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Learn the essentials of antibody conjugation with our user-friendly guide.
Updated August 2, 2023.
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 in our antibody conjugation guide.
Labels used to conjugate antibodies include fluorescent dyes, small molecules (eg, biotin), enzymes (horseradish peroxidase (HRP) or alkaline phosphatase (AP)), proteins (eg, R-PE, APC, streptavidin), latex or gold nanoparticles, and oligonucleotides. The choice of the label will depend on the experimental application, as shown in Table 1.
Table 1. Labels and their most common applications.
|Western blot||HRP, AP, fluorescent dyes|
|Immunofluorescence||Fluorescent dyes, including Alexa Fluor® dyes|
|Fluorescent immunohistochemistry (singleplex)||Fluorescent dyes|
|Fluorescent immunohistochemistry (multiplex)||Fluorescent dyes, oligonucleotides|
|Chromogenic immunohistochemistry||HRP, AP, Biotin|
|Flow cytometry||Fluorescent dyes, such as Alexa Fluor® dyes, and fluorescent proteins, including Phycoerythrin (PE), Allophycocyanin (APC), and tandem dyes|
|ELISA, ELISA-based applications||HRP, Biotin|
|Immuno-PCR, Proximity ligation assay (PLA), Single-cell proteomics||Oligonucleotides|
|Lateral Flow Assay (LFA)||Latex and gold nanoparticles, Fluorescent nanoparticles (Europium)|
|Mass Cytometry (CyTOF®, Imaging Mass Cytometry™)||Metal ion tags|
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 the retention of your antibody.
Our 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.
If you’ve got any questions about our kits or conjugated antibodies, or you’d like to know about our custom conjugation solutions, we’d love to hear from you – get in touch.
Alexa Fluoris a registered trademark of Life Technologies. Alexa Fluor dye conjugates contain(s) technology licensed to Abcam by Life Technologies.