This study focuses on the quantification of inorganic polyphosphate (polyP), a polymer involved in bacterial virulence and stress response. The method utilizes a multiwell plate assay to measure phosphate levels derived from polyP.
Inorganic polyphosphate (polyP) is a linear phosphate polymer involved in bacterial virulence and stress response, stored as cytoplasmic granules.
To quantify polyP, take a multiwell plate with increasing concentrations of inorganic phosphate standards.
Add purified bacterial poly P extract to an empty well.
Introduce a reaction buffer containing exopolyphosphatase enzyme to each well and incubate.
The enzyme cleaves polyP into free inorganic phosphate, converting it into a quantifiable form.
Add a detection reagent containing molybdate and ascorbic acid and incubate.
Molybdate reacts with phosphate to form phosphomolybdate complexes, which are reduced by ascorbic acid into a blue-colored complexes.
Measure the absorbance of the colored complex. The absorbance measured corresponds to blue color intensity which is directly proportional to the phosphate concentration.
Generate the standard curve and calculate the sample's phosphate levels, reflecting the bacteria's original polyP content.
Start by preparing standards containing zero, five, 50, or 200 micromolar potassium phosphate in 50 millimolar pH eight tris-hydrochloride.
Aliquot 100 microliters of each phosphate standard and 100 microliters of extracted polyP samples into separate wells of a clear 96 well plate. Prepare a master mix containing, per sample, 30 microliters of 5X ScPPX reaction buffer, 19 microliters water, and one microliter of purified ScPPX. Add 50 microliters of the master mix to each well of the 96 well plate.
And incubate for 15 minutes at 37 degrees celsius. On the day of detection, prepare a fresh working stock of detection solution by mixing at 9.12 milliliters of detection solution base with 88 milliliters of one molar ascorbic acid, and allow it to come to room temperature before use. Add 50 microliters of detection solution to each sample and standards in the 96 well plate.
To allow color development, incubate the plate at room temperature for approximately two minutes. Use a plate reader to measure absorbance at 882 nanometers and calculate the phosphate concentration of each sample by comparison to the potassium phosphate standard curve. Then convert the phosphate concentrations to nanomoles of polyP derived phosphate in each cell lysate and normalize cellular polyP content to total cellular protein, as described in the manuscript.
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