User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/06/29: 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 part II & SDS-PAGE</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>}}
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==Summary==
==Summary==
===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
===Protein work===
===Protein work===
* SDS-PAGE
* SDS-PAGE
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* Preparing cDNA
* Preparing cDNA
* Real time qPCR
* Real time qPCR
 
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==Notes==
==Notes==
*cAMP precipitations had 50 μL of 4x loading buffer put on them, they should have been boiled for 5 - 10 min. but they were boiled approx. 2 hours
** Hardly any blue is still seen in soluble fraction, probably samples are ruined
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__NOTOC__
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Revision as of 07:21, 29 June 2010

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

cAMP precipitation part II & SDS-PAGE <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

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

Protein work

  • SDS-PAGE
    • RII Overlay
    • Silver / Coomassie staining
    • Immunoblot
      • AKAP250
      • AKAP95
      • AKAP450

RNA work

  • Preparing cDNA
  • Real time qPCR

Notes

  • cAMP precipitations had 50 μL of 4x loading buffer put on them, they should have been boiled for 5 - 10 min. but they were boiled approx. 2 hours
    • Hardly any blue is still seen in soluble fraction, probably samples are ruined


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