An Assay for the Quantification of Inorganic Polyphosphate in Bacteria

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 microlite