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 Alexa Fluor® conjugates exhibit brighter fluorescence and greater photostability than the conjugates of other spectrally similar fluorophores. Alexa Fluor® 488 TFP ester produces a conjugate with excitation/emission of 495/515 nm that is spectrally similar to fluorescein (FITC) and Cy2 conjugates.
Alexa Fluor® dyes are available with fluorescence emissions that span the visible and near-infrared spectrum (see The Alexa Fluor® Dye Series—Note 1.1
in The Molecular Probes® Handbook) and provide the unique combination of water solubility and pH insensitivity between pH 4 and 10 for compatibility in diverse biological environments.
TFP (tetrafluorophenyl) esters are an improvement over the succinimidyl ester (SE or NHS-ester) chemistry typically used to attach fluorophores or haptens to the primary amines of biomolecules. Both reactive chemistries produce the same strong amide bond between the dye or hapten and the compound of interest, but TFP esters are less susceptible to spontaneous hydrolysis during conjugation reactions. Alexa Fluor® TFP esters are stable for several hours at the basic pH typically used for reactions – far outlasting succinimidyl esters.Typical Conjugation Reaction
You can conjugate amine-reactive reagents with virtually any protein or peptide; the provided protocol is optimized for IgG antibodies. You may scale the reaction 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.
The Alexa Fluor® TFP ester is typically dissolved in high-quality, anhydrous dimethylsulfoxide (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. Because the pKa of the terminal amine is lower than that of the lysine epsilon-amino group, you may achieve more selective labeling of the amine terminus 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 Sephadex™ G-25, BioGel® P-30, or equivalent. For much larger or smaller proteins, select a gel filtration media with an appropriate molecular weight cut-off or purify by dialysis.Learn More About Protein and Antibody Labeling