User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/06/29

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cAMP precipitation part II & SDS-PAGE Main project page
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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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