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

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

Related entries

Run 2: Ht31 Dose/Effect Curve & S⁰ D9/D12

• Counting cells 19Feb2010

Same actions

• Putting cells to S⁰

Related topics

• Griffin:_Ultimate_Immunoprecipitation_Guide
  • Promega Guide