Atpase colorimetric assay kit

All ATP hydrolysis assays were performed in the proteoliposomes. E. coli polar lipids (Avanti) were resuspended in 20 mM HEPES-KOH (pH 7.0) to the final concentration of 20 mg mL⁻¹. A final concentration 0.45% of Triton X-100 was added to destabilize the liposomes for 0.5 h at room temperature. The proteins were added to the destabilized liposomes and incubated for 1 hr at 4 °C. The ratio of lipids and proteins was kept at 100:1 (w/w). Triton X-100 was removed by SM-2 Adsorbent Bio-Beads (Bio-Rad) by incubation at 4 °C overnight and repeated for another 2 h. Proteoliposomes were diluted and resuspended twice with the ice cold buffer by 250,000 × g for 1 h at 4 °C. After the final centrifugation, the pellets were resuspended in 20 mM HEPES-KOH, pH 7.0, 50 mM KCl and 2 mM MgCl₂ to 100 μL as one reaction sample. For each reaction sample, 0.2 μM of Spr0694–0695 dimer and 0.4 μM of Spr0693 hexamer or mutated proteins were added.
ATP was added to each sample at a final concentration of 2 mM by three rounds of freezing in liquid nitrogen followed by thawing in a bath sonicator. Reactions were performed at 37 °C for 30 min and the amount of released Pi was quantitatively measured using the ATPase colorimetric Assay Kit (Innova Biosciences) in 96-well plates at OD₆₅₀ mm.

Each portion of WTA was extracted from 10 ml of the S. aureus NCTC8325 strain cultured overnight. The extraction method strictly followed the protocol of the work of Covas et al. (45 (link)). The quantification of WTA was applied by the measure of the phosphate group from WTA using the ATPase colorimetric assay kit (Innova Biosciences) in 96-well plates at OD₆₅₀.

ATPase activities of ABCG25_WT and its mutants in detergent micelle or liposomes were measured using the ATPase Colorimetric Assay kit (Innova Biosciences) in 96-well plates at an optical density of 650 nm. To calculate the ATPase activity in the presence of different concentrations of ATP, 1 μg protein was added to the reaction buffer containing 25 mM HEPES-NaOH (pH 7.4), 150 mM NaCl, 10 mM MgCl₂, with (for ABCG25 in detergent) or without (for ABCG25 in liposomes) 0.01% (w/v) DDM. Then, ATP in different concentrations was supplemented in the solution to start the reaction at 37 °C for 10 min. The amount of released Pi was quantitatively measured with the ATPase Colorimetric Assay kit. For ABA concentration-dependent ATPase activity assay, a stock solution of ABA ((S)-(+)-Abscisic acid, Sigma) was prepared in double-distilled H₂O at 20 mM concentration and then serially diluted to obtain the desired final concentrations. The ABA solution in different concentrations was added into the reaction buffer containing 1 μg protein and the solution mixed for a 20-min incubation on ice. Afterwards, ATP was supplemented into the mixture at a final concentration of 3 mM and reacted at 37 °C for 10 min. All statistical analyses were performed using GraphPad Prism 9.