AbstractA widely used approach for protein conjugation is through the lysine residues reacting with NHS- or other active esters. However, it is a challenge to precisely control the degree of labeling (DoL) due to the instability of active ester and variability of reaction efficiencies. Here, we provide a protocol for better control of aDoL using existing Copper-free Click Chemistry reagents. It is a two-step reaction with one purification in between. Briefly, proteins of interest were first activated with azide-NHS. After removing unreacted azide-NHS, the protein-N3 is then reacted with a limited amount of complementary click tag. Our studies have shown the click tag will fully react with the protein-N3 after 24 h’ incubation, and therefore does not require additional purification steps. As such, the aDoL is equal to the input molar ratio of the click tag and the protein. Furthermore, this approach offers a much simpler and more economical way to perform parallel microscale labeling. Once a protein is pre-activated with N3-NHS, any fluorophore or molecule with the complementary click tag can be attached to the protein by mixing the two ingredients. Quantities of the protein used in the click reaction can be at any desired amount. In one example, we labeled an antibody in parallel with 9 different fluorophores using a total of 0.5 mg of antibody. In another example, we labeled Ab with targeted aDoL value from 2 to 8. In a stability comparison study, we have found the conjugated fluorophore using the suggested click protocol stayed attached to the protein longer than with standard NHS-fluorophore labeling.