User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/03/22: 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;">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>&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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==Materials & Methods==
==Materials & Methods==
===Materials===
*
===Method===
===Method===
====Co-immunoprecipitation part I====
====Co-immunoprecipitation part I====
* Beads were coated with anti-PKA antibodies.
* Beads were coated with anti-PKA antibodies.
* For <b>4</b> samples
* For <b>2</b> samples (D9/D12; Only Beads, Beads with Antibody, Beads w. AB & RIPA and Bead w. AB & Sample)
** 480 μL beads
** 280 μL beads
** Spin down and remove EtOH
** Spin down and remove EtOH
** Wash with excess (400 μL) PBS
** Wash with excess (400 μL) PBS
** Spin down and remove PBS
** Spin down and remove PBS
** Add 400 μL PBS
** Add 1:1 PBS:Beads (280 μL)
** Add 1.8 μL antibody (or not for control)
*** 200 μL for Beads with Antibody, Beads w. AB & RIPA and Bead w. AB & Sample (D9/D12)
** Incubate with antibody ON @ 4 °C
*** 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====
====SDS-PAGE====
* Clean the glass plates (UP, Soap, UP, EtOH and dry)
* Clean the glass plates (UP, Soap, UP, EtOH and dry)
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* 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])
* 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)
* Prepare separating gel (pour until green beam, ~10 mL per gel)
** Add 200 μL of isobutanol OR UP until flooded  
** Add UP until flooded  
** Use [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/24|Pierce determination]] to load equally
* When polymerized (approx. 30 min) remove isolating layer (remove UP with tissue paper)
* When polymerized (approx. 30 min) remove isolating layer (wash 3x with UP when isobutanol, remove UP with tissue paper)
* Prepare [http://cshprotocols.cshlp.org/cgi/content/extract/2006/15/pdb.tab12?text_only=true 5% stacking gel]
* Prepare [http://cshprotocols.cshlp.org/cgi/content/extract/2006/15/pdb.tab12?text_only=true 5% stacking gel]
* Pour until flooding and add combs
* Pour until flooding and add combs
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* Start electrophoresis (40 min. 200 V)
* Start electrophoresis (40 min. 200 V)
** Alternatively start with 100 V until samples reach the separating gel and increase voltage
** Alternatively start with 100 V until samples reach the separating gel and increase voltage
====Western Blot====
====Western Blot====
* Moisten sponges, filter papers and membrane in transfer buffer
* Moisten sponges, filter papers and membrane in transfer buffer
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* Treat with secondary antibody (diluted in 5% TBST-milk solution) (1h, RT)
* Treat with secondary antibody (diluted in 5% TBST-milk solution) (1h, RT)
* Wash 3x 10 min. in TBST buffer  
* Wash 3x 10 min. in TBST buffer  
====Preparing film====
* 1:50 ratio [[wikipedia:Chemiluminescence#Enhanced_chemiluminescence|ECL]] solutions B:A (2 mL per membrane)
* Incubate membrane in ECL solution 5 min, RT (both sides of membrane)
* Place blot in between two overhead sheets
* Take picture (depends on what is available)
<!--{| border="1"
|+'''6-wells'''
!#
!<u>1</u>
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!<u>3</u>
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!A
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!B
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{| border="1"
{| border="1"
|+'''24-wells'''
|+'''Gel loading'''
!#
! align="center" style="background:#f0f0f0;"|#
!<u>1</u>
! align="center" style="background:#f0f0f0;"|<u>1</u>
!<u>2</u>
! align="center" style="background:#f0f0f0;"|<u>2</u>
!<u>3</u>
! align="center" style="background:#f0f0f0;"|<u>3</u>
!<u>4</u>
! align="center" style="background:#f0f0f0;"|<u>4</u>
!<u>5</u>
! align="center" style="background:#f0f0f0;"|<u>5</u>
!<u>6</u>
! align="center" style="background:#f0f0f0;"|<u>6</u>
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! align="center" style="background:#f0f0f0;"|<u>7</u>
!A
! align="center" style="background:#f0f0f0;"|<u>8</u>
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! align="center" style="background:#f0f0f0;"|<u>9</u>
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! align="center" style="background:#f0f0f0;"|<u>10</u>
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! align="center" style="background:#f0f0f0;"|<u>11</u>
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! align="center" style="background:#f0f0f0;"|<u>12</u>
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! align="center" style="background:#f0f0f0;"|<u>13</u>
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! align="center" style="background:#f0f0f0;"|<u>14</u>
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! align="center" style="background:#f0f0f0;"|<u>15</u>
!B
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{| border="1"
|+'''96-wells plate'''
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!01
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!H
!Gel 1
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|Marker<br> (15 μL)
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|D9A 1<br> (all <30 μL)
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|D9A 2<br> (all <30 μL)
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|D9A 3<br> (all <30 μL)
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|D9A 4<br> (all <30 μL)
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|D12A 1<br> (30 μL)
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|D12A 2<br> (30 μL )
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|D12A 3<br> (30 μL)
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|D12A 4<br> (30 μL)
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|D9B 1<br> (30 μL)
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|D9B 2<br> (30 μL)
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|D9B 3<br> (30 μL)
|D9B 4<br> (30 μL)||||
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!I
! align="center" style="background:#f0f0f0;"|Gel 2
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| align="center" style="background:#f0f0f0;"|Marker<br> (15 μL)
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| align="center" style="background:#f0f0f0;"|D12B 1<br> (35 μL)
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| align="center" style="background:#f0f0f0;"|D12B 2<br> (35 μL)
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| align="center" style="background:#f0f0f0;"|D12B 3<br> (35 μL)
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| align="center" style="background:#f0f0f0;"|D12B 4<br> (35 μL)
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| align="center" style="background:#f0f0f0;"|X
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| align="center" style="background:#f0f0f0;"|D9C 1<br> (35 μL)
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| align="center" style="background:#f0f0f0;"|D9C 2<br> (35 μL)
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| align="center" style="background:#f0f0f0;"|D9C 3<br> (35 μL)
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| align="center" style="background:#f0f0f0;"|D9C 4<br> (35 μL)
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| align="center" style="background:#f0f0f0;"|D12C 1<br> (35 μL)
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| 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;"|
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# 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)


