2.5. Quantification of polyP by Malachite Green

PolyP was measured as phosphate after enzymatic digestion of the polymer. RNA/polyP (2–5 μg) were incubated with recombinant Ppx1 and Ddp1 in reaction buffer (20 mM HEPEs pH 6.8; 6 mM MgSO₄; 1 mM DTT; 100 mM NaCl) for 1 h at 37 °C. Phosphate present in 5–10% of digested RNA/polyP, and undigested RNA/polyP (1 μL) were assayed using the malachite green assay. Samples and phosphate standards were distributed in a 96 well plate and the volume adjusted to 100 μL with ddH₂O. Then 100 μL of freshly mixed Molybtate (175 mM (NH₄)₂MoO₄; 2 M H₂SO₄)/Malachite (0.15 malachite green; 1.4 g polyvinyl alcohol (100,000 MW) in 400 mL H₂O) solution (4/3) was added to each well. Absorbance was measured at 650 nm after a 10 min incubation at room temperature. Potassium phosphate standard calibration curve was used to determine the concentration of polyP release phosphate after subtracting the amount of phosphate present in undigested samples.