All liquid samples (anolyte suspension or fresh medium) were filtered through 0.22 μm membranes (Millex, Merck Millipore Ltd., Ireland). Total carbohydrate analysis was performed using a colorimetric phenol/sulphuric acid method (Dubois et al., 1956 (link)). Total phosphate concentration was determined using a phosphate assay kit (Merck, Germany). L -lactate and glycerol were determined using EnzyChrom enzymatic assay kits ECLC-100 and EGLY-100, respectively (BioAssay Systems, USA). The concentrations of glucose and iron were determined using Sigma assay kits GAGO20 and MAK025 (Sigma–Aldrich, USA). The pentosan content in the medium was quantified using a colorimetric method (Finnie et al., 2006 (link)) and xylose concentration was measured using an enzymatic assay kit (Megazyme, Ireland). Acetate, succinate, and propionate in the suspension were determined using 1H NMR spectroscopy as previously described Li et al. (2011) (link). Measurement of pH was performed on 10 ml of anolyte suspension using a pH-meter (Mettler Toledo MP220, Switzerland).
Phosphate assay kit
Manufactured by Merck Group
Sourced in Germany, United Kingdom, United States
The Phosphate Assay Kit is a laboratory tool designed to measure the concentration of phosphate ions in a sample. It provides a quantitative analysis of phosphate levels, which is essential for various applications in biochemistry, cell biology, and environmental studies.
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9 protocols using phosphate assay kit
1
Detailed Carbohydrate and Metabolite Analysis
2
Bacterial Strains and Phosphate Biosensor Assay
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The bacterial strains, plasmids and oligonucleotides used in this work are listed in Table 1 .
For cloning, propagation and phosphate biosensor activity assays, E. coli, and P. putida strains were grown in LB medium or MOPS minimal medium (Neidhardt et al., 1974 (link)) with 0.2% glucose as sole carbon source, with or without KH2PO4. Kanamycin (100 µg ml‐1) was added when necessary. E. coli were grown at 37°C and P. putida KT2440 at 30°C. Phosphate concentrations were calculated using Merck’s phosphate assay kit and following the manufacturer’s instructions.
For cloning, propagation and phosphate biosensor activity assays, E. coli, and P. putida strains were grown in LB medium or MOPS minimal medium (Neidhardt et al., 1974 (link)) with 0.2% glucose as sole carbon source, with or without KH2PO4. Kanamycin (100 µg ml‐1) was added when necessary. E. coli were grown at 37°C and P. putida KT2440 at 30°C. Phosphate concentrations were calculated using Merck’s phosphate assay kit and following the manufacturer’s instructions.
3
Biogeochemical Compound Analysis in Anaerobic Incubations
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Samples for measurements of total and dissolved (0.2 µm-filtered) compounds in the controls and the biotic experiments were taken from serum vials using sterile syringes and needles in the anaerobic chamber. Sulfate samples were immediately sterile-filtered to prevent biological oxidation of sulfide to sulfate and diluted in ddH2O for analysis on an ion chromatograph (Dionex DX-600 IC System) at the Laboratory of Water Geochemistry, IPGP. Dissolved phosphate was quantified using a phosphate assay kit (Sigma-Aldrich) based on the colorimetric malachite green method. Sulfide samples were immediately fixed with zinc acetate solution (5% final concentration) and quantified via the photometric method of Cline75 (link). Total and dissolved (0.2 µm-filtered) iron samples were mixed with HCl (0.5 M final concentration) and stored in the anaerobic chamber until analysis of ferrous and total iron using the ferrozine method76 (link). The pH was monitored on separate liquid samples with a Sensorex© pH electrode inside the anaerobic chamber.
4
Quantitative cGMP Assay Protocol
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To quantitate the level of generated cGMP, Phosphate Assay Kit (MAK168, Sigma‐Aldrich, Gillingham, UK) was used to determine the amount of pyrophosphate. 4.7 μm CaGC was mixed with 0.8 mm GTP in 50 mm HEPES 7.5, 100 mm NaCl and 2.7 mm MgCl2/MnCl2/CaCl2. After 1 h of incubation at 22 °C, the reaction was terminated by adding Master Reaction Mix, followed by 20‐min incubation at 22 °C. Subsequently, fluorescence measurement (excitation 316 nm, emission 456 nm) was taken on a Spectramax M5 plate reader (Molecular Devices, Wokingham, UK). All measurements were done in triplicates.
