To enable direct comparison to some of the previous benchmarking studies, dHis-CuNTA and SLIM were grafted onto the Streptococcus sp. Group G protein G, B1 domain (GB1), an established model protein extensively used for EPR studies. Here, the GB1 construct I6C/K28H/Q32H was used, bearing the dHis motif for coordination to CuNTA in an -helix and a cysteine residue for SLIM labelling in a -sheet (Figure ). GB1 I6C/K28H/Q32H was expressed and purified as described, and labelled with SLIM and CuNTA according to established protocols (for more details see Supplementary Information (SI)). Quantitative labelling with SLIM could be achieved as demonstrated by sample characterisation using continuous wave (CW) EPR spectroscopy (Figure ), electrospray ionization (ESI) mass spectrometry (Figure ) and UV/VIS spectroscopy (Figure ) for concentration determination and estimation of labelling efficiency.