==Results==
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>
*{{todo| WHAT?}}
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==
==Notes==
*{{done}} Ow..
* 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>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

Summary

  • Co-IP Day I
  • Western blot Co-IP
  • Put cells to S0

Materials & Methods

Method

Co-immunoprecipitation part I

  • Beads were coated with anti-PKA antibodies.
  • For 2 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 % (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 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
Gel loading
# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Gel 1 Marker
(15 μL) 		D9A 1
(all <30 μL) 		D9A 2
(all <30 μL) 		D9A 3
(all <30 μL) 		D9A 4
(all <30 μL) 		D12A 1
(30 μL) 		D12A 2
(30 μL ) 		D12A 3
(30 μL) 		D12A 4
(30 μL) 		D9B 1
(30 μL) 		D9B 2
(30 μL) 		D9B 3
(30 μL) 		D9B 4
(30 μL)
Gel 2 Marker
(15 μL) 		D12B 1
(35 μL) 		D12B 2
(35 μL) 		D12B 3
(35 μL) 		D12B 4
(35 μL) 		X D9C 1
(35 μL) 		D9C 2
(35 μL) 		D9C 3
(35 μL) 		D9C 4
(35 μL) 		D12C 1
(35 μL) 		D12C 2
(35 μL) 		D12C 3
(35 μL) 		D12C 4
(35 μL)
  1. Only beads (30 μL)
  2. Beads w. antibody (30 μL)
  3. Beads w. antibody (30 μL) & lysis buffer (200 μL)
  4. Beads w. antibody (30 μL) & sample (200 μL)

A. 23Feb2010 to 25Feb2010
B. 03March2010 to 05March2010
C. 15March2010 to 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. 23Feb2010 to 25Feb2010
B. 03March2010 to 05March2010
C. 15March2010 to 17March2010

Same actions


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