User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/08/10

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ELISA, qPCR Jurkat, SDS-PAGE cAMP precipitation Main project page
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Summary

  • 4.450 mg st-Ht31 was diluted in 159 µL DMSO
  • ELISA
    • NOTE: Plate #1 well C1 received 200 µL of S0 instead of CSE, well D1 received 400 µL and also did not receive CSE. Endvolumes were still 600 µL
1.100810 ' S0+DMSO S0+DMSO S0+DMSO st-Ht31 (50 µM) st-Ht31 (50 µM) st-Ht31 (50 µM)
123456
CTRA
CSE (15%)B
CSE+db-cAMP (500 µM)C
CSE+ mPKI (400 nM)D
02.100810 ' Control 8Pcpt 6Bnz Ht31 Ht31+6Bnz DMSO
123456
CTRA
CTRB
CSEC
CSED
  • SDS-PAGE
4-12% Gradient NuPage Gel loading hTERT D9 + HEK293, HeLa -S & Jurkat see codes 21June2010
# 1 2 3 4 5 6 7 8 9 10 11 12
Gel
cAMP prec. 		Invitrogen Marker
(8 μL) 		Jurkat (LYSATE)
(20 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
  • qPCR Jurkat RNA

Results

  • Ponceau staining cAMP precipitation blot (4-12% NuPage)

  • Coomassie staining of the gel AFTER blot

Discussion

  • AKAP9 is also not amplified in the Jurkat cells, even though these SHOULD express them. Waiting for the AKAP450 immunoblot if positive, talk to Dharmacon (coming tomorrow anyway).

Attachment

qPCR Jurkat expression AKAP12, AKAP9, GAPDH


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