Sulfo cyanine5 maleimide

Rpn4- and yODC-containing substrates were overexpressed, purified (including removal of the His-SUMO tag), labeled with sulfo-cyanine5 maleimide (Lumiprobe, Hunt Valley, MD, USA), and repurified by gel filtration as described previously [14 (link)]. UBL-sGFP-102-His₆ was purified as described previously [15 (link)]. Sequences and molecular weights are given in Supplemental Table S1.

A Gly-Gly-Gly-Cys peptide with C-terminal amidation (Genscript) was dissolved at 173 mM in degassed coupling buffer (50 mM HEPES (pH 7.4), 20 mM TCEP (pH adjusted to 7.5)). Sulfo-Cyanine5 maleimide (Lumiprobe) dissolved in degassed DMSO was mixed in a 2:1 molar ratio (dye:peptide) and incubated overnight at 4 °C. The following morning, the reaction was quenched with 10 mM DTT. The labeling reaction mixture was separated using on a C-18 reverse phase HPLC Column (Beckman Ultrasphere C-18, 4.6 25 cm) that was pre-equilibrated with Buffer 1(filtered MilliQ with 0.1% Trifluoroacetic acid (Sigma-Aldrich)). The protein was eluted at 1.5 ml/min using the following gradient- 0–1 min: 10% buffer 2 (filtered Acetonitrile (Sigma-Aldrich) with 0.1% TFA); 1–16 min: 70% buffer 2; 16–17 min: 90% buffer 2; 17–22 min: 90% buffer 2. The column eluent was monitored at 200 nm and 650 nm. Under these conditions, unlabelled GGGC peptide eluted at ~11 min and GGGC^Cy5 peptide eluted at ~15 min, respectively, under these conditions. The free Cy5 dye was eluted from the column only after washing with 100% buffer 2. The GGGC^Cy5 peptide was lyophilized and stored at −80 °C.