Difference between revisions of "Team:Freiburg/Protocols/Western Blot"
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− | <h1 class="sectionedit1">Western Blot | + | <h1 class="sectionedit1">Western Blot Towbin <i>et al.</i></h1> |
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− | <strong>protocol for Western | + | <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/> | ||
− | <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: | + | <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
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