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

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Coomassie staining
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


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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
 * 5) Put Absorbing material on top of gel to catch excess coomassie

Results

 * 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

Discussion

 * 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

Related topics

 * Silver:_Coomassie_Stain
 * Jacobs:Protocol_Protein_Gel_Staining_Using_Coomassie_Blue_Protein_Stain
 * Sosnick:Rapid_Coomassie_Staining


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