5
Enzymatic Assays for GOGAT and GS
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GOGAT (EC 2.6.1.53) activity was assayed by determining the rate of glutamine-dependent NADH oxidation as described by Groat and Vance (1981) (link) with some modifications. The reaction assay was carried out in a mixture reaction containing 25 mM KH2PO4 buffer (pH 7.5), 2.5 mM 2-oxoglutarate, 1 mM hydroxylamine, 0.1 mM NADH, and 50–100 µg total protein, with the reaction started by the addition of 10 mM of L-glutamine. GOGAT activity was assayed by determining the rate of glutamine-dependent NADH oxidation at 340 nm for 180 s.
GS (EC 6.3.1.2) activity was assayed according to Slawyk and Rodier (1986) (link) with some modification. Enzymatic extract (150 µg of total protein) was incubated in a mixture reaction containing 15 mM magnesium chloride, 0.5 mM EDTA, 20 mM potassium-glutamate, 4 mM ammonic acetate, and 1 mM adenosine triphosphate (ATP) at 30°C for 15 min. The reaction was stopped by the addition of 1 N sulfuric acid and then the product was centrifuged at 11,000 rpm for 2 min. GS activity was determined by measuring the ATP-dependent phosphate concentration. The phosphate concentration was measured with a Phosphate Assay kit (Sigma-Aldrich) at 620 nm. A phosphate standard curve was used to determine the amount of free phosphate in the sample.
GS (EC 6.3.1.2) activity was assayed according to Slawyk and Rodier (1986) (link) with some modification. Enzymatic extract (150 µg of total protein) was incubated in a mixture reaction containing 15 mM magnesium chloride, 0.5 mM EDTA, 20 mM potassium-glutamate, 4 mM ammonic acetate, and 1 mM adenosine triphosphate (ATP) at 30°C for 15 min. The reaction was stopped by the addition of 1 N sulfuric acid and then the product was centrifuged at 11,000 rpm for 2 min. GS activity was determined by measuring the ATP-dependent phosphate concentration. The phosphate concentration was measured with a Phosphate Assay kit (Sigma-Aldrich) at 620 nm. A phosphate standard curve was used to determine the amount of free phosphate in the sample.
6
RalA GTPase Activity Assay
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For estimation of RalA GTPase activity, free phosphate release in solution was analyzed by malachite green–based colorimetric assay using a phosphate assay kit (Sigma-Aldrich) by following the manufacturer’s protocol. GTPase activity assay was performed with immobilized FLAG-HA-RalA (FH-RalA) by addition of recombinant HuR and/or synthetic miRNA in the presence of 1 mM GTP at room temperature for 30 min while shaking. After the reaction, bead-bound RalA was separated by centrifugation, and released phosphate was present in supernatant due to cleavage of GTP by RalA GTPase activity. Tubes containing this supernatant were placed in ice and 50 μl from each set of samples were added in a 96-well clear plate on which 100 μl of malachite green reagent was added. The mix was incubated in the dark for about 30 min for color to develop and absorbance at 620 nm was measured and analyzed to study change in GTPase activity.
7
Quantifying Free Phosphates in Cell Media
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Free phosphate groups in media from HeLa cells stimulated with Stx1, PBS or calcium ionophore were detected using a Phosphate assay kit (Sigma-Aldrich, Saint Louis, MO), as detailed in the supplement.
8
Measuring Maternal and Amniotic Fluid Phosphate
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Maternal urine Pi levels were determined with the Phosphate Assay Kit (Sigma Aldrich, St. Louis, MO, USA; MAK308) and amniotic fluid Pi levels were determined with the QuantiChrom Phosphate Assay Kit (Bioassays Systems, Hayward, CA, USA; DIPI500) according to the manufacturer’s instructions.
9
Plasma Biochemistry Quantification
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Calcium, ionized calcium and intact PTH levels in plasma were quantified by routine clinical biochemistry laboratory methods. For phosphate quantification, the Phosphate Assay Kit (MAK030-1KT, Sigma-Aldrich) was used according to the manufacturer’s instructions.
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