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

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] RII Overlay, Pre-IgG 2nd AB, CSE on HEK293, ASMC to S0, Split cells w. Mileh
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |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

 * 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)

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

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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