User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/08/24: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
(Autocreate 2010/08/24 Entry for User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization)
 
No edit summary
(10 intermediate revisions by the same user not shown)
Line 5: Line 5:
{|{{table}} width="800"
{|{{table}} width="800"
|-
|-
|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;">RII Overlay, Pre-IgG 2<sup>nd</sup> AB, CSE on HEK293, ASMC to S<sup>0</sup>, Split cells w. Mileh</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>}}
|-
|-
Line 11: Line 11:
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
==Summary==
==Summary==
* Short summary
* To Validate all results all sorts of RII overlays were performed with either no addition, addition of AKAP18δ-PP (10 μM) or addition of AKAP18δ-L314E (10 μM).
<!--
* Also Pre-immune test were done to test specificity of antibodies
* As a control to see whether or not effect seen is general in multiple systems HEK293 cells were stimulated for protein analyses
 
==Materials & Methods==
==Materials & Methods==
===Materials===
===Materials===
*  
* 370 MBq/mL [γ<sup>32</sup>P]-ATP (18.5 MBq/500 µCi Hartmann Analytic)
* Blots for RII Overlay
** PKA Co-IP blot [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/07/29|29July2010]] used for RII overlay [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/08/03|03August2010]] (#8)
** Lysate blot #2 [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/08/16|16August2010]] (for AKAP18δ-PP)(#1)
** Lysate blot [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/08/13|13August2010]] (for AKAP18δ-L314E)(#2)
** cAMP precipitation blot (2x) [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/08/23|23August2010]] (for AKAP18δ-L314E & AKAP18δ-PP)(#11(blot1)/#13(blot2))
** Lysate blot (3 fragments) [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/08/18|18August2010]] used for immunoblot [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/08/19|19August2010]] and [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/08/21|21August2010]](#18)
** cAMP precipitation blot (3 fragments) [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/08/10|10August2010]] used for blot [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/08/12|12August2010]] & [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/08/14|14August2010]](#15)


===Method===
===Method===
*
====Western blot Rabbit pre-immune IgG====
* Secondary AB α-Rabbit (1:10.000), 1h @ RT
====RII Overlay====
* As described [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/04/01|01April2010]]
====CSE stimulation HEK293====
* HEK293 put to S<sup>0</sup> yesterday were stimulated with 15% CSE or only S<sup>0</sup> (17.30)
====Put ASMC to S<sup>0</sup>====
* Airway smooth muscle cells D9 P24 were put to S<sup>0</sup> for Carolines experiment
 
==Notes==
==Notes==
*
====RII overlay====
* PKA Co-IP blot was blocked in the isotope lab
* Not all membranes were activated again, while they were maintained under moist conditions
* Column had fallen dry and cracked, this was not noticed until after addition of sample. Separation appeared still succesful
==Results==
==Results==
*{{todo| WHAT?}}
*Rabbit Pre-immune Western blot
[[Image:24082010_WB_Rabbit_Pre-immune_SERUM.tif|500px]]
==Discussion==
==Discussion==
*{{done}} Ow..
* Pre-immune was not only IgG but whole serum this should explain the high amount of signals seen. Dörte is indisposed at the moment so I am unable to ask here where pre-immune IgG is to be found.
<!--
==Related==
==Related==
===Related entries===
===Related entries===

Revision as of 08:49, 24 August 2010

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

RII Overlay, Pre-IgG 2nd AB, CSE on HEK293, ASMC to S0, Split cells w. Mileh <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

  • To Validate all results all sorts of RII overlays were performed with either no addition, addition of AKAP18δ-PP (10 μM) or addition of AKAP18δ-L314E (10 μM).
  • Also Pre-immune test were done to test specificity of antibodies
  • As a control to see whether or not effect seen is general in multiple systems HEK293 cells were stimulated for protein analyses

Materials & Methods

Materials

Method

Western blot Rabbit pre-immune IgG

  • Secondary AB α-Rabbit (1:10.000), 1h @ RT

RII Overlay

CSE stimulation HEK293

  • HEK293 put to S0 yesterday were stimulated with 15% CSE or only S0 (17.30)

Put ASMC to S0

  • Airway smooth muscle cells D9 P24 were put to S0 for Carolines experiment

Notes

RII overlay

  • PKA Co-IP blot was blocked in the isotope lab
  • Not all membranes were activated again, while they were maintained under moist conditions
  • Column had fallen dry and cracked, this was not noticed until after addition of sample. Separation appeared still succesful

Results

  • Rabbit Pre-immune Western blot

Discussion

  • Pre-immune was not only IgG but whole serum this should explain the high amount of signals seen. Dörte is indisposed at the moment so I am unable to ask here where pre-immune IgG is to be found.


Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/08/24&oldid=447792"