Labeling surface proteins with high specificity: Intrinsic limitations of phosphopantetheinyl transferase systems
Fig 3
Comparison of PPTase and HaloTag (HT) labeling.
(A) Using cell-permeable HTL-TMR, saturation is reached by labeling for 15 min with nanomolar concentrations with high specificity (vermillion curve), as very low signal is found in the absence of HT fusion (bluish green curve). (B) In contrast, saturation is not reached by 5 μM of cell-permeable CoA-SiR, even after 30 min incubation with 1 μM Sfp enzyme (vermillion), with about 30% of the signal being non-specific (in the absence of enzyme, bluish green). (C) A HaloTag ligand (HTL) based on Alexa Fluor 660 results in extremely low non-specific signal (absence of HaloTag, inset, bluish green), while the kinetics are only slightly less favorable than those for HTL-TMR (vermillion). (D) Flow cytometry of HEK 293 cells (no HT, no Sfp enzyme) (black), compared with the same cells after non-specific labeling after 15 min incubation with 15 μM of the respective HTL (blue) or CoA derivative (vermillion).