User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/08/21: 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;">Lysate SDS-PAGE, qPCR AKAP5, Western blot AKAP79 and Gravin</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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==Summary==
==Summary==
* SDS-PAGE Lysate samples
* SDS-PAGE Lysate samples
** Forgot antioxidant, added after 1h 11 min.; power failure @ 2h 40 min. → continued to blot (2h, 30V)
** Ponceau staining does not look very nice compared to others
** Gel is coomassie stained
{| border="1"
{| border="1"
|+'''4-12% Gradient NuPage Gel loading LYSATES hTERT D9 + HEK293, HeLa -S & Jurkat see codes [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/06/21|21June2010]]'''
|+'''4-12% Gradient NuPage Gel loading LYSATES hTERT D9 + HEK293, HeLa -S & Jurkat see codes [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/06/21|21June2010]]'''
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! align="center" style="background:#f0f0f0;"|<u>10</u>
! align="center" style="background:#f0f0f0;"|<u>10</u>
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!Gel<br>cAMP prec.
!Gel<br>Lysates
| align="center"|[[Media:HiMark_protein_marker_-_Invitrogen.jpg|Invitrogen Marker]]<br> (8 μL)
| align="center"|[[Media:HiMark_protein_marker_-_Invitrogen.jpg|Invitrogen Marker]]<br> (8 μL)
| align="center"|Jurkat <br>(20 μL)
| align="center"|Jurkat <br>(20 μL)
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** Forgot antioxidant, added after 1h 11 min.; power failure @ 2h 40 min. → continued to blot (2h, 30V)
 
* Prepared cDNA  
* Prepared cDNA  
* qPCR AKAP5 and GAPDH
* qPCR AKAP5 and GAPDH
** Not enough master mix for HEK293 and Jurkat controls
[[Image:AKAP79_expression.jpg|500px]]
[[Image:AKAP79_expression.jpg|500px]]
** Not enough master mix for HEK293 and Jurkat controls
* Western blot 2<sup>nd</sup> Antibody (α-Rabbit) 1:10.000
* Western blot 2<sup>nd</sup> Antibody (α-Rabbit) 1:10.000
** Blot #1 [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/08/16|16August2010]], Loading control was already performed [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/08/19|19August2010]]
[[Image:21082010_Lysates_Immunoblot_AKAP250_and_79.tif|500px]]
<!--
<!--
==Materials & Methods==
==Materials & Methods==

Revision as of 06:49, 23 August 2010

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

Lysate SDS-PAGE, qPCR AKAP5, Western blot AKAP79 and Gravin <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

  • SDS-PAGE Lysate samples
    • Forgot antioxidant, added after 1h 11 min.; power failure @ 2h 40 min. → continued to blot (2h, 30V)
    • Ponceau staining does not look very nice compared to others
    • Gel is coomassie stained
4-12% Gradient NuPage Gel loading LYSATES hTERT D9 + HEK293, HeLa -S & Jurkat see codes 21June2010
# 1 2 3 4 5 6 7 8 9 10
Gel
Lysates 		Invitrogen Marker
(8 μL) 		Jurkat
(20 μL) 		HEK293
(10 μL) 		Biorad Marker
(8 μL) 		C1
(20 μL) 		C2
(20 μL) 		C3
(20 μL) 		S1
(20 μL) 		S2
(20 μL) 		S3
(20 μL)
  • Prepared cDNA
  • qPCR AKAP5 and GAPDH
    • Not enough master mix for HEK293 and Jurkat controls


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