We previously demonstrated that the insertion of the acyl carrier WZ4002 protein tag at the extracellular domain of TrkA makes it possible to specifically label the receptor at the cell surface when the construct is transfected in living cells. The ACP tag belongs to a family of protein and peptide tags, which can be covalently conjugated to virtually any small-probe substituted phosphopantetheinyl arm of Coenzyme A substrate by post-translational modification enzymes named PP transferases. The ACP tag was shown not to interfere with TrkA receptor function. When coupled to various fluorescent probes, this tool made it possible to monitor in living cells single TrkA movements and changes of oligomerization state upon binding of different biologically-relevant ligands including NGF and proNGF. Here we demonstrate the insertion of an 8-amino-acids tag into the sequence of NGF and of 12-amino-acids tags into the sequence of the two NGF receptors, TrkA and P75NTR. These tags derive from in vitro evolution studies committed to the shortening of the ACP and peptidyl carrier protein tags. The tags were inserted by an insertional mutagenesis method, based on a modification of the standard site-directed mutagenesis protocol, that allows their insertion in the protein of interest with no need for any additional linker sequence and thus with minimal interference with protein activity. Here we conjugate biotin to the tags by using PPTases in the presence of CoA-biotin substrates. We demonstrate that upon insertion of these tags, a site-specific biotinylation is achieved for the purified recombinant neurotrophin and for TrkA and P75NTR receptors expressed in the membrane of living cells. Moreover, the properties of orthogonal labeling displayed by the 12-amino-acid A1 and S6 tags are exploited to simultaneously label single TrkA and P75NTR receptors with two spectrally-distinct fluorophores, even when the two receptors are expressed in the same cell. We show that the insertion of such tags in the chosen sites has no measurable impact in NGF functionality or in the correct translocation of TrkA and P75NTR receptors at the cell membrane. Application of these three nanoprobes to the investigation of trafficking and interactions of NGF and its receptors in living cells will be discussed. The proNGF-A4 recombinant construct was expressed and produced in E. coli, collected from inclusion bodies and purified by FPLC ion exchange chromatography with the same procedure adopted for wt recombinant proNGF. ProNGF-A4 purified protein was then digested by trypsin and further purified by FPLC ion exchange chromatography. We were thus able to obtain the purified tagged mature neurotrophin using the same protocol adopted for the wt counterpart. Purified NGF-A4 and proNGF-A4 were incubated with CoA-biotin substrate and AcpS or SfpS PPTases. The same in vitro biotinylation reaction was performed in parallel using untagged wt NGF and wt proNGF as controls. Western blot analyses of all biotinylation reactions are reported in Fig. 2. Data show that specific biotin labeling was achieved for NGF-A4 and proNGF-A4 reacted with AcpS. In order to verify if the modified neurotrophin still maintains its biological function.