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| Line 132: |
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| | </ol> | | </ol> |
| | </div> | | </div> |
| − | <div id="phosphate assay">
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| − | <h2 id="phosphate assay"> Phosphate Assay</h2>
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| − | <h3> Reagent preparation</h3>
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| − | <ul>
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| − | <li>
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| − | <spam style="font-color:orange">Phosphate Reagent:</spam> 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.
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| − |
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| − | </li>
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| − | <li>
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| − | <span style="font-color:orange">Phosphate Standard: </span>500uL of 10mM Phosphate standard. Ready to use as supplied. Equilibrate to room temperature before use. Store at room temperature. Actually Clear.
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| − | </li>
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| − | </ul>
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| − | <h3> Standard preparation</h3>
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| − | <h4>Materials</h4>
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| − | <ul>
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| − | <li>
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| − | <span style="color:green">Phosphate Standard
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| − |
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| − | </li>
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| − | <li>
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| − | <span style="color:blue">Phosphate Reagent
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| − |
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| − | </li>
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| − | <li>Distilled Water</li>
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| − | </ul>
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| − | </h4>
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| − | <h4>Procedure</h4>
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| − | <ol>
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| − | <li> Dilute 5ul of supplied Phosphate Standard into 495ul of dH2O in a 1.5ml Eppie. Label ‘S’ </li>
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| − | <li> Make further dilutions to construct the curve. Suggested values are below. Make these in 1.5ml Eppies</li>
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| − | </ol>
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| − | <table>
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| − | <tr>
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| − | <td>Volume S/ ul</td>
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| − | <td>Volume dH2O/ ul</td>
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| − | <td>Concentration/ nMol/200ul(well)</td>
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| − | </tr>
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| − | <tr>
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| − | <td>0</td>
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| − | <td>600</td>
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| − | <td>0</td>
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| − | </tr>
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| − | <tr>
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| − | <td>30</td>
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| − | <td>570</td>
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| − | <td>1</td>
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| − | </tr>
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| − | <tr>
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| − | <td>60</td>
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| − | <td>540</td>
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| − | <td>2</td>
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| − | </tr>
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| − | <tr>
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| − | <td>90</td>
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| − | <td>510</td>
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| − | <td>3</td>
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| − | </tr>
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| − | <tr>
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| − | <td>120</td>
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| − | <td>480</td>
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| − | <td>4</td>
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| − | </tr>
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| − | <tr>
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| − | <td>150</td>
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| − | <td>450</td>
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| − | <td>5</td>
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| − | </tr>
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| − | </table>
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| − | <ol>
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| − | <li>Pipette 200ul of each curve sample into a well.</li>
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| − | <li>Add 30ul of Phosphate Reagent to each well</li>
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| − | <li>Leave to equilibrate for 30 mins at room temperature. N.B the reagent is light sensitive so store in a dark room while equilbrating</li>
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| − | </ol>
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| − | <h3>Sample preparation</h3>
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| − | <p>N.B. Make sure our sample is diluted to ensure the readings are within the standard value range.</p>
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| − | <p>
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| − | <u>Steps for cell (adherent or suspension) samples:</u>
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| − | </p>
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| − | <ol>
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| − | <li>Harvest the amount of cells necessary for each assay (initial recommendation = 2 x 10^6 cells).</li>
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| − | <li>Wash cells with cold TBS (kept in 4oC fridge, stable for 3 months). </li>
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| − | <li>Resuspend the cell pellet in 150 µL of Assay Buffer (TBS) on ice. </li>
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| − | <li>Homogenize cells quickly by pipetting up and down a few times. </li>
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| − | <li>Sonicate cells for 50 seconds 3X at high setup (one cycle = 30 s sonication – 10 s break – 10 s sonication – 10 s break). </li>
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| − | <li>Centrifuge sample for 15 minutes at 4°C at top speed using a cold microcentrifuge to remove any insoluble material. </li>
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| − | <li>Collect supernatant and transfer to a clean tube. </li>
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| − | </ol>
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| − | <ul>
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| − | <li>Keep on ice</li>
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| − | </ul>
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| − | <p>
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| − | <u>Tris-Buffered Saline (TBS) Recipe</u>
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| − | </p>
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| − | <ol>
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| − | <li>50 mM Tris-Cl, pH 7.5</li>
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| − | <li>150 mM NaCl</li>
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| − | <li>To prepare, dissolve 6.05 g and 8.76 g NaCl in 800 mL of H2O. </li>
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| − | <li>Adjust pH to 7.5 with 1 M HCl </li>
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| − | <li>Tris Make volume up to 1 L with H2O.</li>
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| − | <p>N.B. TBS is stable at 4°C for 3 mo.</p>
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| − | </ol>
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| − | <h3>Assay procedure and detection</h3>
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| − | <p> Equilibrate all materials and prepared reagents to room temperature prior to use. It is recommended to assay all standards, controls and samples in duplicate.</p>
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| − | <p>Set up Reaction wells: </p>
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| − | <ol>
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| − | <li>Standard wells = 200 µL standard dilution. </li>
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| − | <li>Sample wells = 1 – 100 µL samples (adjust volume to 200 µL/well with ddH2O). </li>
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| − | <li>Background control sample wells = 1 – 100 samples (adjust volume to 200 µL/well with ddH2O). </li>
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| − | <li>Add 30 µL of Phosphate Reagent to standard, sample and background control wells. </li>
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| − | <li>Mix and incubate at room temperature for 30 minutes protected from light. </li>
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| − | <li>Measure output at OD = 650 nm on a microplate reader.</li>
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| − | </ol>
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| − | </div>
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| − | </div>
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| − | <!-- <h3> Glassmilk recipe </h3>
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| − | <ol> <li>Stir 400g silica 325 in 800 ml of dH2O</li>
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| − | <li>Leave to settle for 90 minutes</li>
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| − | <li>Remove supernatant and centrifuge 6000 rpm for 10 minutes</li>
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| − | <li>Resuspend pellet in 250 ml dH2O</li>
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| − | <li>Add conc. Nitric Acid to 50%</li>
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| − | <li>Stir and heat to a boil gently</li>
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| − | <li>Stir to RT gently</li>
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| − | <li>Centrifuge @ 6000 rpm for 10 minutes (make sure you use either glass or polypropylene tubes, polystyrene tubes can’t withstand over 3000g) </li>
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| − | <li>Resuspend in 250 ml dH2O</li>
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| − | <li>Repeat spinning and washing until pH is neutral</li>
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| − | <li>Resuspend pellet to make 50% by volume slurry</li>
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| − | </ol>
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| − | <p>Aliquot and store indefinitely at RT</p>
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| − | -->
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