Team:Calgary/Notebook/Protocols/LargeScaleProteinExpressionAndPurification

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<h1>Large Scale Protein Expression &#38; Purification</h1>
<h1>Large Scale Protein Expression &#38; Purification</h1>
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<p>Insert Text Here</p>
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<h2>Reagents and Materials</h2>
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<ul>
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<li>Autoclaved 1 L LB in 2.8 L flask </li>
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<li> Plates of protein producing bacteria being grown</li>
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<li>Shaker</li>
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<li>1 mM IPTG</li>
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<li>Appropriate antibiotics</li>
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<li>Homogenizer</li>
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<li>Shaker</li>
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<li>Shaker</li>
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</ul>
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<h2>Protocol</h2>
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<ol>
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<li>Culture protein-producing bacteria in the LB with appropriate antibiotics </li>
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<li>Grow for 12 hours at 37 C and check OD</li>
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<li>Induce with IPTG to a final concentration of 0.1mM when OD is 0.6</li>
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<li>Let it grow for 4 hours at 30 C</li>
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<li>Spin down the cells for 1 hour at 20 000 rpm</li>
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<li>Homogenize the cells with a homogenizer. Add proteinase inhibitor cocktail </li>
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<li>Take crude lysate and run it through the AKTA, an automatic His tag affinity chromatography machine. </li>
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<li>Run the purified elutions on SDS-PAGE gel</li>
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<li>Concentrate the proteins and store the proteins in 10 mM HEPES buffer, pH 8.0</li>
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</ol>
</section>
</section>
</html>
</html>

Revision as of 01:12, 28 September 2013

Large Scale Protein Expression & Purification

Reagents and Materials

  • Autoclaved 1 L LB in 2.8 L flask
  • Plates of protein producing bacteria being grown
  • Shaker
  • 1 mM IPTG
  • Appropriate antibiotics
  • Homogenizer
  • Shaker
  • Shaker

Protocol

  1. Culture protein-producing bacteria in the LB with appropriate antibiotics
  2. Grow for 12 hours at 37 C and check OD
  3. Induce with IPTG to a final concentration of 0.1mM when OD is 0.6
  4. Let it grow for 4 hours at 30 C
  5. Spin down the cells for 1 hour at 20 000 rpm
  6. Homogenize the cells with a homogenizer. Add proteinase inhibitor cocktail
  7. Take crude lysate and run it through the AKTA, an automatic His tag affinity chromatography machine.
  8. Run the purified elutions on SDS-PAGE gel
  9. Concentrate the proteins and store the proteins in 10 mM HEPES buffer, pH 8.0

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