User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/07/03

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Silver staining attempt #3 Main project page
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Summary

  • Performed silver staining for third time, suspected that the dH2O washing steps were too prolonged and therefore had too much opportunity to wash away the reagents. Washing steps were now just rinsing steps, including the 10% Ethanol/5% Acetic acid. The fixing step in 40% Ethanol/10% acetic acid was 1 step for 45 min.

Notes

  • The biorad picture machine had some kind of bubbles in the way of the lens, photography was performed manually

Results

  • Silver staining

Discussion

  • Staining was successful, for next week Farmer's reducer should be used to clear background staining
  • HeLa cells did not show any bands, probably the concentration again?
  • HEK293 shows expected bands, although not clear RII, AKAP79 (79 kDa), AKAP149 (149 kDa) and Gravin (250 kDa)
  • hTERT ASMC appears to show the same bands as seen in HEK293 although 250 and higher is just to much background to be sure
  • RII subunits are more yellow then brown
  • HiMark is not a nice marker, 240 is even higher than the 250 band of Precission plus and 160 kDA band is always a smear. Probably not made for Bis-acrylamide gels? Example marker is on NuPage
  • Gradient gel might be a nice idea for higher bands?


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