Fast micro equilibrium dialyzer

The binding rate of SCP2 and 15(S)–HpETE (a monohydroperoxy polyunsaturated fatty acid in cells) was determined by a Fast Micro-Equilibrium Dialyzer (Harvard Apparatus) and LC-MS/MS (AB sciex, API 4000 Q-Trap). Recombinant SCP2 (5 μM; Sangon, Shanghai, China) and 15(S)–HpETE (5 μM; Glpbio, CA, USA) were mixed with or without ScpI2 (5 μM) in phosphate buffered saline (PBS; Corning, NY, USA) and then filled in one chamber. The other chamber was filled with blank PBS. A 3.5 kDa dialysis membrane (Viskase, USA) was applied to separate the chambers which were dialyzed for 2 h at 37 °C in a shaker. A mixture of SCP2 (5 μM) and GSH (5 μM; Macklin, Shanghai, China) served as negative control. After dialysis, the solution from both sides was subjected to compound extraction and LC-MS/MS analysis.

Equilibrium dialysis measurements were performed with a Fast-Micro-Equilibrium Dialyzer™, with 25 μl chambers, and a Fast-Micro-Equilibrium Dialyzer 1 kDa MW cut-off membrane™ (Harvard Apparatus, Boston, MA, USA), in RB. Experiments were performed overnight at 4 °C. The concentration of free ligand was determined at the beginning of the experiment by measuring the absorbance at 259 nm (ε_ex,coeff = 15,400 M⁻¹ cm⁻¹, T = 25 °C), and then determined again at the end of incubation from the chamber of the free ligand only. Bound ligand concentration was determined using the mass conservation equation, [N]_i = 2[N]_f + n[RecBCD], where [N]_i is the concentration of the nucleotide at the beginning of the dialysis and [N]_f is the concentration at the end of the experiment, from the ligand-only chamber. In the high concentration regime, [RecBCD] was held at 45 μM and [ADP]_i or [AMPpNp]_i at 1 mM, with a 1:1 ratio to MgCl₂. For the low concentration regime, we held [RecBCD] at 17 μM and nucleotides at 200 μM. We performed control experiments without RecBCD to estimate the time sufficient to reach equilibrium (overnight incubation) and to validate the level of nucleotide’s stickiness to the membrane (Supplementary Fig. 17). The total loss of nucleotides across the membrane was less than 0.5%.