User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/07/10: 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;">Gravin Co-IP gel</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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* Destaining gel remains that was coomassie stained after blotting the cAMP precipitation samples
* Destaining gel remains that was coomassie stained after blotting the cAMP precipitation samples
* Putting the AKAP250 immunoprecipitation samples to gel and blot them
* Putting the AKAP250 immunoprecipitation samples to gel and blot them
* Gradient (4-12%) SDS-PAGE (NuPage) for cAMP Precipitations
* Gradient (4-12%) SDS-PAGE (NuPage) for AKAP250 Co-IP Precipitations
{| border="1"
{| border="1"
|+'''Gel loading hTERT D9 + HEK293 & HeLa -S see codes [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/06/21|21June2010]]'''
|+'''Gel loading hTERT D9 + HEK293 & HeLa -S see codes [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/06/21|21June2010]]'''

Revision as of 05:37, 29 July 2010

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

Gravin Co-IP gel <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

  • Destaining gel remains that was coomassie stained after blotting the cAMP precipitation samples
  • Putting the AKAP250 immunoprecipitation samples to gel and blot them
  • Gradient (4-12%) SDS-PAGE (NuPage) for AKAP250 Co-IP Precipitations
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
  • After this gel has been blot it will be blocked and then together with the other blot treated with antiAKAP250 antibody

Notes

  • During wash steps of co-ip centrifuge steps were performed at 4400 rpm instead of 2500 rpm until the third wash step
  • Supernatant of ON incubation for co-ip was saved, "NAIP 250 9.7.10" stored @ -20 °C
  • Made new MES NuPage buffer

Results

  • Coomassie staining was put together with the ponceau S staining of yesterday
  • Ponceau staining of AKAP250 Co-IP blot

Discussion

  • Very peculiar that the Ponceau staining gives the exact same image as the cAMP precipitations.


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