User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/08/24

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RII Overlay, Pre-IgG 2nd AB, CSE on HEK293, ASMC to S0, Split cells w. Mileh Main project page
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Summary

  • To Validate all results all sorts of RII overlays were performed with either no addition, addition of AKAP18δ-PP (10 μM) or addition of AKAP18δ-L314E (10 μM).
  • Also Pre-immune test were done to test specificity of antibodies
  • As a control to see whether or not effect seen is general in multiple systems HEK293 cells were stimulated for protein analyses

Materials & Methods

Materials

Method

Western blot Rabbit pre-immune IgG

  • Secondary AB α-Rabbit (1:10.000), 1h @ RT

RII Overlay

CSE stimulation HEK293

  • HEK293 put to S0 yesterday were stimulated with 15% CSE or only S0 (17.30)

Put ASMC to S0

  • Airway smooth muscle cells D9 P24 were put to S0 for Carolines experiment

Notes

RII overlay

  • PKA Co-IP blot was blocked in the isotope lab
  • Not all membranes were activated again, while they were maintained under moist conditions
  • Column had fallen dry and cracked, this was not noticed until after addition of sample. Separation appeared still succesful

Results

  • Rabbit Pre-immune Western blot

Discussion

  • Pre-immune was not only IgG but whole serum this should explain the high amount of signals seen. Dörte is indisposed at the moment so I am unable to ask here where pre-immune IgG is to be found.


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