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

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Putting cells to S0, Co-immunoprecipitation
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 * style="background-color: #F2F2F2" align="center"|  |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

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

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

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


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