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Antibody conjugation, also known as antibody labeling, is the process of chemically linking an antibody to a specific tag. There are several common methods to conjugate antibodies, although they can have complex protocols and usually require a certain level of skill and expertise.
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.
|ELISA||Enzymes (horseradish peroxidase (HRP) or alkaline phosphatase (AP), Biotin|
|Flow cytometry||Phycoerythrin (PE), Allophycocyanin (APC), or tandem dyes, and Alexa Fluor®|
|Immunocytochemistry and immunofluorescence||Fluorophores|
|Immunohistochemistry||Enzymes (HRP or AP), Biotin|
|Western blot||Enzymes (HRP or AP), fluorescent dyes|
Traditional covalent conjugation, such as the NHS ester and two-tag methods, usually involve attaching the label to lysine residues. These 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 covalent conjugation, including eliminating the need for separation steps to ensure retention of your antibody.
Our guide to antibody conjugation describes three common conjugation methods to help you decide which technique will enable you to complete your experiment.
Alexa Fluoris a registered trademark of Life Technologies. Alexa Fluor dye conjugates contain(s) technology licensed to Abcam by Life Technologies.