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 RII Overlay, Pre-IgG 2nd AB, CSE on HEK293, ASMC to S0, Split cells w. Mileh
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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
- 370 MBq/mL [γ32P]-ATP (18.5 MBq/500 µCi Hartmann Analytic)
- Blots for RII Overlay
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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RII Overlay, Pre-IgG 2nd AB, CSE on HEK293, ASMC to S0, Split cells w. Mileh
