Sulfo cy3 dbco

Tail insertion was measured by tracking Forster resonance energy transfer (FRET) between Cy3-labeled base and Cy5-labeled substrate, while enforcing single-turnover conditions using 1,10-phenanthroline (oPA), an inhibitor of Rpn11 that stalls the proteasome at the site of ubiquitin linkage (Worden et al., 2017 (link)). Base containing Rpt1^I191AzF was labeled with Sulfo-Cy3-DBCO (Click Chemistry Tools) as described above. A 30 mM oPA stock was made in GF buffer. Reactions were performed in the Auto SF120 using 2× reconstituted holoenzyme and 2× Sulfo-Cy3 labeled substrate. Final concentrations were 100 nM Cy3-Rpt1 base, 400 nM core, 600 nM lid, 750 nM Rpn10, 3 mM oPA, and 3 μM substrate. An excitation wavelength of 550 nm was used, and the Cy3 and Cy5 emission channels were monitored simultaneously.
The kinetics of tail insertion were determined by fitting the quenching of the Cy3 signal. The amplitudes listed in Table 1 were calculated by normalizing the amplitudes of the fast-phases to the initial fluorescence, and then comparing those to the normalized amplitude of titin-I27^V15P-23-K-35-Cy5. All measurements used in this calculation were from traces collected on the same day.