Team:Calgary/Notebook/Protocols/LargeScaleProteinExpressionAndPurification

From 2013.igem.org

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<ol>
<li>Culture protein-producing bacteria in the LB with appropriate antibiotics </li>
<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>
+
<li>Grow for 12 hours at 37°C and check OD</li>
<li>Induce with IPTG to a final concentration of 0.1mM when OD is 0.6</li>
<li>Induce with IPTG to a final concentration of 0.1mM when OD is 0.6</li>
-
<li>Let it grow for 4 hours at 30 C</li>
+
<li>Let it grow for 4 hours at 30°C</li>
<li>Spin down the cells for 1 hour at 20 000 rpm</li>
<li>Spin down the cells for 1 hour at 20 000 rpm</li>
<li>Homogenize the cells with a homogenizer. Add proteinase inhibitor cocktail </li>
<li>Homogenize the cells with a homogenizer. Add proteinase inhibitor cocktail </li>

Revision as of 01:19, 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
  • AKTA
  • Proteinase Inhibitor Cocktail
  • Shaker
  • 10mM HEPES buffer

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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