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

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Co-stimulation part II, (Stimulation hTERT cells v2) <html><img src="/images/9/94/Report.png" border="0" /></html> 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 & Methods

Materials

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

  • Spin down beads prepared 23Feb2010
    • Remove liquid
  • Add PBS to wash
  • Spin down and remove liquid
  • Prepare
  1. Only beads (100 μL)
  2. Beads w. antibody (100 μL)
  3. Beads w. antibody (100 μL) & lysis buffer (200 μL)
  4. Beads w. antibody (100 μL) & sample (200 μL)

(possible also beads (100 μL) with only sample (200 μL))

  • Incubate?
  • Wash?
  • Add 75 μL 4x loading buffer
  • Boil samples and put on gel

Results

  • WHAT?

Conclusion

  • DONE Ow..

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