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

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{|{{table}} width="800"
{|{{table}} width="800"
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">ELISA, qPCR Jurkat, SDS-PAGE cAMP precipitation</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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| align="center" style="background:#f0f0f0;"|'''8Pcpt'''
| align="center" style="background:#f0f0f0;"|'''8Pcpt'''
| align="center" style="background:#f0f0f0;"|'''6Bnz'''
| align="center" style="background:#f0f0f0;"|'''6Bnz'''
| align="center" style="background:#f0f0f0;"|'''Ht31'''
| align="center" style="background:#f0f0f0;"|'''Ht31+6Bnz'''
| align="center" style="background:#f0f0f0;"|'''Ht31+6Bnz'''
| align="center" style="background:#f0f0f0;"|'''DMSO'''
| align="center" style="background:#f0f0f0;"|'''DMSO'''
| align="center" style="background:#f0f0f0;"|'''Ht31'''
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*qPCR Jurkat RNA
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==Materials & Methods==
==Materials & Methods==
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==Discussion==
==Discussion==
*{{done}} Ow..
* 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).
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==Attachment==
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<a href="http://openwetware.org/wiki/Image:FILE.xls">
<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">
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</html> [[Media:FILE.xls| Excel TITLE]]
</html> [[Media:10082010_qPCR_AKAP12,_AKAP9.xls|qPCR Jurkat expression AKAP12, AKAP9, GAPDH]]
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__NOTOC__
__NOTOC__

Revision as of 02:10, 20 August 2010

<html><style type="text/css"> .todo { color: red } .done { color: green} </style></html>

ELISA, qPCR Jurkat, SDS-PAGE cAMP precipitation <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

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)

  • 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

<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


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