| Detailed Description |
| Molecular Weight |
701.8 |
| Ex/Em |
430/545 nm |
| Molar Extinction Coefficient |
15,000cm-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 430 is a bright green fluorescent dye that is highly suitable for use with 405 nm violet lasers. AF 430 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 430 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 will exhibit brighter fluorescence and higher photostability compared to conjugates with other fluorophores of 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 according to the amount of protein used; 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 at room temperature in 0.1–0.2 M sodium bicarbonate buffer (pH 8.3) 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 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. |