User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/07/09

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Gravin Co-IP, cAMP precipation gel Main project page
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Summary

  • AKAP250 immunoprecipitation
  • Gradient (4-12%) SDS-PAGE (NuPage) for cAMP Precipitations
Gel loading hTERT D9 + HEK293 & HeLa -S see codes 21June2010
# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Gel
cAMP prec.
X X Invitrogen Marker
(8 μL)
HeLa -S
(20 μL)
HEK293
(20 μL)
Biorad Marker
(8 μL)
C1
(20 μL)
C2
(20 μL)
C3
(20 μL)
S1
(20 μL)
S2
(20 μL)
S3
(20 μL)
X X X

Notes

  • HeLa cells had less sample put too gel, both HEK293 and HeLa are very slimy
  • Forgot to lyse the ordered HEK293 cells
  • Blot was blocked ON in 1% BSA in TBS-T
  • Gel was Coomassie stained ON

Results

  • Overnight autoradiograph of RII overlay did not change anything of the image of yesterday
  • Ponceau of cAMP pull down SDS-PAGE

Discussion

  • Ponceau shows bands at higher altitude, maybe look for AKAP-Lbc next to AKAP250?
  • Anoying that both markers are not running equal on every height, but HiMark at least has nice sharp band @ 160 kDa on NuPage gels. Although transfer was less perfect
  • Coomassie staining shows not a 100% efficiency of transfer, and it shows bands in the control lanes that were not visible on Ponceau staining. Hopefully something did transfer


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