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

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RII Overlay, Pre-IgG 2^nd 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
- PKA Co-IP blot 29July2010 used for RII overlay 03August2010 (#8)
- Lysate blot #2 16August2010 (for AKAP18δ-PP)(#1)
- Lysate blot 13August2010 (for AKAP18δ-L314E)(#2)
- cAMP precipitation blot (2x) 23August2010 (for AKAP18δ-L314E & AKAP18δ-PP)(#11(blot1)/#13(blot2))
- Lysate blot (3 fragments) 18August2010 used for immunoblot 19August2010 and 21August2010(#18)
- cAMP precipitation blot (3 fragments) 10August2010 used for blot 12August2010 & 14August2010(#15)

Method

Western blot Rabbit pre-immune IgG

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

RII Overlay

As described 01April2010

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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