Alexa Fluor™ 532 is a bright yellow dye with excitation ideally suited for the frequency-doubled Nd:YAG laser line. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor™ 532 dye is water soluble and pH-insensitive from pH 4 to pH 10. In addition to reactive dye formulations, we offer Alexa Fluor™ 532 dye conjugated to a variety of antibodies, peptides, proteins, tracers, and amplification substrates optimized for cellular labeling and detection (learn more).

The NHS ester (or succinimidyl ester) of Alexa Fluor™ 532 is the most popular tool for conjugating this dye to a protein or antibody. NHS esters can be used to label to the primary amines (R-NH₂) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The resulting Alexa Fluor™ conjugate will exhibit brighter fluorescence and greater photostability than the conjugates of other spectrally similar fluorophores.

Detailed information about this AlexaFluor™ NHS ester:

• Fluorophore label: Alexa Fluor™ 532 dye
• Reactive group: NHS ester
• Reactivity: Primary amines on proteins and ligands, amine-modified oligonucleotides
• Ex/Em of the conjugate: 530/555 nm
• Extinction coefficient: 81,000 cm^-1M^-1
• Molecular weight: 723.8

Typical Conjugation Reaction

You can conjugate amine-reactive reagents with virtually any protein or peptide (the provided protocol is optimized for IgG antibodies). You can 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 dimethylformamide (DMF) or 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. We offer several purification kits optimized for different quantities of antibody conjugate:

• Antibody Conjugate Purification Kit for 0.5-1 mg (A33086)
• Antibody Conjugate Purification Kit for 20-50 μg (A33087)
• Antibody Conjugate Purification kit for 50-100 μg (A33088)

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 our Antibody Labeling kits 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 stand by the quality of our work. We are ISO 13485:2000 certified.