Team:Warsaw/Calendar-Main/8 September 2008

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<h4>Michał L.</h4>
<h4>Michał L.</h4>
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<ol>
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<li>200 ml of overnight <i>E. coli</i> culture was spun down at 6000 RPM, 10 min at 4&deg;C</li>
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<li>Pellet was resuspended in 20 ml of sonication buffer A (50 mM phosphate buffer pH 7.2, 100 mM NaCl, 10 mM imidazole) </li>
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<li>ssupernatant containing A-alpha was mixed with NiNta bead in proportion 10:1 and placed on rotating platform for 30 min at 4&deg;C</li>
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<li>The mixture was sonicated in 20 pulses: 90 W pulse duration - 20 s, pause 40 s. </li>
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<li>Post-sonication debris were spun down at 13200 RPM, 20 min at 4&deg;C</li>
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<li>Clear supernatant was mixed with NiNta bead in proportion 10:1 and placed on rotating platform for 30 min at 4&deg;C</li>
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<li>Bead was spun down at 6000 RPM, 5 min at 4&deg;C</li>
<li>Bead was spun down at 6000 RPM, 5 min at 4&deg;C</li>
<li>Supernatant was discarded and equal amount of Elution buffer 1 (Sonication buffer A suplemented with 50 mM imidazole) was placed on top of the bead</li>
<li>Supernatant was discarded and equal amount of Elution buffer 1 (Sonication buffer A suplemented with 50 mM imidazole) was placed on top of the bead</li>

Revision as of 21:27, 19 October 2008

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Purification of proteins: A-alpha

Piotr

Electrophoresis in polyacrylamide gel (12 %) of sonicate containing A-alpha

Purification of A-alpha with His-tag

Michał L.

  1. ssupernatant containing A-alpha was mixed with NiNta bead in proportion 10:1 and placed on rotating platform for 30 min at 4°C
  2. Bead was spun down at 6000 RPM, 5 min at 4°C
  3. Supernatant was discarded and equal amount of Elution buffer 1 (Sonication buffer A suplemented with 50 mM imidazole) was placed on top of the bead
  4. Bead was spun down at 6000 RPM, 5 min at 4°C
  5. Supernatant was collected for further analysis and equal amount of Elution buffer 2 (Sonication buffer A supplemented with 100 mM imidazole) was placed on top of the bead
  6. The above two steps were repeated for elution buffers 3 and 4 (150 mM and 200 mM imidazole)
  7. All fractions obtained in this procedure were resolved on 8% SDS-PAGE gel. It turned out that optimum imidazole concentration for elution of His-tagged A protein is 150 mM

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