I have troubles quantifying my tau aggregates"purified" by sequential protein extraction from HEK293 cells, especially with the sarkosyl-insoluble fraction. The pellet was resuspended in 1x PBS, and therefore should not interfere with the several BCA and TCA assays I´ve done so far. For all other fractions I can measure a decent protein concentration, but for the sarkosyl-insoluble fractions I get negative values in comparison to the blank. Interestingly, I can see a band I believe to be specific for tau on a western blot, so there seems to be some protein. Also I have confirmed the presence of aggregates by Thiazine Red staining. What am I doing wrong here?
Thank you very much for your help!
You could start from the following:
Article Quantitative Methods for the Detection of Tau Seeding Activi...
Presumably, the sarkosyl-insoluble fraction will not dissolve in PBS, and aggregates will prevent forming a homogenous protein solution. Thereby it is very likely not to see any response at protein assays. I do not have any experience with tau proteins but in the case of obtaining a clear protein solution and choosing a convenient protein assay (compatible with used solubilization reagents), you will probably solve the problem. You need to work around the different solubilization reagents such as strong chaotropes and/or different detergents. You may dissolve the aggregates with Leamnli sample buffer, 2D rehydration buffers (both without tracking dye) and perform RC-DC protein assay...
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