Difference between revisions of "Team:Heidelberg/Your Aptabody"

 
 
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#REDIRECT [[Team:Heidelberg/Software/Contact]]
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<h1>AptaBody Western Blot Protocol</h1>
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<ol>
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<li>Run SDS-PAGE and do Western Blot transfer following common protocols,</li>
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<li>Prepare 5% milk or BSA in TBS-T.</li>
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<li>After blotting, wash the membrane with TBS-T three times for 5 min each,</li>
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<li>Block the membrane for 1 h with 5% milk in TBS-T<br /><strong>Note:</strong> For very fast results you can skip the blocking step. However, the background signal may be higher.</li>
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<li>After blocking, wash membrane with TBS-T three times for 5 min each</li>
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<li>Prepare the Anti-His-tag reaction solution:
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<ol>
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<li>Fill up the  “His-Tag AptaBody” with 50 µl H<sub>2</sub>O</li>
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<li>Heat up the “His-Tag AptaBody” at 95 °C for 5 min</li>
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<li>Fill up the “Catalyst for AptaBody” with 50 µl H<sub>2</sub>O</li>
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<li>Mix equal volumes of both solutions</li>
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<li>Let reaction solution stand for 10 min</li>
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</ol></li>
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<li>Add 15 µl Anti-His-tag reaction solution to 5 ml TBS-T in a Falcon tube.</li>
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<li>Carefully put the membrane into the tube and let it roll at room temperature for at least 1 h.</li>
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<li>Wash the membrane in TBS-T three times for 5 min.</li>
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<li>Prepare ECL Western blotting substrate according to manufacturer's protocol.</li>
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<li>Pipet 50 µl H<sub>2</sub>O onto a clear plastic film, add the membrane avoiding any air bubbles, and pipet another 50 µl H<sub>2</sub>O onto the membrane. Put another sheet of clear plastic on top of the membrane.</li>
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<li>Detect chemiluminescence with the proper imaging system.</li>
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<img class="img-responsive" src="https://static.igem.org/mediawiki/2015/thumb/b/b9/AptabodytextFig7.png/800px-AptabodytextFig7.png">
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<div class="imagewrappercaption">
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<strong>
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Figure 1: Detection of His-tagged protein in cell lysates using Anti-His I AptaBody.
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<p class="basictext">
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AptaBody Western Blot to detect his-tagged T7-polymerase (T7-His) in <i>E. coli</i> cell lysates. The blots were incubated with Anti-His I AptaBody over night. DNA or RNA do not interfere with the AptaBody and do not influence the readout.
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Latest revision as of 03:03, 19 September 2015

AptaBody Western Blot Protocol

  1. Run SDS-PAGE and do Western Blot transfer following common protocols,
  2. Prepare 5% milk or BSA in TBS-T.
  3. After blotting, wash the membrane with TBS-T three times for 5 min each,
  4. Block the membrane for 1 h with 5% milk in TBS-T
    Note: For very fast results you can skip the blocking step. However, the background signal may be higher.
  5. After blocking, wash membrane with TBS-T three times for 5 min each
  6. Prepare the Anti-His-tag reaction solution:
    1. Fill up the “His-Tag AptaBody” with 50 µl H2O
    2. Heat up the “His-Tag AptaBody” at 95 °C for 5 min
    3. Fill up the “Catalyst for AptaBody” with 50 µl H2O
    4. Mix equal volumes of both solutions
    5. Let reaction solution stand for 10 min
  7. Add 15 µl Anti-His-tag reaction solution to 5 ml TBS-T in a Falcon tube.
  8. Carefully put the membrane into the tube and let it roll at room temperature for at least 1 h.
  9. Wash the membrane in TBS-T three times for 5 min.
  10. Prepare ECL Western blotting substrate according to manufacturer's protocol.
  11. Pipet 50 µl H2O onto a clear plastic film, add the membrane avoiding any air bubbles, and pipet another 50 µl H2O onto the membrane. Put another sheet of clear plastic on top of the membrane.
  12. Detect chemiluminescence with the proper imaging system.
Figure 1: Detection of His-tagged protein in cell lysates using Anti-His I AptaBody.

AptaBody Western Blot to detect his-tagged T7-polymerase (T7-His) in E. coli cell lysates. The blots were incubated with Anti-His I AptaBody over night. DNA or RNA do not interfere with the AptaBody and do not influence the readout.