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

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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
Lysates
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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