E coli alkaline phosphatase

Bulk tRNAs were prepared as previously described using acid buffered-phenol
(phenol saturated with 50 mM sodium acetate, pH 5.8) and alcohol precipitation
20 (link). Nucleosides were prepared as
described in 61 (link) by hydrolyzing bulk tRNA
with 10 units of Nuclease P1 (Sigma) overnight at 37°C, with the addition of
0.01 units of phosphodiesterase I (Sigma) and 3 µL E. colialkaline phosphatase (Sigma). The hydrolyzed nucleosides were further purified
by filtering through a 5 kD MWCO filter (Millipore) (to remove enzymes), dried
in a CentriVap Concentrator, and suspended in 20 µL of water prior to analysis
by HPLC or LC-MS/MS.

Sarkosyl-insoluble pellets from 0.5 to 1 g of AD, PiD, CBD, PSP, CBD + PSP and MAPT cortices were suspended in 0.1 mL of 30 mM Tris (pH 7.5) containing 10 mM CaCl₂ by sonication. Trypsin was added at a final concentration of 0.5–2 mg/mL and incubated at 37 °C for 1 h. The digestion was stopped by adding 2 % SDS for SDS-PAGE, or 6 M guanidine HCl for further purification, which was performed by size-exclusion chromatography on a TSK gel SW-3000 column [2.1 × 300 mm, TOSOH] equilibrated with 6 M guanidine HCl in 10 mM phosphate (pH 6.0) and on a reverse-phase HPLC with an Aquapore RP300 column (2.1 × 100 mm). For dephosphorylation, the sarkosyl-insoluble pellets or the trypsin-resistant cores of tau filaments were suspended in 50 mM Tris–HCl containing 6 M guanidine HCl, then dialyzed overnight at 4 °C against 50 mM Tris–HCl (pH 8.8), and centrifuged at 20,400g for 10 min. The supernatants were incubated with E. coli alkaline phosphatase (10 U/mL; type III, Sigma) at 67 °C for 2 h.