User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/03/01: Difference between revisions
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** 4 mL Tween20 | ** 4 mL Tween20 | ||
** Mix | ** Mix | ||
* 1x ELFO buffer | |||
* 10x ELFO buffer | |||
** 30.26 g/L TRIS | |||
** 187.60 g/L Glycin | |||
** 10 g/L SDS | |||
===Method=== | ===Method=== | ||
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* Bring gels into the electrophoresis elaboration and pour ELFO buffer in the inner space to check for leakage | * Bring gels into the electrophoresis elaboration and pour ELFO buffer in the inner space to check for leakage | ||
* Remove combs and rinse slots with buffer | * Remove combs and rinse slots with buffer | ||
* Fill slots with sample (40 μL) and marker (15 μL) | * Fill slots with sample (40 μL) and [http://www.cellsignal.com/products/7720.html marker] (15 μL) | ||
* Start electrophoresis (40 min. 200 V) | * Start electrophoresis (40 min. 200 V) | ||
** Alternatively start with 100 V until samples reach the separating gel and increase voltage | ** Alternatively start with 100 V until samples reach the separating gel and increase voltage | ||
====Western Blot==== | ====Western Blot==== | ||
* Moisten sponges, filter papers and membrane in transfer buffer | * Moisten sponges, filter papers and membrane in transfer buffer | ||
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** White clamp | ** White clamp | ||
* Put sandwhich in the transferring system (black to black) and fill it with transferbuffer | * Put sandwhich in the transferring system (black to black) and fill it with transferbuffer | ||
** DON'T FORGET ICE BLOCK! | |||
* Transfer proteins to membrane for 1 hour @ 100 V on ice while stirring | * Transfer proteins to membrane for 1 hour @ 100 V on ice while stirring | ||
* After transfer check if marker is visible on membrane (successful transfer) | * After transfer check if marker is visible on membrane (successful transfer) | ||
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* Wash 3x 10 min. in TBST buffer (first can be less) | * Wash 3x 10 min. in TBST buffer (first can be less) | ||
* Treat with secondary antibody (diluted in 5% TBST-milk solution) (1h, RT) | * Treat with secondary antibody (diluted in 5% TBST-milk solution) (1h, RT) | ||
* Wash 3x 10 min. in TBST buffer | * Wash 3x 10 min. in TBST buffer | ||
====Preparing film==== | ====Preparing film==== | ||
* 1: | * 1:1 ratio [[wikipedia:Chemiluminescence#Enhanced_chemiluminescence|ECL]] solutions (2 mL per membrane) | ||
* Incubate membrane in ECL solution 5 min, RT (both sides of membrane) | * Incubate membrane in ECL solution 5 min, RT (both sides of membrane) | ||
* Place blot in between two overhead sheets | * Place blot in between two overhead sheets | ||
* Take picture (depends on what is available) | * Take picture (depends on what is available) | ||
==Results== | ==Results== | ||
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==Conclusion== | ==Conclusion== | ||
*{{done}} Ow.. | *{{done}} Ow.. | ||
==Related== | |||
===Related topics=== | |||
[[Western blot]] | |||
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__NOTOC__ | __NOTOC__ |
Revision as of 05:16, 25 March 2010
<html><style type="text/css"> .todo { color: red } .done { color: green} </style></html>
Western blot | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
Summary
Materials & MethodsMaterials
MethodSDS-PAGE
Western Blot
Preparing film
Results
--> PICTURES ANOUK Conclusion
RelatedRelated topics |