Fluorolabeling of the PPTase-Related Chemical Tags: Comparative Study of Different Membrane Receptors and Different Fluorophores in the Labeling Reactions

Choosing the Probe for Single-Molecule Fluorescence Microscopy

“…In general, the authors recommended blue-excitation fluorophores such as Alexa 488 or Atto 488 for their few or no interactions with different kinds of lipids; for smFRET, they selected the blue–red pair Alexa 488 and Alexa 647 and the green–red pair Atto 532 and Alexa 647 [ 26 ]. Various fluorophores were compared also for single-molecule tracking exploiting ACP-derived tags [ 28 ] or SNAP-tag [ 29 ] fused proteins. The first study selected Abberior STAR 635p as the best candidate in labeling the tropomyosin receptor kinase A (TrkA) receptor, considering aspecific interactions, mean fluorescence intensity and photostability; however, the authors stated that this choice is not universal for labeling receptors, and indeed they obtained different results in dye comparisons on a different receptor [ 28 ].…”

“…Various fluorophores were compared also for single-molecule tracking exploiting ACP-derived tags [ 28 ] or SNAP-tag [ 29 ] fused proteins. The first study selected Abberior STAR 635p as the best candidate in labeling the tropomyosin receptor kinase A (TrkA) receptor, considering aspecific interactions, mean fluorescence intensity and photostability; however, the authors stated that this choice is not universal for labeling receptors, and indeed they obtained different results in dye comparisons on a different receptor [ 28 ]. The second work selected Dy 549 and CF 640 as the best choices to label the epidermal growth factor receptor (EGFR) considering aspecific interactions and photostability.…”

“…For all the described reasons, it is clear that minimizing aspecific interactions is another essential step in choosing the fluorescent probe for single-molecule microscopy. Some studies have reported comparisons of aspecific interactions for different dyes, in particular for single-molecule applications [ 26 , 27 , 28 , 29 , 85 , 102 ] ( Table 2 ). The first consideration is that all of them observed high variability in the behavior of the different dyes and identified several ones to exclude since they could impair or falsify single-molecule measurements.…”

Axonal plasticity in response to active forces generated through magnetic nanopulling

“…A total of 90-100 mg purified YBBR-NGF (kindly donated by Prof. A. Cattaneo, Bio@SNS Lab, Pisa) was incubated with 73 µM CoA-Alexa647, 17µM Sfp Synthase, 36 mM MgCl 2 in DPBS up to 270 µl final volume for 30 min at 37°C; the excess of free fluorophore was removed by desalt spin-column and the fluoNGF obtained was stored at 4°C for a maximum of 10 days. This reaction enabled the NGF C-terminus to be stoichiometrically labelled with Alexa647 fluorophore using a method first described in Yin and colleagues (Yin et al, 2005) and optimized as in (Amodeo et al, 2020), to finally achieve the covalent binding of two fluorophores per neurotrophin dimer. At DIV3 (after overnight stimulation), DRG cultures were prepared for time-lapse studies.…”

DNA-Templated Glycan Labeling for Monitoring Receptor Spatial Distribution in Living Cells

“…For this purpose, receptors are tagged either with genetically encoded labels (e.g., green fluorescent protein, SNAP/CLIP tag, ACP/PCP tag, etc.) or receptor-specific antibodies conjugated to fluorophores. − The development of genetically encoded labels has initially raised great hopes for visualization of receptor trafficking in living cells. − However, their time-consuming genetic manipulation procedure and overexpressed recombinant constructs are still problematic. , Nowadays, some researchers have envisioned that labeling with DNA tags would provide receptors with generic landing platforms for the generation of fluorescent signals using DNA assembly, thus opening new opportunities for monitoring receptor spatial distribution. − In this case, DNA tags are conjugated with a recognition element, such as an antibody or an aptamer, to attach DNA tags to a specific receptor. Due to the noncovalent interaction between a receptor and a recognition element, DNA tags would dissociate from the receptor and will be difficult to realize long-term stable trafficking …”