Sephacryl s 100 resin

E. coli Topoisomerase I (EcTopoI) was purified as previously described^{28 (link)} and stored at −20°C at 2 mg/mL in storage buffer (50 mM Tris-HCl pH8, 1mM DTT, 50 mM KCl, 50% glycerol). To create fluorescently labeled EcTopoI, the purified protein was dialyzed into de-gassed labeling buffer (50 mM Tris-HCl pH 7, 150 mM KCl, 1 mM EDTA). Protein was then incubated overnight at 4°C with 10-fold molar excess of Alexa Fluor 555 maleimide (A20346, Thermo Fisher, Waltham, MA). After incubation, protein and dye were purified over a Sephacryl S-100 resin (17-1194-01, GE Healthcare Life Sciences) to remove excess dye. After purification mass spectrometry confirmed primarily single labeling of the protein, although the location of the label is unknown. Bulk activity assays confirmed relaxation activity for Alexa 555 labeled EcTopoI, although at approximately five times lower rate than wild type EcTopoI. For the experiments, EcTopoI was first diluted 20-fold in buffer (50 mM Tris-HCl pH8, 150 mM KCl, 1mM EDTA) and stored on ice until needed. Immediately prior to flowing protein into the flow cell, EcTopoI was further diluted to 40 nM in the single molecule fluorescence oxygen scavenging buffer, described below. For experiments with Alexa 555 labeled EcTopoI, the protein was diluted to 2 nM.