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

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Owwnotebook icon.pngELISA, qPCR Jurkat, SDS-PAGE cAMP precipitation <html><img src="/images/9/94/Report.png" border="0" /></html> 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)
1 2 3 4 5 6
CTR A
CSE (15%) B
CSE+db-cAMP (500 µM) C
CSE+ mPKI (400 nM) D
02.100810 ' Control 8Pcpt 6Bnz Ht31 Ht31+6Bnz DMSO
1 2 3 4 5 6
CTR A
CTR B
CSE C
CSE D
  • 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)

File:100810 ponceau cAMP & lysate2s.TIF

  • Coomassie staining of the gel AFTER blot

File:SDS-page after blot 100810.tif

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

<html> <a href="http://openwetware.org/wiki/Image:10082010_qPCR_AKAP12,_AKAP9.xls">

  <img src="http://openwetware.org/images/f/f8/Owwnotebook_icon.png" alt="" width="50" height="50">

</a> </html> qPCR Jurkat expression AKAP12, AKAP9, GAPDH