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

From OpenWetWare

< User:Wilfred J. Poppinga | Notebook | cAMP compartmentalization | 2010 | 08
Revision as of 14:18, 23 August 2010 by Wilfred J. Poppinga (Talk | contribs)
(diff) ←Older revision | Current revision (diff) | Newer revision→ (diff)
Jump to: navigation, search

SDS-PAGE cAMP precipitation, pre-IgG antibody Main project page
Previous entry      Next entry

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


Retrieved from "http://www.openwetware.org/wiki/User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/08/23"
Personal tools