Conjugated Primary Antibodies

Labeling chemistries often exploit primary amine groups to covalently attach labels to antibody molecules. The N-terminus of polypeptide chains and the side chain of lysine amino acid residues contain primary amines. It is possible to link labels to antibodies by using chemical groups that react with amines. The four main chemical approaches for antibody labeling are summarized below:   

1. NHS Esters: In the case of fluorescent dyes, it is typical to purchase an activated form of the dye with an inbuilt NHS ester (also called a ‘succinimidyl ester’). Linking the activated dye to an antibody by chemical conjugation occurs under physiological or alkaline conditions to generate stable amide bonds. Excess reactive dye is removed by one of several possible methods, often column chromatography before the labeled antibody is used in an immunoassay.

   Find out more about reactivity of fluorescent dyes.

2. Heterobifunctional Reagents: If the label is a protein molecule (e.g. HRP, alkaline phosphatase, or phycoerythrin), then the antibody labeling procedure is complicated by the fact that both the antibody and label contain multiple amine groups. To restrict conjugation to the antibody, lysine residues on the antibody are modified to create a new reactive group (X), while lysine residues on the label are modified to create another reactive group (Y). A ‘heterobifunctional reagent’ is used to introduce the Y groups, which subsequently react with X groups when the antibody and label are mixed, thus creating heterodimeric conjugates. There are many variations on this theme. 
3. Carbodiimides: Carbodiimides are commonly used to conjugate antibodies to carboxylated particles (e.g. latex particles, magnetic beads) and to other carboxylated surfaces, such as microwell plates or chip surfaces. Carbodiimides are rarely used to attach dyes or protein labels to antibodies, although they are important in the production of NHS-activated dyes (see above).These reagents are used to create covalent links between amine- and carboxyl-containing molecules. Carbodiimides activate carboxyl groups, and the activated intermediate is then attacked by an amine (e.g. provided by a lysine residue on an antibody). 
4. Sodium Periodate: Although this chemical is not compatible with the majority of labels, it is applicable to HRP, the most popular diagnostic enzyme. Periodate activates carbohydrate chains on the HRP molecule to create aldehyde groups, which are capable of reacting with lysine residues on antibody molecules. Since HRP itself has very few lysine residues, it is relatively easy to create antibody-HRP conjugates without significant HRP polymerization. 

Why DyLight®? 
DyLight® Fluorescent Dyes are a family of high-intensity, photostable fluorescent tags for labeling antibodies and other molecular probes, which may be used for flow cytometry, ELISA, fluorescence microscopy, and other array platforms.  DyLight® dyes have absorption maxima ranging from 350nm-800nm, covering the entire visible light spectrum as well as several key near-IR and IR wavelengths. DyLight® dyes are trusted and relied on by researchers around the globe.

DyLight® Antibody Labeling Kits
Lightning-Link® Rapid DyLight® Antibody Labeling Kits combine the quick labeling efficiency of Lightning-Link® antibody labeling technology with the trust and stability of the DyLight® dye brand.  Label your antibody or protein directly with a Lightning-Link® Rapid DyLight® Antibody Labeling Kit - 10 different DyLight® dyes are available.

A tandem dye, also called a FRET pair or FRET dye, is a pair of covalently linked fluorescent molecules, comprising a donor and an acceptor molecule. The donor molecule emits energy that is absorbed by the acceptor molecule through a phenomenon known as Förster- or fluorescence-resonance energy transfer (FRET).  FRET is a technique widely used in biology, to study for example protein-protein interactions, membrane fluidity and receptor-ligand binding.

To learn more about tandem dyes read our Tandem Dye Conjugated Antibodies FAQs.