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
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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)
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- 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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SDS-PAGE cAMP precipitation, pre-IgG antibody
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