User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/06/25

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Coomassie staining

  1. Run SDS-PAGE (8%)
  2. Fix in fixing solution (40% EtOH, 10% Acetic Acid) 10 min. @ RT
  3. Stain with staining solution (0.025% Coomassie (G-250), 10% acetic acid) for 30 min. @ RT (Heat in Microwave to speed up process)
  4. Destain in destaining solution (10% acetic acid) for as long a necessary
    1. Put Absorbing material on top of gel to catch excess coomassie

Gel loading hTERT D9 + HEK293 see codes 21June2010
# 1 2 3 4 5 6 7 8 9 10
cAMP prec.
Biorad Marker
(8 μL)
(20 μL)
Invitrogen Marker
(8 μL)
(20 μL)
(20 μL)
(20 μL)
(20 μL)
(20 μL)
(20 μL)


  • Sample C1 was loaded a little less then 20 μL probably with some of the beads in there because of a lack of sample volume


  • The amount of protein in the samples apears to be very low, bands around the size of the RII subunits (50 ~ 55 kDa) can be barely seen. Also between 116 - 150 kDa a faint band is seen.
  • 500 kDa band appears to reside in the stacking gel still, next time run gel further (although I usually don't make such large stacking gels)

25062010 cAMP precipitation Coomassie.png

  • For better resolution gel will be destained over the weekend


  • It was mentioned that the Perkin-Elmer device used for the Bradford method for protein concentration is somehow defect and not giving proper measurements. This might have led to the wrong protein concentrations and a too high dilution of the samples.
  • Protein concentration needs to be re-determined
  • Perhaps the bands seen ~116 kDa are Epac2, also due to the fact that they are downregulated after CSE induction


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