User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/05/07

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Summary

  • cAMP precipitation of D9 P28 treated with 15% CSE for RII overlay. New way of disrupting cells was used that would be more gently than the previous method.

Materials & Methods

Materials

  • Potter buffer
    • 250 mM Sucrose (8.56 g sucrose/sacharose in 100 mL)
    • 3 mM imidazole (300 μL 1 M imidazole in 100 mL)
  • Potter lysis buffer (per mL)
    • 1 mL Potter buffer
    • 8 μL protease inhibitor mix
    • 1 μL NaF
    • 1 μL NaVO3
    • 12.5 μL PMSF
  • 8-AHA-cAMP coated agarose beads
  • Cold PBS

Method

Disrupting cells

  1. Put cells on ice
  2. Remove medium
  3. Wash twice with 6 mL cold PBS
  4. Add 0.5 mL Potter lysis buffer
  5. Scrape off cells and pool in 15 mL tube
  6. Using Potter-Elvehjem Homogenizer for 10-20 strokes @ 700 RPM gently homogenize cells
  7. Leave cells on ice @ 4 °C for 15 min.
  8. Centrifuge 15 min. @ 3000 x g and collect supernatant

Incubating lysates

  1. incubate 1.5 mL of each sample with 29.3 mg cAMP for 15-30 min. while rotating @ 4 °C
  2. Collect 100 μL of these samples
  3. Put 1.5 mL of each sample on 30 uL of 8-AHA-cAMP beads and incubate 2 – 4 h while rotating @ 4 °C
Washing beads: cAMP precipitation
  • Spin beads 5 min. 2100xg @ 4 °C
  • Collect supernatant (= NAC)
  • Add 500 μL Potter buffer
  • Spin 5 min. 2100xg @ 4 °C
  • Remove supernatant with vacuum pump
  • Add 500 μL Potter buffer
  • Spin 5 min. 2100xg @ 4 °C
  • Remove supernatant with vacuum pump
  • Add 500 μL Potter buffer
  • Spin 5 min. 2100xg @ 4 °C
  • Remove supernatant with vacuum pump and syringe
  • Add 50 μL of 4x sample buffer
  • Spin 5 min. 2100xg @ 4 °C

Samples on gel

  1. 75 μL lysate was mixed with 25 μL 4x sample buffer
  2. Samples were incubated @ 95 °C for 5 min.
  3. Samples were put to 8% gel together with remaining lysates on Sunday

Notes

  • 50 μL of beads were used instead of 30 μL


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