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

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 & Methods

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)

Method

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

• 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)

(possible also beads (30 μL) with only sample (200 μL))

• Incubate ON @ 4 °C

Stimulation hTERT (BSc students

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

Results

Related entries

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