| Detailed Description |
| Molecular Weight |
1100 |
| Ex/Em |
668/698 nm |
| Molar Extinction Coefficient |
132,000 cm-1M-1 |
| Reactive Group |
NHS Ester |
| Reactivity |
Primary amines on proteins, ligands, and amine-modified oligonucleotides |
| Transportation and Storage |
Room temperature or wet ice, -20°C, protected from light |
| Introduction |
AF 660 is a bright, photostable far-red fluorescent dye whose excitation spectrum is highly compatible with 633 nm or 647 nm laser lines. AF 660 dye is used to generate stable signals in imaging and flow cytometry analyses, featuring water solubility and pH insensitivity (pH 4 - pH 10). The fluorescence of this long-wavelength AF dye is invisible to the human eye but can be easily detected by most imaging systems. The NHS ester (or succinimide ester) of AF 660 is a commonly used tool for conjugating the dye to proteins or antibodies. NHS esters can be used to label primary amines (R-NH?) in proteins, amine-modified oligonucleotides, and other amine-containing molecules. The resulting AF conjugates exhibit brighter fluorescence and higher photostability compared to conjugates of other fluorophores with similar spectra. |
| Typical Conjugation Reaction |
You can conjugate amine-reactive reagents to nearly any protein or peptide (the provided protocol is optimized for IgG antibodies). The reaction can be scaled proportionally for any amount of protein; however, for optimal results, the protein concentration should be at least 2 mg/mL. We recommend using three different molar ratios of reactive reagent to protein to achieve three distinct levels of labeling.
AF NHS esters are typically dissolved in high-quality anhydrous dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) (D12345), and the reaction is carried out in 0.1–0.2 M sodium bicarbonate buffer (pH 8.3) at room temperature for 1 hour. Since the pKa of terminal amines is lower than that of the ε-amino groups in lysine, you can use a buffer with a near-neutral pH to achieve more selective labeling of the amine termini. |
| Conjugate Purification |
Typically, gel filtration columns (such as Sephadex™ G-25, BioGel™ P-30, or equivalent columns) are used to separate labeled antibodies from free AF dyes. For larger or smaller proteins, select a gel filtration medium with an appropriate molecular weight cutoff or perform purification via dialysis. |