Hi everyone,
We perform a conformational stability assay to look at conformational differences between aggregates of amyloid beta. We digest with increasing amounts of GdnHCl (up to 6 M) and then further digest with 20 ug/mL PK. I can't really examine any loading controls because 1) PK reduces the amount of proteins I can examine, and 2) most proteins will become increasingly solubilized with increasing GdnHCl. Any ideas on how I can show that I'm not just sneakily loading decreasing amounts of sample?
Thanks!
Prepare a series of amyloid beta solutions containing equal amounts of peptide and the desired amounts of GuHCl, all with the same volume (substitute water for GuHCl for the less concentrated GuHCl solutions). Then, all the solutions will have the same concentration of peptide, and a loading control will be unnecessary. This assumes you can run the gel without first precipitating the proteins. Perhaps this will not be possible if the differences in GuHCl concentration between samples mess up the gel. In that case, after the PK digestion, bring all the samples to the same GuHCl concentration and volume by adding GuHCl in the necessary amounts to the samples that started with lower GuHCl concentrations.
You can try a dot blot for overall protein of the samples prior to PK digestion. Dot blot can be stained with Ponceu C. As I understand, since you do GuHCl treatment, you do precipitate after PK treatment, since otherwise you couldnt load the material onto a gel?
Also, you might be able to use several other stability assays -
1) Different temperatures in the presence of SDS (1-2%)
2) Different concentrations of GuHCl or Urea in the presense of thioflavin, its binding will disappear once the aggregates are disassembled (ive seen this in an article, though I do have some doubts on the interaction of ThT and the amyloid being able to withstand high chaotrope concentrations)
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