User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/04/08

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cAMP precipitation for RII overlay & New cells Main project page
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Summary

  • Short summary

Materials & Methods

Materials

  • RIPA (Lysis) Buffer (per 1 mL)
    • 1.06 mg β-Glycerolphosphate
    • 1 mL RIPA buffer
    • 8 μL Protease inhibitor mix
    • 1 μL NaF (1 M)
    • 10 μL NaVO3 (10 mM)
    • 12.5 μL PMSF
  • cAMP coated agarose beads

Method

cAMP precipitation & Preparations RII overlay

  • Cells were lysed as described 24Feb2010 without determining protein concentration
  • 900 μL was put to cAMP coated agarose beads and incubated @ 4 °C ON while mixing
  • 75 μL was mixed with 25 μL 4x sample buffer and incubated @ 95 °C for 5 min.
    • Samples were put to -20 °C together with remaining lysate

Taking cells from -80 °C part II

  • D9 P21 had media removed and was washed twice with warm PBS
  • 10 mL warm DMEM S+ was added
  • D12 P26 was left to grow without disturbance due to lack of attachment

Notes

  • Stimulation was stopped after 25 hours

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