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

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.