amine-modified oligonucleotides
Ex/Em of the conjugate: 646/660 nm
Extinction coefficient: 102,000 cm-1M-1
Molecular weight: 643.45

Typical Conjugation Reaction
Amine-reactive reagents can be conjugated with virtually any protein or peptide; the provided protocol is optimized for IgG antibodies. The reaction can be scaled for any amount of protein, but the concentration of the protein should be at least 2 mg/mL for optimal results. We recommend trying three different degrees of labeling, using three different molar ratios of the reactive reagent to protein.

The BODIPY™ NHS ester is typically dissolved in high-quality anhydrous dimethylformamide (DMF) or dimethylsulfoxide (DMSO), and the reaction is carried out in 0.1-0.2 M sodium bicarbonate buffer, pH 8.3, at room temperature for 1 hour. Because the pKa of the terminal amine is lower than that of the lysine epsilon-amino group, you may achieve more selective labeling of the amine terminus using a buffer closer to neutral pH.

Conjugate Purification
Labeled antibodies are typically separated from free BODIPY™ dye using a gel filtration column, such as Sephadex™ G-25, BioGel™ P-30, or equivalent. For much larger or smaller proteins, select a gel filtration medium with an appropriate molecular weight cut-off or purify by dialysis. We offer several purification kits optimized for different quantities of antibody conjugate:
Antibody Conjugate Purification Kit for 0.5-1 mg (A33086)
Antibody Conjugate Purification Kit for 20-50 μg (A33087)
Antibody Conjugate Purification kit for 50-100 μg (A33088)

Learn More About Protein and Antibody Labeling
We offer a wide selection of Molecular Probes™ antibody and protein labeling kits to fit your starting material and your experimental setup. See our Antibody Labeling kits or use our Labeling Chemistry Selection Tool for other choices. To learn more about our labeling kits, read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in The Molecular Probes™ Handbook.

We’ll Make a Custom Conjugate for You
If you can’t find what you’re looking for in our online catalog, we’ll prepare a custom antibody or protein conjugate for you. Our custom conjugation service is efficient and confidential, and we stand by the quality of our work. We are ISO 13485:2000 certified.