User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/08/23

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] SDS-PAGE cAMP precipitation, pre-IgG antibody
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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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Summary

 * Short summary

Method

 * Blocking blot of 21August2010 for pre-IgG antibodies
 * Blocking occured ~8 hours @ RT
 * Rabbit Pre-Immune serum taken just before immunisation was used(ZDE10030 animal 122 antibody (Eurogentech antibody service)) 1:1000 and incubated ON @ 4°C
 * SDS-PAGE of cAMP precipitation samples 21June2010
 * 10 min. 70 °C → RT
 * 5 min. 95 °C (no DTT in this loading bufer) → RT
 * 1 min. 95 °C
 * 200 V
 * Western blot, 2h @ 30 V
 * Put HEK293 (p17) to S0
 * Weighed AKAP18δ-L314E (3.21 mg) and dissolved it (111 μL DMSO) for used in RII overlay

Results

 * Ponceau staining of the cAMP precipitation blot

Discussion

 * cAMP precipitation samples are not easily seen with Ponceau S staining, we will proceed in using it


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