User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/03/23: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Co-IP Part II, hTERT Stimulation | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Co-IP Part II, hTERT Stimulation & Western blot cont'd</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> </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> </html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}} | ||
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* Stimulation | * Stimulation | ||
** ELISA | ** ELISA | ||
** VASP | ** <del>VASP</del> | ||
* Western blot, secondary antibody | * Western blot, secondary antibody | ||
==Materials & Methods== | ==Materials & Methods== | ||
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# Beads w. antibody (30 μL) & lysis buffer (200 μL) | # Beads w. antibody (30 μL) & lysis buffer (200 μL) | ||
# Beads w. antibody (30 μL) & sample (200 μL) | # Beads w. antibody (30 μL) & sample (200 μL) | ||
# Beads (30 μL) with only sample (200 μL)) | |||
* Incubate ON @ °C | * Incubate ON @ 4 °C | ||
====hTERT stimulation==== | ====hTERT stimulation==== | ||
* Remove medium form 24-wells plate with ASMC's grown ON in DMEM (S<sup>0</sup>) | * Remove medium form 24-wells plate with ASMC's grown ON in DMEM (S<sup>0</sup>) | ||
* Rinse twice with warm PBS and remove buffer | * Rinse twice with warm PBS and remove buffer | ||
* Add 200 μL DMEM S<sup>0</sup> with either HT31 or | * Add 200 μL DMEM S<sup>0</sup> with either HT31 or S<sup>0</sup> (see schedule below) | ||
** Incubate 20 min. | ** Incubate 20 min. | ||
* Add 400 μL DMEM S<sup>0</sup> to CTR (lane A) | * Add 400 μL DMEM S<sup>0</sup> to CTR (lane A) | ||
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===Western blot, secondary Antibody=== | ===Western blot, secondary Antibody=== | ||
* Wash 3x 10 min. in TBST buffer (first can be less) | * Wash 3x 10 min. in TBST buffer (first can be less) | ||
* Treat with secondary antibody (diluted in 5% TBST-milk solution) ( | * Treat with secondary antibody (anti-Mouse) (diluted in 5% TBST-milk solution) (1.5h, RT) | ||
* Wash 3x 10 min. in TBST buffer | * Wash 3x 10 min. in TBST buffer | ||
====Preparing film==== | ====Preparing film==== | ||
* 1: | * 1:1 ratio [[wikipedia:Chemiluminescence#Enhanced_chemiluminescence|ECL]] solutions (2 mL per membrane) | ||
* Incubate membrane in ECL solution 5 min, RT (both sides of membrane) | * Incubate membrane in ECL solution 5 min, RT (both sides of membrane) | ||
* Place blot in between two overhead sheets | * Place blot in between two overhead sheets | ||
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==Notes== | ==Notes== | ||
* 6-wells put to S<sup>0</sup> [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/22|22March2010]] were passed on to Anouk to continue --> VASP experiment cancelled due to time limitation | * 6-wells put to S<sup>0</sup> [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/22|22March2010]] were passed on to Anouk to continue --> VASP experiment cancelled due to time limitation | ||
* For Pierce determination new standard was made, <u>only</u> concentration 750, 500 & 250 μg/mL | |||
==Results== | ==Results== | ||
* | *Co-IP Blots of samples pulled down using PKA antibody coated beads, blots were treated with anti-AKAP antibody | ||
[[Image:230310_CoIP_AKAP_Gel_1.JPG|500px]]<br> | |||
Gel 1<br> | |||
[[Image:230310_CoIP_AKAP_Gel_2.JPG|500px]]<br> | |||
Gel 2<br> | |||
# Only beads (30 μL) | |||
# Beads w. antibody (30 μL) | |||
# Beads w. antibody (30 μL) & lysis buffer (200 μL) | |||
# Beads w. antibody (30 μL) & sample (200 μL) | |||
A. [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/23|23Feb2010]] to [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/23|25Feb2010]]<br> | |||
B. [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/03|03March2010]] to [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/05|05March2010]]<br> | |||
C. [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/15|15March2010]] to [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/17|17March2010]] | |||
==Conclusion== | ==Conclusion== | ||
* | * All Co-IP samples incubated with sample (and antibody) showed a double band it is unclear wether or not it is at the right height. The used antibody was directed against AKAP179, 86 kDa | ||
==Related== | ==Related== | ||
===Related entries=== | ===Related entries=== | ||
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===Same actions=== | ===Same actions=== | ||
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/24| Cell lysis & Co-IP Day II]] | *[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/24| Cell lysis & Co-IP Day II]] | ||
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/16| Cell | *[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/16| Cell stimulation]] | ||
Revision as of 09:08, 23 March 2010
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Summary
Materials & MethodsMaterials
MethodDetermination of protein concentration
Co-immunoprecipitation
hTERT stimulation
CTR = control, CSE = Cigarette Smoke extract, 8-pcpt = 8-pcpt-2'-o-me-camp Western blot, secondary Antibody
Preparing film
Notes
Results
A. 23Feb2010 to 25Feb2010 Conclusion
RelatedRelated entriesSame actions
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