Alexa 488 maleimide

Three milligrams of THIOMAB capped with cysteine and/or glutathione was first uncapped as described previously by being incubated with a 20 molar equivalents of dithiothreitol for 16 h at room temperature.^{19 (link)} Following reduction, the Ab was purified using a 1 mL cation exchange column. The column was equilibrated in 20 mM succinate (pH 5.0), and the sample was diluted in the same buffer prior to loading to allow binding to the column. Following a 10 mL wash at pH 5.0, the Ab was eluted using Tris buffer [50 mM Tris and 150 mM NaCl (pH 7.5)].
After preparation of reduced thiols in THIOMAB, a 6× equivalent (40 μM) of CuSO₄ was added to catalyze reoxidation of cysteines, including the creation of inter-Ab disulfides. This reaction mixture was incubated for 2 h at room temperature and resulted in a small amount of visible precipitate. Immediately following oxidation, a 6× equivalent of Alexa 488 maleimide (Thermo Fisher, A10254) was added to the sample from a 5 mg/mL stock in dimethyl sulfoxide (DMSO) and incubated for 3 h at room temperature. An additional buffer exchange was required after SEC to remove free dye using a 0.5 mL desalting column with a 7K molecular weight cutoff. Afterward, the ratio of dye to Ab was 0.2 for monomer and 0.1 for dimer as determined by absorbance at 280 and 493 nm.

All MBP-Nup62 constructs were labelled with Alexa-488 maleimide (ThermoFisher Scientific, PI78601) as described in (5 (link)). For assays examining phase separation of MBP-FUS with MBP-Nup62 domain constructs (Figure 2), MBP-FUS full length was labelled with Alexa-555 maleimide (ThermoFisher Scientific, PI78601) as described in (5 (link)). For assays examining phase separation of MBP-FUS domain constructs with MBP-Nup62 full length (Figure 5), all MBP-FUS constructs were labelled with Alexa-555 NHS ester (ThermoFisher Scientific, PI78601) at primary amines due to the lack of cysteines in MBP-FUS QGSY and RGG3 that can react with maleimide. Labelling reactions were buffer exchanged twice serially into the respective storage buffers (20 mM Tris/Cl, 150 mM NaCl, pH 7.4 for MBP-Nup62 and 20 mM sodium phosphate, 1M NaCl, 25% glycerol, pH 7.4 for MBP-FUS) using spin desalting columns (ThermoFisher Scientific, PI78601) to remove unreacted dye molecules.