User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/08/17

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] ON Blot lysate samples
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


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Summary

 * According to the NuPage blotting manual adding 0.01% SDS might improve transfer to the membrane
 * Volume of blotting chamber is ~200 mL so adding 100 μL of 20% SDS solution should be fine
 * Blot of 16August2010 is repeated (only one gel)
 * NOTE: Only 10 µL for HEK293 cells and de Untreated samples a smaller amount of sample left (about 70-80% of CSE treated)
 * This gel will be blotted ON @, 30 V using the NuPage system while on ice in the cold room (4°C)
 * Blots blocked yesterday were treated with α-RIIα, α-AKAP95 (mouse) and α-AKAP450 to see what we can find in lysates despite the fact that transfer was not complete

Results

 * Ponceau staining of membrane and Gel after 15h blotting
 * Ponceau of the second membrane

Discussion

 * Transfer looks still incomplete, and hardly anything seems to have leaked through the first membrane


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