AAT Bioquest

What are the steps involved in labeling an antibody using an antibody labeling kit?

Posted April 18, 2023


There are 7 common steps involved in labeling an antibody using an antibody labeling kit. Step 1 is choosing the labeling method (e.g. fluorescent labeling, enzyme labeling) that is necessary for the experiment. Step 2 is the preparation of the antibody, where the antibody being labeled (which is purified from serum or hybridoma cell culture) is placed in the correct buffer. Step 3 is preparing the labeling reagent, which is unique to the selected labeling method and usually supplied in lyophilized form. Step 4 involves the mixing of the antibody and labeling reagent at a specific molar ratio. After the mixture is completed, it must be incubated for the necessary amount of time to allow covalent binding between the labeling reagent and antibody. Step 5 is the purification of the labeled antibody (to remove unbound labeling reagent), which is primarily done using size exclusion chromatography, centrifugation, or dialysis membrane. For step 6, one should check the labeling efficiency to ensure the labeled antibody is appropriate for the desired application and the experiment was successful. This can be done through measuring the degree of labeling, measuring stability of the labeled antibody, or by carrying out assays to ensure the labeled antibody keeps its ability to bind to the target antigen. Step 7 is the storing of the antibody, which should be stored under conditions in the instructions given in the kit. 

Additional resources

Simple, Rapid Chemical Labeling and Screening of Antibodies with Luminescent Peptides

Antibody and Protein Labeling

ReadiLink™ Rapid iFluor® 488 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*