User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/05/27

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cAMP precipitation & AKAP79 immunodetection Main project page
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Summary

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

Immunoblot

  • Wash blot incubated ON 3x 10 min. with blocking solution
  • Incubate with secondary antibody 1 h @ RT
    • anti-mouse
    • 1:10.000 dilution in blocking solution
  • Wash 3x 10 min. with blocking solution

Preparing film

  • 1:1 ratio ECL solutions (2 mL per membrane)
  • Incubate membrane in ECL solution 5 min, RT (both sides of membrane)
  • Place blot in between plastic
  • Take picture (depends on what is available)

Stripping blot

  • 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

Immunoblot

  • Block membrane in 1% BSA TBST solution for >1 h @ RT
  • Incubate with primary antibody ON @ 4 °C
    • Antibodies used on membrane
    • Rabbit monoclonal antibody - AKAP79 (Upstate)
    • 1:1000 dilution in blocking solution

Lysing HEK293 cells

  • RIPA (Lysis) Buffer (per 1 mL)
    • 1.06 mg β-Glycerolphosphate
    • 1 mL RIPA buffer
    • 8 μL Protease inhibitor mix
    • 1 μL NaF (1 M)
    • 10 μL NaVO3 (10 mM)
    • 12.5 μL PMSF
  • Cells were lysed as described 24Feb2010 without determining protein concentration

Materials & Methods

Materials

Method

Notes

  • HEK293 cells might have been a nice control to take along with the AKAP79 blot
  • Antibody check would have been nice to compare to the old antibody

Results

  • AKAP79 immunoblot

Discussion

  • DONE Ow..


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