Whether it’s a western blot analysis or profiling the tumor microenvironment using multiplex imaging, we rely on antibodies conjugated to various labels to visualize and understand protein structure, function, localization, and other protein-protein interactions.

This comprehensive guide covers the ins and outs of antibody conjugation, including the difference between direct and indirect assays, common labels used, different chemistries behind antibody labeling, and methods you can use to conjugate your primary antibody.

What is antibody conjugation?

Antibody conjugation, also known as antibody labeling, is the process of linking an antibody to a specific tag or label. Most immunoassays require antibodies either directly conjugated to a specific label or detected by a conjugated secondary antibody to provide a measurable signal.

When conjugated, a primary antibody allows for the direct detection of the target antigen of interest without needing a secondary antibody. Using direct detection has pros and cons, which are explored below.

Why conjugate your primary antibody?

Due to their specificity, antibodies are widely used to detect and quantify antigens in numerous applications such as flow cytometry, ELISA, western blotting, immunohistochemistry, and lateral flow. The antibody which binds to the antigen is known as the ‘primary antibody’. However, to provide measurability, most immunoassays must also directly or indirectly have a label incorporated. Labels can include enzymes, fluorochromes, or biotin (table 1).

Table 1. Common immunoassay techniques and their associated labels.

How to conjugate antibodies?

Traditional covalent conjugation techniques, such as the NHS ester and two-tag methods, usually involve attaching the label to lysine residues. These conjugation protocols typically require knowledge of chemistry and experience in separation techniques, such as fixed-column chromatography, to remove low molecular weight activation reagents for the label and/or antibody.

Alternatively, simpler, three-step conjugation kits only require the addition of your antibody to a tube containing your desired label. These kits can help avoid common issues associated with the covalent conjugation process, including eliminating the need for separation steps to ensure retention of your antibody.

For more information about quick & easy conjugation kits, read our page on Lightning-Link^® kits.

Our guide to antibody conjugation describes three common conjugation methods to help you decide which technique will enable you to complete your experiment.

Download our full guide to find out more about:

• NHS ester conjugation
• Two-tag conjugation
• Conjugation kits

Alexa Fluor® is a registered trademark of Life Technologies. Alexa Fluor® dye conjugates contain(s) technology licensed to Abcam by Life Technologies.