User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/08/20

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Stripping blots, qPCR AKAP8, refresh medium new cells, 2nd AB Main project page
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Summary

  • Stripping solution
    • 50 mL dH2O
    • 5 mL Tris-Cl (1.0 M, pH 6.8) (Stacking gel buffer SDS-PAGE)
    • 1 g SDS
    • 150 mg DTT (Add later)
  1. Prewarm buffer (w/o DTT) for 30 min. to 70 °C in waterbath
  2. Add DTT just before adding membrane
  3. Completely immerse membrane
  4. Incubate 30 min @ 70 °C
  5. Wash 3x with dH2O or TBS-T
  6. Membrane can now be blocked again
  • qPCR AKAP8
    • 25 μL
  • Western blot
    • Primary antibodies were put on blot #1 made 16August2010 and used for AKAP450 and AKAP95 blot 18August2010
      • Gravin 1:5000 (Rabbit, antibody ZDE10030 animal 122 antibody (Eurogentech antibody service))
      • AKAP79 1:1000 (Rabbit, Upstate)

Results

  • qPCR AKAP8

  • Western blot AKAP95, AKAP450, RIIα

Attachment

Results qPCR AKAP8

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