4.4. Tau Immunoprecipitation

Cell lysates were diluted in 10 mM PBS, pH 7.4, containing 0.1% TritonX-100, to obtain a solution of 10 µg of proteins/100 µL. Samples were then incubated overnight at 4 °C under orbital shaking, with 1 µg of anti-HA tag mouse monoclonal antibody (Enzo Life Sciences Inc., Lausen, Switzerlandy) or anti-Green Fluorescent Protein (GFP) mouse monoclonal antibody (B-2, Santa Cruz Biotechnology Inc., Heidelberg Germany). At the end of incubation, samples were mixed with 20 µL of Protein A and protein G Resin beads (Genespin Srl, Milan, Italy), then incubated for 2 h at 4 °C under orbital shaking. Samples were centrifuged at 150× g for 5 min at 4 °C, the supernatant was collected and centrifuged again at 100× g for 5 min at 4 °C. The supernatants were then analyzed by SDS-PAGE and Western blot to determine the amount of tau in immunoprecipitated samples (output). Thirty µg of cell lysates were analyzed in the same experimental condition to determine the level of tau before immunoprecipitation (input). Twenty µL of input and output samples were analyzed as described before and incubated overnight with anti-human Tau rabbit polyclonal antibody (1:1000) or anti-vinculin mouse monoclonal antibody (1:5000). Peroxidase-conjugated anti-mouse and anti-rabbit IgG (1:10,000) were used as secondary antibodies. Input and output samples were also employed for C. elegans experiments.