Team:Warsaw/Calendar-Main/8 September 2008

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<h3>Our webpage has a completely new layout</h3>
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<h4>Emilia</h4>
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<h3>Purification of proteins: A-alpha</h3><h4>Piotr</h4>
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<h3>Purification of proteins: A-alpha</h3><h4>Piotr</h4>
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<p>Electrophoresis in polyacrylamide gel (12 %) of sonicate containing A-alpha</p>
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<p>Electrophoresis in polyacrylamide gel (12 %) of sonicate containing A-alpha (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/8_September_2008#fig1">Fig. 1.</a>).</p>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/4/49/September_8_th.jpg" width=150 /></a><var><b>Fig. 1.</b> Sonicate containing A-alpha protein fusion<br>
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The arrow shows place of our overexpressed protein:<br>
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1. Marker<br>
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2. sonicate A-alpha with induction<br></var>
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<h3>Purification of A-alpha with His-tag</h3>
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<h3></h3>
<h4>Michał L.</h4>
<h4>Michał L.</h4>
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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>Supernatant 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>
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<li>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</li>
<li>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</li>
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Latest revision as of 19:38, 26 October 2008

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Our webpage has a completely new layout

Emilia

Purification of proteins: A-alpha

Piotr

Electrophoresis in polyacrylamide gel (12 %) of sonicate containing A-alpha (Fig. 1.).

Fig. 1. Sonicate containing A-alpha protein fusion
The arrow shows place of our overexpressed protein:
1. Marker
2. sonicate A-alpha with induction

Michał L.

  1. Supernatant 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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