- Run SDS-PAGE (8%)
- Fix in fixing solution (40% EtOH, 10% Acetic Acid) 10 min. @ RT
- Stain with staining solution (0.025% Coomassie (G-250), 10% acetic acid) for 30 min. @ RT (Heat in Microwave to speed up process)
- Destain in destaining solution (10% acetic acid) for as long a necessary
- Put Absorbing material on top of gel to catch excess coomassie
Gel loading hTERT D9 + HEK293 see codes 21June2010
- 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)
- 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