User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/02/24

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Co-immunoprecipitation part II, (Stimulation hTERT cells v2)
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


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Summary
Conditions
 * CTR
 * CSE
 * CSE + 8-pcpt
 * CSE + 6-Bzn
 * 8-pcpt
 * 6-Bnz

With a dose response range of 0 - 50 μg/mLug Ht31, instead of HT31P S0 was used.

Materials

 * RIPA buffer (per mL)
 * 1.06 mg β-glycerolphosphate
 * 1 mL RIPA buffer
 * 1 μL Apoprotein (1 mg/mL)
 * 1 μL Leupeptin (1 mg/mL)
 * 1 μL Pepstatin (A) (1 mg/mL)
 * 5 μL Na3VO4
 * 5 μL NaF (200 mM)
 * PBS
 * Coomassie (Bradford) Protein Assay Kit
 * BCA Protein Assay Reagent (bicinchoninic acid)

Determination of protein concentration

 * Use cells put on S0 23Feb2010
 * Put cells on ice
 * Remove medium
 * Wash twice with 5 mL cold PBS
 * Add 1 mL RIPA buffer
 * Scrape of cells and collect in 1.5 mL tube
 * Sonicate (4x short pulse)
 * Take 10 μL of undiluted and a 5x diluted samples for Bradford assay (Pierce determination)

Co-immunoprecipitation
(possible also beads (30 μL) with only sample (200 μL))
 * resuspend beads prepared 23Feb2010
 * Prepare for all donors (D9 & D12)
 * 1) Only beads (30 μL)
 * 2) Beads w. antibody (30 μL)
 * 3) Beads w. antibody (30 μL) & lysis buffer (200 μL)
 * 4) Beads w. antibody (30 μL) & sample (200 μL)
 * Incubate ON @ 4 °C

Stimulation hTERT (BSc students

 * HT31P dose response curve (0 - 50 μM)
 * HT31 (200 μM) /S0 Basal

Results


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

 * Counting cells 19Feb2010
 * Putting to S0 23Feb2010

Same actions

 * Stimulation hTERT cells

Related topics

 * Cell_and_tissue_lysis_hub
 * Protocol_Total_Protein_Isolation_using_RIPA_buffer.html
 * Jacobs:Protocol_Total_Protein_Isolation_using_RIPA_buffer_2
 * Jacobs:Protocol_Total_Protein_Isolation_Using_RIPA_Lysis_Buffer


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