Increasing the amount of EBX 2a to 50 equivalents allowed to doubly modify the protein after 24 hours of incubation (conditions B). Again, only Y31, Y42, Y48 and Y71 were functionalized. Finally, under denaturing conditions, the 4 reactive tyrosine residues could be labelled (conditions B a ). Interestingly, when another mutant streptavidin (66 kDa) was used, a better conversion was observed (See Supporting Information). The incomplete conversion observed indicated that the tyrosines of the protein investigated so far were not fully accessible. We therefore decided to investigate the functionalization of trastuzumab (148 kDa), a monoclonal antibody used to treat breast cancer, containing a high number of tyrosine residues including some with high accessibility and reactivity. Gratefully, and despite the glycans present on the heavy chains, under mild conditions without denaturation, trastuzumab could be efficiently labelled with an average of 2 EBX and complete conversion after 4 hours to give 9. A longer reaction time and higher excess of EBX allowed to reach a higher degree of bioconjugation (see Supporting Information for details).