| Detailed Description |
| Molecular Weight |
1400 |
| Ex/Em |
702/723 nm |
| Molar Extinction Coefficient |
205,000cm-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 700 is a bright, photostable near-infrared dye that can be excited using a xenon arc lamp, far-red diode laser, or dye-pumped laser with an operating spectral range of 675–700 nm. The AF 700 dye is used to generate stable signals in imaging and flow cytometry analysis, and it exhibits water solubility and pH insensitivity (over the pH range of 4 to 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 700 is a widely used tool for conjugating this 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, but 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 different degrees 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 the terminal amine is lower than that of the lysine ε-amino group, you can use a buffer with a near-neutral pH for more selective labeling of the amine terminus. |
| 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.
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