System gold 166 uv detector

For TIRF microscopy experiments, 3M was dialyzed into 10 mM sodium acetate pH 5 and was labeled with Alexa Fluor^® 555 succinimidyl ester (Molecular Probes, Eugene, OR) following the manufacturer’s protocol (MP 30007). The average labeling efficiency was 9±1 fluorophores per protein molecule and was measured using UV-visible spectroscopy at 280 nm and 555 nm, following the manufacturer’s protocol. After labeling, the protein was dialyzed back into 10 mM L-histidine pH 5 using a Slide-A-Lyzer 10,000 MWCO dialysis cassette (Thermo Scientific, Rockford, IL) before TIRF microscopy experiments were conducted.
After labeling and dialysis, labeled 3M was analyzed by size exclusion chromatography (SEC) using a TSK-GEL G3000SW_XL column with guard column (TOSOH Biosciences, Montgomeryville, PA). The flowrate was 0.6 mL/min, and the mobile phase was 0.2 M potassium phosphate monobasic, 0.2 M potassium chloride, and 0.1 g/L sodium azide at pH 7. The absorbance was monitored at 280 nm using a Beckman Coulter (Fullerton, CA) System Gold 166 UV detector. SEC analysis confirmed that labeled 3M was in a monomeric state.