Alexa Fluor™ 420 NHS Ester (Succinimidyl Ester)

Alexa Fluor 420 NHS Ester is a fluorescence-based amine-reactive dye specifically optimized for multiplex immunohistochemistry and spatial biology. With excitation/emission maxima of 420/500 nm, this dye is a good match for the CFP filter on fluorescence microscopes.

Alexa Fluor dyes are reactive molecules that can be used to add a fluorescent label to the primary amines (R-NH₂) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The resulting conjugates exhibit bright fluorescence and excellent photostability.

Typical conjugation reaction

Amine-reactive Alexa Fluor 420 NHS Ester can be conjugated with virtually any protein, antibody, or peptide. 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.

Alexa Fluor 420 NHS Ester is typically dissolved in high-quality, anhydrous dimethylsulfoxide (DMSO) (Cat. No. D12345), and the reaction is carried out in 0.1–0.2 M sodium bicarbonate buffer, pH 8.3, at room temperature for 15 minutes. Because the pKa of the terminal amine is lower than that of the lysine epsilon-amino group, more selective labeling of the amine terminus may be achieved using a buffer closer to neutral pH.

Conjugate purification

Labeled antibodies are typically separated from free Alexa Fluor dye using a gel filtration column, such as those included in the Antibody Labeling Flex Kit for 0.5-1 mg of antibody (Cat. No. A88072).