1. Harvesting Samples for Polyphosphate Extraction
- Grow bacteria under the conditions of interest for determining polyP content. For this protocol, grow Lactobacillus reuteri overnight at 37 °C without shaking in malic enzyme induction (MEI) medium without cysteine (MEI-C).
- Harvest enough cells by centrifugation in a 1.5 mL microcentrifuge tube to total 50 – 100 µg of cellular protein (see step 3 below). For E. coli, this is 1 mL of a log phase culture at an A600 of 0.2 - 0.4. For L. reuteri, this is 1 mL of an overnight culture. Adjust as necessary for other species of interest.
- Completely remove the supernatant from the cell pellets.
- Resuspend the cell pellets in 250 µL of GITC lysis buffer (4 M guanidine isothiocyanate, 50 mM Tris-HCl, pH 7) and lyse by incubation at 95 °C for 10 min. Store lysates at -80 °C.
NOTE: Be consistent with lysis time, since extended incubation at high temperature may result in degradation of polyP by hydrolysis. Lysates can be stored indefinitely at -80 °C.
CAUTION: Guanidine isothiocyanate is a chaotropic salt and should be handled with gloves and disposed of as hazardous waste.
2. Measuring the Protein Content of Cell Lysates
- Prepare bovine serum albumin (BSA) standards containing 0, 0.1, 0.2, and 0.4 mg mL-1 of BSA in GITC lysis buffer.
NOTE: It is important to make the BSA standards in GITC, since GITC influences the color development of the Bradford assay. BSA standards can be stored indefinitely at -20 °C. - Aliquot 5 µL of thawed, well-mixed cell lysates and of BSA standards to separate wells in a clear 96-well plate.
- Add 195 µL of Bradford reagent to each well and measure absorbance at 595 nm (A595) in a plate reader (see Table of Materials).
- Calculate the amount of protein in each well by comparison to the BSA standard curve, using the formula y = mx + b, where y is A595, x is µg of BSA, m is the slope of the standard curve, and b is the y-intercept of the standard curve. Multiply the resulting value by 0.05 to determine the total mg of protein in each sample.
3. Extracting Polyphosphate
- Add 250 µL of 95% ethanol to each GITC-lysed sample and vortex to mix.
- Apply that mixture to a silica membrane spin column and centrifuge for 30 s at 16,100 x g.
- Discard the flow-through, then add 750 µL of 5 mM Tris-HCl (pH 7.5), 50 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA), 50% ethanol, and centrifuge for 30 s at 16,100 x g.
- Discard the flow-through and centrifuge for 2 min at 16,100 x g.
- Place the column in a clean 1.5 mL microfuge tube and add 150 µL of 50 mM Tris-HCl (pH 8).
- Incubate at room temperature for 5 min, then elute polyP by centrifuging for 2 min at 8,000 x g.
NOTE: If desired, the eluates can be stored at -20 °C for at least 1 week.
4. Digesting Polyphosphate
- Prepare standards containing 0, 5, 50, or 200 µM potassium phosphate in 50 mM Tris-HCl (pH 8).
NOTE: Potassium phosphate standards can be stored indefinitely at room temperature. - Aliquot 100 µL of each phosphate standard and of extracted polyP samples into separate wells of a clear 96-well plate.
- Prepare a master mix containing (per sample): 30 µL of 5x ScPPX reaction buffer (100 mM Tris-HCl, 25 mM MgCl2, 250 mM ammonium acetate, pH 7.5)13, 19 µL of H2O, and 1 µL of purified ScPPX (1 mg mL-1).
- Add 50 µL of the master mix to each well of the 96-well plate. Incubate for 15 min at 37 °C.
NOTE: If desired, the digested polyP samples can be stored at -20 °C indefinitely.
5. Detecting Free Phosphate
- Prepare detection solution base by dissolving 0.185 g of antimony potassium tartrate in 200 mL of water, adding 150 mL of 4 N H2SO4, then adding 1.49 g of ammonium heptamolybdate. Stir to dissolve and then bring to a final volume of 456 mL. Filter the solution to remove particulates and store protected from light at 4 °C for up to 1 month.
- Prepare a stock solution of 1 M ascorbic acid. Store protected from light at 4 °C for up to 1 month.
- Prepare a fresh working stock of detection solution each day by mixing 9.12 mL of detection solution base with 0.88 mL of 1 M ascorbic acid. Allow the detection solution to come to room temperature before use.
- Add 50 µL of detection solution to each sample and standard in the 96-well plate and incubate at room temperature for about 2 min to allow color development.
- Measure absorbance at 882 nm with a plate reader and calculate the phosphate concentration of each sample by comparison to the potassium phosphate standard curve.
CAUTION: The detection solution contains toxic salts and strong acids. Wear gloves and treat excess solution as toxic waste.
6. Calculating Cellular Polyphosphate Content
- Convert the phosphate concentrations determined in step 5.5 to nanomoles of polyP-derived phosphate in each entire cell lysate according to the following formula:
nmol polyP = 1.5 x (µM phosphate / 10)
NOTE: The standard curve-based method in step 5 determines the concentration of phosphate (in µM) in each 100 µL polyP sample aliquoted in step 4.2. To convert this concentration to a number of nmol, 100 µL is divided by 106 to give a volume in L, multiplied by the concentration (µmol L-1), then multiplied by 1,000 (the number of nmol in a µmol). This reduces to dividing the concentration by 10. The total extract volume prepared in step 3 is 150 µL, so it is necessary to multiply the resulting value by 1.5 to calculate the nmol of phosphate present in the entire extract. - Normalize cellular polyP content to total cellular protein by dividing nmol polyP by the mg total protein in each sample determined in step 1.4. PolyP levels are expressed in terms of the concentration of individual polyP-derived free phosphate.
NOTE: In some cases, the amount of polyP-derived phosphate present in a sample may fall outside the linear range of the phosphate standard curve. If the levels of polyP are very high, the excess polyP-containing eluate from step 3.6 can be diluted 1:10 or 1:100, as necessary, and then measured again as described in steps 4 through 6.
