Ab80579

Tau was immunoprecipitated from whole lysates of cortex and hippocampus or from PSD preparations with tau5 antibody free of bovine serum albumin (BSA) and azide (ab80579, Abcam), and a cross-link immunoprecipitation kit (Thermo-Pierce) following the manufacturer's instructions. Elution buffer (30 μl) was used for the second elution step of the cross-link immunoprecipitation protocol. Columns were used up to twice without noticeable loss of immunoprecipitation efficiency. For experiments using in-gel trypsin digestion of proteins before mass spectrometry, the immunoprecipitated proteins were run on a 4–12% Bis-Tris gel and stained with SafeStain (Life Technologies). A broad band around 50 kDa was then cut out for mass spectrometry analysis. For experiments using in-solution trypsin digestion before mass spectrometry, immunoprecipitated samples were neutralized with 1 M Tris, pH 9.6, containing 25 μM Thiamet G and phosphatase inhibitors. For quantitative experiments, 3 μM trichostatin A and 10 mM niacinamide were added to the neutralization solution. For the initial assignment of tau modifications, we used 1 mg of whole lysate of cortex and hippocampus and 30 μg of tau5. For quantitative immunoprecipitation experiments, we used 215 μg of whole lysate of cortex and hippocampus or the entire PSD fraction from one mouse (approximately 215 μg), and 10–20 μg of tau5 antibody.