User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/04/21

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cAMP precipitation, stimulation for ELISA Main project page
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Summary

  • cAMP precipitation (D9 P24)
    • including negative control, 19.5 mg of cAMP (BioLog) per mL lysate (1.8 mL was used)
      • incubation was well over 30 minutes while shaking at 4 °C
    • Samples are incubated with 8-HA-cAMP coated beads ON at 4 °C
  • 24-wells stimulation (D9 P24)
    • Not certain if concentrations are the same compared to Groningen
      • 50 mM db-cAMP was diluted, 20 μL in 10 mL H2O
      • cBIMPs, MBC & 8Pcpt all 5 mM were diluted 200 μL in 10 mL H2O
      • Ht31 10 mM was used as provided
    • All where added as described 16Feb2010 with the cBIMPs, MBC and db-cAMP stocks being prepared as was 6-Bnz. However both cBIMPS and MBC where put into one vial, 300 μL each per 2 mL medium.
      • First 20 min. incubation was extended to ~1 h due to circumstances
      • Second 25 min. incubation was also extended the same
      • Smoke was added, but the machine malfunctioned due to there not coming any bubbles in the media. Tar was smeared troughout the tube (instead of 1 location) so to create a bigger hole. Smoke did come into the falcon tube for the first cigarette but appeared not to come from the media. Because it didn't bubble. After some modifications with the medium end of the tube the device seemed to work better than ever with smoke pumping through the medium and filling the tube.
  • Splitting cells
  • Preparing new media
    • +3.5 g/L glucose, 17.5 g per 100 mL H2O filter sterilized (0.2 μM) and 10 mL is added per 500 mL medium
  • Stimulation used DMEM with 4.5 g/L


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