User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/08/23: Difference between revisions

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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;">SDS-PAGE cAMP precipitation, pre-IgG antibody</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>}}
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==Materials & Methods==
==Materials & Methods==
===Materials===
 
*
 
===Method===
===Method===
* Blocking blot of [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/08/21|21August2010]] for pre-IgG antibodies
* Blocking blot of [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/08/21|21August2010]] for pre-IgG antibodies
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** Gel was burned?? after 1h 13 min. and due to current change machine stopped running
** Gel was burned?? after 1h 13 min. and due to current change machine stopped running
** SDS-PAGE was stopped at 2 h 2 min., burning discoloring was spreading
** SDS-PAGE was stopped at 2 h 2 min., burning discoloring was spreading
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==Results==
==Results==
*{{todo| WHAT?}}
*Ponceau staining of the cAMP precipitation blot
[[Image:23082010_Ponceau_staining_cAMP_precipitation_blot_-1.tif||350px]]
[[Image:23082010_Ponceau_staining_cAMP_precipitation_blot_-2.tif||350px]]
 
==Discussion==
==Discussion==
*{{done}} Ow..
*cAMP precipitation samples are not easily seen with Ponceau S staining, we will proceed in using it
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__NOTOC__
__NOTOC__

Revision as of 11:18, 23 August 2010

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

SDS-PAGE cAMP precipitation, pre-IgG antibody <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

  • Short summary

Materials & Methods

Method

  • Blocking blot of 21August2010 for pre-IgG antibodies
    • Blocking occured ~8 hours @ RT
    • Rabbit Pre-Immune serum taken just before immunisation was used(ZDE10030 animal 122 antibody (Eurogentech antibody service)) 1:1000 and incubated ON @ 4°C
  • SDS-PAGE of cAMP precipitation samples 21June2010
    • 10 min. 70 °C → RT
    • 5 min. 95 °C (no DTT in this loading bufer) → RT
    • 1 min. 95 °C
    • 200 V
4-12% Gradient NuPage Gel loading hTERT D9 + HEK293 & HeLa -S see codes 21June2010
# 1 2 3 4 5 6 7 8 9 10
Gel
cAMP prec. 		Invitrogen Marker
(8 μL) 		HeLa -S
(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)
  • Western blot, 2h @ 30 V
  • Put HEK293 (p17) to S0
  • Weighed AKAP18δ-L314E (3.21 mg) and dissolved it (111 μL DMSO) for used in RII overlay

Notes

  • SDS-PAGE
    • Gel was burned?? after 1h 13 min. and due to current change machine stopped running
    • SDS-PAGE was stopped at 2 h 2 min., burning discoloring was spreading

Results

  • Ponceau staining of the cAMP precipitation blot

Discussion

  • cAMP precipitation samples are not easily seen with Ponceau S staining, we will proceed in using it


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