Team:York/Protocols/PhosphateAssay

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Phosphate Assay

Reagent preparation

• Phosphate Reagent: 15 ml of colorimetric dye. Ready to use as supplied. Equilibrate to room temperature before use. There may be a small amount of precipitate visible which does not affect the assay performance. Store at room temperature. Actually orange.
• Phosphate Standard: 500uL of 10mM Phosphate standard. Ready to use as supplied. Equilibrate to room temperature before use. Store at room temperature. Actually Clear.

Standard preparation

Materials

• Phosphate Standard
• Phosphate Reagent
• Distilled Water

Procedure

1. Dilute 5ul of supplied Phosphate Standard into 495ul of dH2O in a 1.5ml Eppie. Label ‘S’
2. Make further dilutions to construct the curve. Suggested values are below. Make these in 1.5ml Eppendorfs

Volume S/ ul	Volume dH2O/ ul	Concentration/ nMol/200ul(well)
0	600	0
30	570	1
60	540	2
90	510	3
120	480	4
150	450	5

1. Pipette 200ul of each curve sample into a well.
2. Add 30ul of Phosphate Reagent to each well
3. Leave to equilibrate for 30 mins at room temperature. N.B the reagent is light sensitive so store in a dark room while equilbrating

Sample preparation

N.B. Make sure our sample is diluted to ensure the readings are within the standard value range.

Steps for cell (adherent or suspension) samples:

1. Harvest the amount of cells necessary for each assay (initial recommendation = 2 x 10^6 cells).
2. Wash cells with cold TBS (kept in 4oC fridge, stable for 3 months).
3. Resuspend the cell pellet in 150 µL of Assay Buffer (TBS) on ice.
4. Homogenize cells quickly by pipetting up and down a few times.
5. Sonicate cells for 50 seconds 3X at high setup (one cycle = 30 s sonication – 10 s break – 10 s sonication – 10 s break).
6. Centrifuge sample for 15 minutes at 4°C at top speed using a cold microcentrifuge to remove any insoluble material.
7. Collect supernatant and transfer to a clean tube.

• Keep on ice

Tris-Buffered Saline (TBS) Recipe

1. 50 mM Tris-Cl, pH 7.5
2. 150 mM NaCl
3. To prepare, dissolve 6.05 g and 8.76 g NaCl in 800 mL of H2O.
4. Adjust pH to 7.5 with 1 M HCl
5. Tris Make volume up to 1 L with H2O.

N.B. TBS is stable at 4°C for 3 mo.

Assay procedure and detection

Equilibrate all materials and prepared reagents to room temperature prior to use. It is recommended to assay all standards, controls and samples in duplicate.

Set up Reaction wells:

1. Standard wells = 200 µL standard dilution.
2. Sample wells = 1 – 100 µL samples (adjust volume to 200 µL/well with ddH2O).
3. Background control sample wells = 1 – 100 samples (adjust volume to 200 µL/well with ddH2O).
4. Add 30 µL of Phosphate Reagent to standard, sample and background control wells.
5. Mix and incubate at room temperature for 30 minutes protected from light.
6. Measure output at OD = 650 nm on a microplate reader.