User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/03/01: 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;">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===
===Materials===
*  
* Transfer buffer (1 L)
** 700 mL H<sub>2</sub>O (Cold)
** 100 mL 10x Transfer buffer (Cold)
*** 0.25 M TRIS (30.3 g/L)
*** 1.92 M Glycin (144.1 g/L)
*** 1.0% SDS (10 g/L)
**200 mL Methanol (Cold)
** MIX and Store @ 4 °C
* TBST buffer (4 L)
** 3.6 L UltraPure H<sub>2</sub>O
** 400 mL 10x TBS
*** for 1 L
*** 60.5 g/L TRIS
*** 87.6 g/L NaCl
*** pH 7.6
** 4 mL Tween20
** Mix
* 1x ELFO buffer
* 10x ELFO buffer
** 30.26 g/L TRIS
** 187.60 g/L Glycin
** 10 g/L SDS


===Method===
===Method===
{| border="1"
====SDS-PAGE====
|+'''6-wells'''
* Clean the glass plates (UP, Soap, UP, EtOH and dry)
!#
* Prepare elaboration, look out for leakage!
!<u>1</u>
* 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])
!<u>2</u>
* Prepare separating gel (pour until green beam, ~10 mL per gel)
!<u>3</u>
** Add 200 μL of isobutanol OR UP until flooded
|-
** Use [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/24|Pierce determination]] to load equally
!A
* 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]
|
* 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
!B
** 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 [http://www.cellsignal.com/products/7720.html marker] (15 μL)
|-
* Start electrophoresis (40 min. 200 V)
|}
** Alternatively start with 100 V until samples reach the separating gel and increase voltage
{| border="1"
 
|+'''24-wells'''
====Western Blot====
!#
* Moisten sponges, filter papers and membrane in transfer buffer
!<u>1</u>
* Prepare sandwich
!<u>2</u>
** Black clamp
!<u>3</u>
** Sponge
!<u>4</u>
** Whatman paper
!<u>5</u>
** Gel
!<u>6</u>
** Membrane
|-
** Whatman paper
!A
** Sponge
|
** White clamp
|
* Put sandwhich in the transferring system (black to black) and fill it with transferbuffer
|
** DON'T FORGET ICE BLOCK!
|
* 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
!B
* 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)
!C
* Wash 3x 10 min. in TBST buffer
|
 
|
====Preparing film====
|
* 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)
|
* Place blot in between two overhead sheets
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* Take picture (depends on what is available)
|-
!D
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|+'''96-wells plate'''
|#
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==Results==
==Results==
*{{todo| WHAT?}}
*{{todo| WHAT?}}
--> PICTURES ANOUK


==Conclusion==
==Conclusion==
*{{done}} Ow..
*{{done}} Ow..
 
==Related==
 
===Related topics===
[[Western blot]]
<!-- ##### 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. ##### -->
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|}


__NOTOC__
__NOTOC__

Revision as of 05:16, 25 March 2010

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

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

  • Short summary

Materials & Methods

Materials

  • Transfer buffer (1 L)
    • 700 mL H2O (Cold)
    • 100 mL 10x Transfer buffer (Cold)
      • 0.25 M TRIS (30.3 g/L)
      • 1.92 M Glycin (144.1 g/L)
      • 1.0% SDS (10 g/L)
    • 200 mL Methanol (Cold)
    • MIX and Store @ 4 °C
  • TBST buffer (4 L)
    • 3.6 L UltraPure H2O
    • 400 mL 10x TBS
      • for 1 L
      • 60.5 g/L TRIS
      • 87.6 g/L NaCl
      • pH 7.6
    • 4 mL Tween20
    • Mix
  • 1x ELFO buffer
  • 10x ELFO buffer
    • 30.26 g/L TRIS
    • 187.60 g/L Glycin
    • 10 g/L SDS

Method

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)
  • When polymerized (approx. 30 min) remove isolating layer (wash 3x with UP when isobutanol, 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
    • DON'T FORGET ICE BLOCK!
  • 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

Preparing film

  • 1:1 ratio ECL solutions (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)

Results

  • WHAT?

--> PICTURES ANOUK

Conclusion

  • DONE Ow..

Related

Related topics

Western blot


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