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. 2019 Jul 22;12:2237–2242. doi: 10.2147/IDR.S181906

Figure 2.

Figure 2

Polyphosphate kinase fluorescence enzymatic assay using DAPI. (A) PolyP kinase activity was measured using DAPI to quantify polyP production. Purified E. coli PPK1-His6-tag (16,5 µg/ml) was added to the reaction mix: 50 mM Hepes-KOH pH 7.2, 5 mM MgCl2, 50 mM ammonium sulfate, 1 mM ATP, 1 µg/ml poliP45, 2 mM phosphate creatine and 20 µg/ml creatine kinase. At each time reaction was stopped by adding 50 μM DAPI to 20 μL aliquots and fluorescence was read using SynergyTM Multi-Mode Microplate Reader (Biotek, Winooski, VT, USA) using excitation and emission filters of 400/10 nm and 540/25 nm respectively. (B) PPK activity of the purified PPK1-His6-Tag was assayed at different substrate (ATP) concentrations.