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

From OpenWetWare
Jump to navigationJump to search

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

cAMP precipitation part II & SDS-PAGE Main project page
Previous entry      Next entry

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


Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/06/29&oldid=1004987"