Figure 5: Sulfonation on the HaloTag ligand v2.0 (HTL.2) for improved labelling with sticky ATTO 647N.

A) Chemical structure of ATTO 647N, with a N-methyl amidated additional four carbon linker on the 3 position, which disallows proper dye:HTP secondary interactions. B) Sulfonation protocol on second version HaloTag ligand HTL.2 yields double sulfonated S₂HTL.2. C) qTOF full protein mass spectrometry of recombinant HTP labelled with ATTO 647N-HTL.2 and ATTO 647N-S₂HTL.2. E) HTP-SNAP- mGluR2 transfected HEK293 cells, labelled with BG-Sulfo549 (1 uM) and different concentrations of ATTO 647N-HTL.2 for 10 minutes prior to fixation and imaging gives rise to unspecific signal. F, G) Zoom ins and brightness contrast adjusted images from (E). H-J) As for (E-G) but with different concentrations of ATTO 647N-SHTL leads to image improvements by removing unspecific signals. K-M) As for (E-G) but with different concentrations of ATTO 647N-S₂HTL.2 allows clear membrane labelling even at 1 nM.