Red loading buffer

Full-length TrxA and a C-terminal SsrB fragment encompassing residues 137–212 expressed as GST fusions from pGEX6P1 (GE Healthcare Biosciences, Fairfield, CT) were purified as described previously for DksA (Henard et al., 2014 (link)). Where indicated, 25 µM recombinant SsrB was treated with 250 µM H₂O₂ for 1 h at 37°C. Selected samples were treated wit h 25 µM TrxA for 30 min. The specimens were mixed with 3× Red loading buffer (New England Biolabs, Ipswich, MA) in the absence of reducing agents. The samples were loaded into 4–20% SDS-PAGE gels (Bio-Rad) and electrophoresed at 125V on ice. Proteins were visualized by Coomassie blue staining.

50 µM recombinant protein was treated with 500 µM spermine NONOate, 500 µM GSNO, 500 µM H₂O₂, or 500 µM ONOO⁻ for 1 h at 37°C. The specimens were then mixed with 3× Red loading buffer (New England Biolabs, Ipswich, MA) lacking reducing agents. 10 µL (~10 µg protein) of the samples were loaded into a 12% (v/v) SDS-PAGE gels and electrophoresed at 125V on ice. Proteins were visualized by Coomassie blue staining.