User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/03/22: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Co-IP & Western blot</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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==Materials & Methods== | ==Materials & Methods== | ||
=== | ===Method=== | ||
* | ====Co-immunoprecipitation part I==== | ||
* Beads were coated with anti-PKA antibodies. | |||
* For <b>2</b> samples (D9/D12; Only Beads, Beads with Antibody, Beads w. AB & RIPA and Bead w. AB & Sample) | |||
** 280 μL beads | |||
** Spin down and remove EtOH | |||
** Wash with excess (400 μL) PBS | |||
** Spin down and remove PBS | |||
** Add 1:1 PBS:Beads (280 μL) | |||
*** 200 μL for Beads with Antibody, Beads w. AB & RIPA and Bead w. AB & Sample (D9/D12) | |||
*** 80 μL for Only Beads (D9/D12) | |||
*** (30 μL for each condition) | |||
** Add 1:100 (PKA RIIβ) antibody (or not for control), 2 μL | |||
** Incubate ON @ 4 °C | |||
=== | ====SDS-PAGE==== | ||
* Clean the glass plates (UP, Soap, UP, EtOH and dry) | |||
* Prepare elaboration, look out for leakage! | |||
! | * Choose % of gel, big proteins require higher % ([http://cshprotocols.cshlp.org/cgi/content/extract/2006/15/pdb.tab11?text_only=true here 10% SDS gel]) | ||
* Prepare separating gel (pour until green beam, ~10 mL per gel) | |||
** Add UP until flooded | |||
* When polymerized (approx. 30 min) remove isolating layer (remove UP with tissue paper) | |||
* Prepare [http://cshprotocols.cshlp.org/cgi/content/extract/2006/15/pdb.tab12?text_only=true 5% stacking gel] | |||
* Pour until flooding and add combs | |||
** Dilute samples with the buffer which they are stored in until an equal amount is reached (20 ng in 30 μL) | |||
** Add 10 μL 4x loading buffer | |||
** Boil samples for 5 min. and cool samples on ice | |||
* Bring gels into the electrophoresis elaboration and pour ELFO buffer in the inner space to check for leakage | |||
* Remove combs and rinse slots with buffer | |||
* Fill slots with sample (40 μL) and marker (15 μL) | |||
* Start electrophoresis (40 min. 200 V) | |||
** Alternatively start with 100 V until samples reach the separating gel and increase voltage | |||
====Western Blot==== | |||
* Moisten sponges, filter papers and membrane in transfer buffer | |||
* Prepare sandwich | |||
** Black clamp | |||
** Sponge | |||
** Whatman paper | |||
** Gel | |||
** Membrane | |||
** Whatman paper | |||
** Sponge | |||
** White clamp | |||
* Put sandwhich in the transferring system (black to black) and fill it with transferbuffer | |||
* Transfer proteins to membrane for 1 hour @ 100 V on ice while stirring | |||
* After transfer check if marker is visible on membrane (successful transfer) | |||
** Alternatively Ponceau S staining is possible (not used @ this lab) | |||
** Remove LEFT UPPER corner to mark what is what | |||
* Block membrane with 5% milk solution in TBST buffer (ON, 4°C) | |||
** Alternatively 2 h @ RT | |||
* Treat with primary antibody (diluted in 5% TBST-milk solution) (ON, 4°C) | |||
** GAPDH 1:2000 | |||
** VASP 1:1000 | |||
** AKAP 1:1000 | |||
* Wash 3x 10 min. in TBST buffer (first can be less) | |||
* Treat with secondary antibody (diluted in 5% TBST-milk solution) (1h, RT) | |||
* Wash 3x 10 min. in TBST buffer | |||
{| border="1" | {| border="1" | ||
|+''' | |+'''Gel loading''' | ||
|# | ! align="center" style="background:#f0f0f0;"|# | ||
! | ! align="center" style="background:#f0f0f0;"|<u>1</u> | ||
! | ! align="center" style="background:#f0f0f0;"|<u>2</u> | ||
! | ! align="center" style="background:#f0f0f0;"|<u>3</u> | ||
! | ! align="center" style="background:#f0f0f0;"|<u>4</u> | ||
