The NHS esters (also known as succinimidyl esters) of the 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® 594 NHS ester produces a conjugate with excitation/emission of 590/617 nm that is spectrally similar to Texas Red.
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.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® NHS 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
We offer a wide selection of Molecular Probes® antibody and protein labeling kits to fit your starting material and your experimental setup. See Antibody Labeling from A to Z
or use our Labeling Chemistry Selection Tool
for other choices. To learn more about our labeling kits, read Kits for Labeling Proteins and Nucleic Acids—Section 1.2
in The Molecular Probes® Handbook.We’ll Make a Custom Conjugate for You
If you can’t find what you’re looking for in our online catalog, we’ll prepare a custom antibody or protein conjugate
for you. Our custom conjugation service is efficient and confidential, and we are ISO 9001:2000 certified.