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

Description

• 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-NH 2 ) 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.