Difference between revisions of "Team:UiOslo Norway/Experiments/Solubility Assay"

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<p></br></p>
 
<p></br></p>
  
 +
<h3> Cell lysis using glass beads </h3>
 
<p>
 
<p>
 
<ul>
 
<ul>
−
<li><p>Resuspend cell pellet in 600 ul PBS buffer (pH 7.4) </p></li>
+
<li><p>Resuspend cell pellet in 600 µl PBS buffer (pH 7.4) </p></li>
 
<li><p>Add glass beads and disrupt cells by vortexing</p></li>
 
<li><p>Add glass beads and disrupt cells by vortexing</p></li>
 
<li><p>Centrifuge at 16.000 x g for 5 minutes</p></li>
 
<li><p>Centrifuge at 16.000 x g for 5 minutes</p></li>
 
<li><p>Transfer soluble fraction into a new tube and resuspend insoluble fraction in 600 ul PBS buffer (pH 7.4)</p></li>
 
<li><p>Transfer soluble fraction into a new tube and resuspend insoluble fraction in 600 ul PBS buffer (pH 7.4)</p></li>
 
</p>
 
</p>
 +
</ul>
 +
<h3> Cell lysis using sonication </h3>
 +
 +
<p>
 +
<ul>
 +
<li><p>Resuspend the cell pellet in precooled imidazolebuffer. </p></li>
 +
<li><p>Sonicate 3x for 30 sec at a amplitude of 60. Incubate on ice for 1 minute between each sanitation step.</p></li>
 +
<li><p>Centrifuge the cell lysate at 13.000 rpm for 30 minutes at 4 °C.</p></li>
 +
<li><p>Keep the supernatant for further experiments</p></li>
 +
</p>
 +
<p><b>Imidazole buffer</b></br>
 +
</br>
 +
50 mM Tris-HCl pH 8 </br>
 +
100 mM NaCl</br>
 +
10 mM  beta-Mercaptoethanol</br>
 +
10 mM        Imidazole </br>
 +
  
 
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<div class="clear"></div>

Revision as of 12:07, 10 September 2015

Solubility Assay:

Back to Protocols


Cell lysis using glass beads

  • Resuspend cell pellet in 600 µl PBS buffer (pH 7.4)

  • Add glass beads and disrupt cells by vortexing

  • Centrifuge at 16.000 x g for 5 minutes

  • Transfer soluble fraction into a new tube and resuspend insoluble fraction in 600 ul PBS buffer (pH 7.4)

Cell lysis using sonication

  • Resuspend the cell pellet in precooled imidazolebuffer.

  • Sonicate 3x for 30 sec at a amplitude of 60. Incubate on ice for 1 minute between each sanitation step.

  • Centrifuge the cell lysate at 13.000 rpm for 30 minutes at 4 °C.

  • Keep the supernatant for further experiments

    • Imidazole buffer

    50 mM Tris-HCl pH 8
    100 mM NaCl
    10 mM beta-Mercaptoethanol
    10 mM Imidazole


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