| Detailed Description |
| Molecular Weight |
819.8 |
| Ex/Em |
590/617 nm |
| Molar Extinction Coefficient |
92,000 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 594 is a bright red dye. AF 594 dye is used to generate stable signals in imaging and flow cytometry analysis, and it exhibits water solubility and pH insensitivity (pH 4 to pH 10). The NHS ester (or succinimide ester) of AF 594 is a commonly 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 display brighter fluorescence and higher photostability compared to conjugates with other fluorophores that have similar spectral properties. |
| 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 |
Labeled antibodies are typically separated from free AF dyes using gel filtration columns (such as Sephadex™ G-25, BioGel™ P-30, or equivalent columns). For larger or smaller proteins, select a gel filtration medium with an appropriate molecular weight cutoff or purify via dialysis. |