User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/06/22: 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;">cAMP precipitation</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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==Summary==
==Summary==
===SDS-PAGE===
*Prepare 6x 8% gels
===Washing beads: cAMP precipitation===
===Washing beads: cAMP precipitation===
*Spin beads 5 min. 2100xg @ 4 °C
*Spin beads 5 min. 2100xg @ 4 °C
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*1% Sodium deoxycholate (1,03 g (97%))
*1% Sodium deoxycholate (1,03 g (97%))
*0.1% SDS (1 g SDS)
*0.1% SDS (1 g SDS)
 
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==Notes==
==Notes==
*One gel was too small, probably added water to fast (to remove airbubbles from top). I only needed 5 so it's okay.
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__NOTOC__
__NOTOC__

Revision as of 02:05, 22 June 2010

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

cAMP precipitation <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

SDS-PAGE

  • Prepare 6x 8% gels

Washing beads: cAMP precipitation

  • Spin beads 5 min. 2100xg @ 4 °C
  • Collect supernatant (= NAC)
  • Add 500 μL Potter buffer
  • Spin 5 min. 2100xg @ 4 °C
  • Remove supernatant with vacuum pump
  • Add 500 μL Potter buffer
  • Spin 5 min. 2100xg @ 4 °C
  • Remove supernatant with vacuum pump
  • Add 500 μL Potter buffer
  • Spin 5 min. 2100xg @ 4 °C
  • Remove supernatant with vacuum pump and syringe
  • Add 50 μL of 4x sample buffer
  • Spin 5 min. 2100xg @ 4 °C

RIPA BUFFER (100 mL)

  • 50 mM Tris-HCl (5 mL 1 M Tris pH 7.4)
  • Adjust to pH 7.4
  • 150 mM NaCl (0.87 g NaCl)
  • 1 mM EDTA (37.22 mg EDTA)
  • 1% Triton x-100 (1 mL Tritox x-100)
  • 1% Sodium deoxycholate (1,03 g (97%))
  • 0.1% SDS (1 g SDS)

Notes

  • One gel was too small, probably added water to fast (to remove airbubbles from top). I only needed 5 so it's okay.


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