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

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==Notes==
==Notes==
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==Results==
==Results==
*{{todo| WHAT?}}
*Ponceau staining of membrane and Gel after 15h blotting
[[Image:17082010_lysates_Ponceau_staining.tif|350px]][[Image:Coomassie_gel_after_blot_18.8.10.tif|350px]]
* Ponceau of the second membrane
[[Image:17082010_lysates_Ponceau_staining_2nd.tif|350px]]


==Discussion==
==Discussion==
*{{done}} Ow..
*Transfer looks still incomplete, and hardly anything seems to have leaked through the first membrane
 
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==Related==
==Related==
===Related entries===
===Related entries===

Revision as of 08:34, 20 August 2010

<html><style type="text/css"> .todo { color: red } .done { color: green} </style></html>

ON Blot lysate samples <html><img src="/images/9/94/Report.png" border="0" /></html> 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)
4-12% Gradient NuPage Gel loading LYSATES hTERT D9 + HEK293, HeLa -S & Jurkat see codes 21June2010
# 1 2 3 4 5 6 7 8 9 10
Gel
cAMP prec. 		Invitrogen Marker
(8 μL) 		Jurkat
(20 μL) 		HEK293
(10 μL) 		Biorad Marker
(8 μL) 		C1
(20 μL) 		C2
(20 μL) 		C3
(20 μL) 		S1
(<20 μL) 		S2
(<20 μL) 		S3
(<20 μL)
  • 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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