In the two channels quantified for each cell. Cells transfected with both receptors were similarly stained with the two different S-Qdots upon labeling ; single transfections performed as a control yielded labeling BAY 73-4506 molecular weight largely dominated by the Qdots added after the reaction with the tag-specific PPTase. These data demonstrate that the use of A1 and S6 tags leads to orthogonal fluorolabeling of TrkA and P75NTR receptors co-expressed in living cells. The experimental study of molecular interactions occurring between NGF and its receptors requires means to label them independently, simultaneously, and with controlled stoichiometry. In this work we developed a toolbox for this aim. We describe here a method for the introduction of three different tags into the sequence of NGF and of TrkA and P75NTR receptors. The chosen tags belong to the ACP and PCP families and bear a serine residue as the site of covalent transfer of the CoA PP arm by PPTase enzymes. In all experiments presented here, the CoA PP arm is substituted with biotin so that we achieve site-specific biotinylation of NGF and its receptors. We wish to stress, however, that virtually any small-probe carried by CoA PP arms can be coupled to the three proteins. NGF is labeled in vitro, after purification of the proneurotrophin expressed in E. coli. On the other hand, TrkA and P75NTR are labeled in living cells that express the tagged receptors. PPTases and CoA-biotin substrate are added to the cell medium and do not permeate the cell membrane, so that only the receptor pool exposed at the cell surface is actually biotinylated. Fluorolabeling of the two receptors at the cell membrane is achieved by addition of two spectrally-distinct S-Qdots to the cell medium, and their subsequent visualization at the single-receptor level. Our strategy fulfills all the recommended criteria to achieve the specific labeling of proteins of interest that are involved in molecular interactions. First of all these tags are small, being shortened versions of the ACP and PCP tags : A4 tag fused to NGF is 8 amino-acid long, while A1 and S6 tags fused to either TrkA or P75NTR are 12 amino-acid long. Our insertional mutagenesis method makes it possible to insert tags with no need for any additional flanking or linker sequence in virtually any site of the protein of interest. This is particularly relevant for the case of TrkA and P75NTR, since their tag insertion site is neither the N-terminus nor the Cterminus of the receptors, but is downstream the signal of localization to the plasma membrane. Traditional tag-cloning procedures would in this case result in the insertion of additional amino acids thus compromising the effective gain resulting from tag shortening. Although the choice of not inserting any linker sequence may in principle hinder accessibility of the tags for the labeling reaction, this seems not to be the case for most of our constructs. We provided unambiguous biochemical evidence that tag biotinylation occurs both for the neurotrophin and for its receptors. Generally, non-specific biotinylation of proteins is achieved by chemical conjugation of reactive biotin derivatives to amine, thiol or carboxyl groups of proteins.