Hi all! I've been trying to prepare sarkosyl insoluble tau from both human confirmed AD cases and triple transgenic mouse samples. I'm having problems seeing anything in the insoluble fraction, and therefore must be doing something basic wrong. I've been using the Forest and Davies protocol:
http://www.ncbi.nlm.nih.gov/pubmed/22976073
I think there are three possible places I might be going wrong.
1) Initial homogenisation of tissue - I've tried both sonication and teflon rotor homogenisation. The pellet after a low speed spin (6000xg) is always huge.
2) Choice of antibodies - I've tried the Dako total tau, tau46, and a couple of phospho site antibodies
3) Solubilisation of the final pellet before running western blot - I've been boiling in Laemlli buffer and have also tried 8M urea.
There is protein in my sark insoluble fraction, a good sized pellet and enough protein to see by Ponceau on a gel. But nothing in those lanes labels with any of the tau antibodies. In the sark soluble fraction I get an enormous signal with the Dako antibody, but the majority of the signal seems to be around 50 kDa.
Any advice about this prep would be much appreciated, I can't help but think that I'm doing something very fundamental wrong. Happy to answer more questions about what I'm doing too!
Thank you!
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