User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/05/28

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Immunoblot AKAP79 (upstate), SDS-PAGE, Prep. siRNA Main project page
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Summary

Immunoblot

  • Wash blot incubated ON 3x 10 min. with blocking solution
  • Incubate with secondary antibody 1 h @ RT
    • anti-rabbit
    • 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

SDS-PAGE

  • HEK lysate, hTERT RIPA & Potter lysate & cAMP beads/NAC
  • Put samples to 8% polyacrylamide gel
# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
SampleMarkerCSE beadsCSE NACCSE Lysate PotterCSE Lysate RIPAxHEK293 RIPA lysatemarkerS0 beadsS0 NACS0 Lysate PotterS0 Lysate RIPAxxx

siRNA prep

  • Splitting cells into 12-wells?

Split new cells

  • For weekend

Notes

  • Other people had some issues with SDS-PAGE today, so this was cancelled and will be continued next week

Results

  • AKAP79 immunoblot

Discussion

There doesn't seem to be a clear band around 79 kDa. However, the bands between the two AKAP79 antibodies seem to be similar. Perhaps there is something that is not really unspecific binding. It might also be that the stripping was not succesful and that the first antibody is still shown. These bands may however appear with each antibody and not just these. AKAP-KL antibody was looked for but not found, this might have been a nice addition, also to check for the latter theory.


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