Difference between revisions of "Team:Freiburg/Protocols/Western Blot"

 
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<h1 class="sectionedit1">Western Blot towbin et al.</h1>
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<strong>protocol for Western blot towbin et al.</strong><br/>
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<strong>protocol for Western Blot Towbin <i>et al.</i></strong><br/>
 
<em>remarks concerning the protocol (author, adapted from etc.)</em>
 
<em>remarks concerning the protocol (author, adapted from etc.)</em>
 
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<a class="media" href="https://static.igem.org/mediawiki/2015/4/4b/Freiburg_wiki-symbols-orga.png" title="wiki:symbols:orga.png"><img alt="" class="media" src="https://static.igem.org/mediawiki/2015/4/4b/Freiburg_wiki-symbols-orga.png"/></a> material: chemicals, used kits, …<br/>
 
<a class="media" href="https://static.igem.org/mediawiki/2015/4/4b/Freiburg_wiki-symbols-orga.png" title="wiki:symbols:orga.png"><img alt="" class="media" src="https://static.igem.org/mediawiki/2015/4/4b/Freiburg_wiki-symbols-orga.png"/></a> material: chemicals, used kits, …<br/>
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<a class="media" href="https://static.igem.org/mediawiki/2015/b/bb/Freiburg_wiki-symbols-alarm2.png" title="wiki:symbols:alarm2.png"><img alt="" class="media" src="https://static.igem.org/mediawiki/2015/b/bb/Freiburg_wiki-symbols-alarm2.png"/></a> duration: … min<br/>
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<a class="media" href="https://static.igem.org/mediawiki/2015/b/bb/Freiburg_wiki-symbols-alarm2.png" title="wiki:symbols:alarm2.png"><img alt="" class="media" src="https://static.igem.org/mediawiki/2015/b/bb/Freiburg_wiki-symbols-alarm2.png"/></a> duration: pipapo… <br/>
 
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Latest revision as of 23:01, 13 September 2015

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Western Blot Towbin et al.

protocol for Western Blot Towbin et al.
remarks concerning the protocol (author, adapted from etc.)

material: chemicals, used kits, …
duration: pipapo…

  • cut PVDF membrane and 4 whatman-papers in gel size
  • PVDF membranes have to be activated in 100% MeOH before use
  • Rinse membranes with blocking ubffer, Whatman-paper soaked in blocking buffer
  • stack 2 whatman, membrane, gel and 2 whatman into the blotting apparatus
  • (make sure there are no bubbles after adding each layer)
  • blot for 1h (thin gels) (0.8mA/cm^2 Gel)
  • block membrane ~ 30 min in blocking buffer
  • 1st antibody incubation: 1h, RT or overnight 4°C
  • wash membrane 3x 10 min in 1x TBS-T
  • 2nd antibody incubation: 1h, RT or overnight 4°C
  • wash membrane 3x 10min in 1y TBS-T

Staining with ECL:

  • put membrane into prepared foil
  • mix ECL-solution 1:1
  • put mix on membrane; assure good distribution of the mix over the whole membrane
  • incubation: 1min RT
  • perform the read-out

Solutions: 10x Transfer buffer:

  • 30.275g Tris base
  • 144g Glycine
  • bring up the volume to 1L with ddH2O

1xTransfer buffer:

  • 700ml ddH2O
  • 100ml 10x Transfer buffer
  • 200ml Methanol

10x TBS

  • 1M Tris/HCl pH 7.5
  • 1.5M NaCl

Blocking Buffer:

  • 5% dry milk powder in 1x TBS-T
,