User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/02/23

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Putting cells to S0, Co-immunoprecipitation Main project page
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Summary

Cells were put to S0 as was supposed to be done yesterday. Beads for Co-immunoprecipitation were prepared.

Materials & Methods

Materials

  • DMEM S0
  • DMEM S+
  • Cells prepared 19Feb2010
  • PBS
  • HT4 cells (neuroblastoma cell line)

Method

Putting cells to S0

  • Cells were put to S0 as described 15Feb2010
    • Cells were washed three times with PBS

Splitting HT4 cells

  • Split HT4 cells as described for hTERT cells
    • Cells of three flasks were resuspended in total volume of 18 mL (3 times 1 mL Trypsin + 15 mL DMEM S+)
    • 3 mL of suspension was diluted in 42 mL of DMEM S+
    • Cells were plated out 15 mL per flask

Co-immunoprecipitation part I

  • Beads were coated with anti-PKA antibodies.
  • For 4 samples
    • 480 μL beads
    • Spin down and remove EtOH
    • Wash with excess (400 μL) PBS
    • Spin down and remove PBS
    • Add 400 μL PBS
    • Add 1.8 μL antibody (or not for control)
    • Incubate with antibody ON @ 4 °C

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