Team:Warsaw/Calendar-Main/3 September 2008

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<h3>Purification of proteins: A-alpha</h3><h4>Piotr, Emilia</h4>
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<h3>Purification of proteins: A-alpha</h3><h4>Piotr, Emilia</h4>
<p><ol><li>Samples were resuspended in PBS, sonicated and centrifuged.</li>  
<p><ol><li>Samples were resuspended in PBS, sonicated and centrifuged.</li>  
<li>Lysis buffer was added separately to pellet and supernatant, then samples were boiled for 10 min. </li>
<li>Lysis buffer was added separately to pellet and supernatant, then samples were boiled for 10 min. </li>
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<li>Lysates loaded on 12% polyacrylamide gel (Fig. 1.) (amount relating to 100 μl of OD=1.0 culture).</li>
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<li>Lysates were loaded on 12% polyacrylamide gel (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/3_September_2008#fig1">Fig. 1.</a>) (amount relating to 100 μl of OD=1.0 culture).</li>
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<li>Gel stained with Coomassie Blue. Optimal induction conditions chosen.</li>
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<li>Gel was stained with Coomassie Blue. Optimal induction conditions chosen.</li>
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<li>A-alpha inoculated for overnight culture (Rosetta strain).</li></ol></p>
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<li>A-alpha in Rosetta strain was cultured overnight.</li></ol></p>
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<img src="https://static.igem.org/mediawiki/2008/b/bd/September_3_rd.jpg" width=300 /><var>Fig. 1. <b>Supernatants from A-alpha induction with various IPTG concentrations<br>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/b/bd/September_3_rd.jpg" width=300 /></a><var><b>Fig. 1.</b> Supernatants from A-alpha induction with various IPTG concentrations<br>
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The arrow shows place of our overexpressed protein:</b><br>  
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The arrow shows place of our overexpressed protein:<br>  
1.  22&deg;C 0.5 mM IPTG 5 h<br>
1.  22&deg;C 0.5 mM IPTG 5 h<br>
2.  Marker<br>
2.  Marker<br>

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Purification of proteins: A-alpha

Piotr, Emilia

  1. Samples were resuspended in PBS, sonicated and centrifuged.
  2. Lysis buffer was added separately to pellet and supernatant, then samples were boiled for 10 min.
  3. Lysates were loaded on 12% polyacrylamide gel (Fig. 1.) (amount relating to 100 μl of OD=1.0 culture).
  4. Gel was stained with Coomassie Blue. Optimal induction conditions chosen.
  5. A-alpha in Rosetta strain was cultured overnight.

Fig. 1. Supernatants from A-alpha induction with various IPTG concentrations
The arrow shows place of our overexpressed protein:
1. 22°C 0.5 mM IPTG 5 h
2. Marker
3. 22°C 0.5 mM IPTG overnight
4. 22°C 1 mM IPTG 5 h
5. 22°C 1 mM IPTG overnight
6. 37°C 0.5 mM IPTG 5 h
7. 37°C 0.5 mM IPTG overnight
8. 37°C 1 mM IPTG 5 h
9. 37°C 1 mM IPTG overnight
10. Marker


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