Team:Calgary/Sandbox/Notebook/Protocols/FreezeThawProtocolforProteinSamples

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<h1>Freeze-Thaw Cell Lysis Protocol for Protein Samples</h1>

<h2>Reagents and Materials</h2>
<ul>
<li>Lysis Buffer (50mM NaH2PO4 + 500mM NaCl)/li>
<li>PMSF (phenylmethylsulfonyl fluoride)</li>
<li>Lysozyme</li>
<li>DTT (dithiothreitol)</li>
<li>Falcon tubes</li>
<li>Dry ice</li> 
</ul>
<h2>Protocol</h2>
<ol>
<li>Centrifuge cells at max for 10 minutes. Discard supernatant</li>
<li>Add 250 µL Lysis Buffer, 2.5 µL PMSF, 5 µL Lysozyme, and 1 µL DDT. Re-suspend pellet</li>
<li>Leave on ice for 30 min</li>
<li>Put samples in dry ice for ~ 3 minutes, and then transfer to 37°C water bath for ~3 minutes. Repeat this cycle 6 times. </li>
<li>Centrifuge at max for 10 minutes</li>
<li>Transfer supernatant to a 1.5 mL microcentrifuge tube. Re-suspend the pellet in 250 µL of Lysis Buffer and transfer to a separate microcentrifuge tube</li>
<li>Run a Bradford Assay on the samples if the protein amount in the gel must be constant</li>
<li>Add appropriate amount of dye and sample (40 µL total). If Bradford was not performed, use 30 µL of protein sample and 10 µL of 4x dye</li>
<li>Boil the sample/dye mixture for 5 minutes at 95°C</li>
<li>Load 30 - 40 µL of sample into gel. Run gel at 100V until the sample moves out of the wells, then run at 150V</li>
</ol>

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