! | ! align="center" style="background:#f0f0f0;"|<u>5</u> | ||
! | ! align="center" style="background:#f0f0f0;"|<u>6</u> | ||
! | ! align="center" style="background:#f0f0f0;"|<u>7</u> | ||
! | ! align="center" style="background:#f0f0f0;"|<u>8</u> | ||
! align="center" style="background:#f0f0f0;"|<u>9</u> | |||
! align="center" style="background:#f0f0f0;"|<u>10</u> | |||
! align="center" style="background:#f0f0f0;"|<u>11</u> | |||
! align="center" style="background:#f0f0f0;"|<u>12</u> | |||
| | ! align="center" style="background:#f0f0f0;"|<u>13</u> | ||
! | ! align="center" style="background:#f0f0f0;"|<u>14</u> | ||
! align="center" style="background:#f0f0f0;"|<u>15</u> | |||
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! | |||
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! | |||
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! | |||
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! | |||
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! | |||
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! | |||
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|- | |- | ||
! | !Gel 1 | ||
| | |Marker<br> (15 μL) | ||
| | |D9A 1<br> (all <30 μL) | ||
| | |D9A 2<br> (all <30 μL) | ||
| | |D9A 3<br> (all <30 μL) | ||
| | |D9A 4<br> (all <30 μL) | ||
| | |D12A 1<br> (30 μL) | ||
| | |D12A 2<br> (30 μL ) | ||
| | |D12A 3<br> (30 μL) | ||
| | |D12A 4<br> (30 μL) | ||
| | |D9B 1<br> (30 μL) | ||
| | |D9B 2<br> (30 μL) | ||
| | |D9B 3<br> (30 μL) | ||
|D9B 4<br> (30 μL)|||| | |||
|- | |- | ||
! | ! align="center" style="background:#f0f0f0;"|Gel 2 | ||
| | | align="center" style="background:#f0f0f0;"|Marker<br> (15 μL) | ||
| | | align="center" style="background:#f0f0f0;"|D12B 1<br> (35 μL) | ||
| | | align="center" style="background:#f0f0f0;"|D12B 2<br> (35 μL) | ||
| | | align="center" style="background:#f0f0f0;"|D12B 3<br> (35 μL) | ||
| | | align="center" style="background:#f0f0f0;"|D12B 4<br> (35 μL) | ||
| | | align="center" style="background:#f0f0f0;"|X | ||
| | | align="center" style="background:#f0f0f0;"|D9C 1<br> (35 μL) | ||
| | | align="center" style="background:#f0f0f0;"|D9C 2<br> (35 μL) | ||
| | | align="center" style="background:#f0f0f0;"|D9C 3<br> (35 μL) | ||
| | | align="center" style="background:#f0f0f0;"|D9C 4<br> (35 μL) | ||
| | | align="center" style="background:#f0f0f0;"|D12C 1<br> (35 μL) | ||
| | | align="center" style="background:#f0f0f0;"|D12C 2<br> (35 μL) | ||
| align="center" style="background:#f0f0f0;"|D12C 3<br> (35 μL) | |||
| align="center" style="background:#f0f0f0;"|D12C 4<br> (35 μL) | |||
| align="center" style="background:#f0f0f0;"| | |||
|- | |- | ||
|} | |} | ||
# 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]] | |||
== | ==Notes== | ||
* | * No ice block was put in transfer system for the first 24 min. (of 60 min. in total) | ||
* Membrane was blocked for 1.5 h @ RT (14.22 - 15.52) | |||
==Related== | |||
===Related entries=== | |||
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]] | |||
===Same actions=== | |||
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/01|Western blot]] | |||
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/23|Co-IP day I]] | |||
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> |
Revision as of 01:07, 23 March 2010
<html><style type="text/css"> .todo { color: red } .done { color: green} </style></html>
Co-IP & Western blot | <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> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||||||||||||||||||||||||||||||||
Summary
Materials & MethodsMethodCo-immunoprecipitation part I
SDS-PAGE
Western Blot
A. 23Feb2010 to 25Feb2010 Notes
RelatedRelated entriesA. 23Feb2010 to 25Feb2010 Same actions |