User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/07/06

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Summary

cAMP precipitation

  • Beads were washed as described 09April2010
  • 50 μL of NuPage LDS sample buffer + DTT was added to the beads (800 μL LDS sample buffer + 200 μL 1 M DTT)
    • Beads were put to 70 °C for 10 min.
  • Samples were run on NuPage prefabricated Bis-Tris gradient (4-12%) gel in MES buffer (with 500 μL Antioxidant in inner chamber (~200 mL)). 200 V.
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
  • Proteins were transferred using the NuPage system (transfer buufer +10% methanol), 30 V for 1.5 h
    • All of the markers was transferred to blot, to check gel was Coomassie stained after blotting
  • cAMP precipitation Ponceau S staining


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