Team:Warsaw/Calendar-Main/11 August 2008

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<h3>Checking for presence of A on the cell membrane<br>
 
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Piotr</h3>
 
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<li>Pouring bacteria by drops onto nitrocellulose</li>
 
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<li>Drying</li>
 
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<li>Blocking</li>
 
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<li>Anti-A antibody binding</li>
 
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<li>Washing</li>
 
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<li>Anti-rabbit antibody binding</li>
 
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<li>Developing with BCIP and NBT</li>
 
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[photo of the gel is to be placed here]
 
<h3>Checking whether degradation of the fusions with OmpA is caused by Lon and OmpT proteases (present in <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a>)</h3>
<h3>Checking whether degradation of the fusions with OmpA is caused by Lon and OmpT proteases (present in <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a>)</h3>

Revision as of 14:16, 12 October 2008

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Checking whether degradation of the fusions with OmpA is caused by Lon and OmpT proteases (present in TOP10)

Piotr

Test was conducted in E.coli Rosetta strain expressing omp_omega_A_alfa (with and without induction) and omp_A_alfa.

  1. Spinnign
  2. Suspending
  3. Adding of lysis buffer
  4. Boiling
  5. Putting into poliacrylamide gel
  6. Transfer onto nitrocellulose
  7. Blocking
  8. Anti-A antibody binding
  9. Washing
  10. Anti-rabbit antibody binding
  11. Developing with BCIP and NBT

[photo of the gel is to be placed here]

We didn't observe differences in espression and degradation in Rosettas nor in TOP10. Therefore we suppose that degradation of the fusions is caused by other factor than Lon and OmpT proteases.

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