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

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SDS-PAGE cAMP precipitation, pre-IgG antibody Main project page
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Summary

  • Short summary

Materials & Methods

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
4-12% Gradient NuPage Gel loading hTERT D9 + HEK293 & HeLa -S see codes 21June2010
# 1 2 3 4 5 6 7 8 9 10
Gel
cAMP prec.
Invitrogen Marker
(8 μL)
HeLa -S
(20 μL)
HEK293
(10 μL)
Biorad Marker
(8 μL)
C1
(20 μL)
C2
(20 μL)
C3
(20 μL)
S1
(<20 μL)
S2
(<20 μL)
S3
(<20 μL)
  • 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

Notes

  • SDS-PAGE
    • Gel was burned?? after 1h 13 min. and due to current change machine stopped running
    • SDS-PAGE was stopped at 2 h 2 min., burning discoloring was spreading

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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