Phospho-Tau (Ser396) (PHF13) Mouse mAb #9632

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	-Mouse (1:4000)

Protocol tips
-Mouse (1:4000)

Publication protocol

For fly larvae, dissected body-wall muscles were homogenized in lysis buffer (50 mM Tris pH 8.0, 1% Triton X-100, 150 mM NaCl, 2 mM Na3VO4, 10 mM NaF, 60 mM β-glycerolphosphate, 10% glycerol, protease inhibitor cocktail), and then centrifuged at 13,000 g for 20 minutes at 4 °C. Supernatants were boiled in SDS sample buffer. For 293T cultures, cells were washed in ice cold 1 × PBS and lysed in CHAPS lysis buffer (CLB: 50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.3% CHAPS). After two free-thaw cycles, cell lysates were centrifuged at 13,000 rpm 4 °C for 10 min. Supernatant was used for BCA protein assay, immunoprecipitation or WB. Western blot was performed using the following primary antibodies: rabbit anti-PAR1 antibody21,39 at 1:3000; polyclonal rabbit anti-Tau (pS262) antibody (44750G, Life Technologies) at 1:2500; monoclonal mouse anti-Tau (pS396) (PHF13) (#9632, Cell Signaling) at 1:4000; monoclonal rabbit anti-Tau (pS214) (ab170892, Abcam) at 1:4000; monoclonal rabbit anti-Tau (pS202) (ab108387, Abcam) at 1:4000; polyclonal rabbit anti-human total Tau antibody (A0024, Dako) at 1:5000; monoclonal mouse anti-total GSK-3β (3D10) (#9832, Cell Signaling) at 1:2000; polyclonal rabbit anti-total CDK5 (#2506, Cell Signaling) at 1:2500; monoclonal mouse anti-HA antibody (H3663, Sigma) at 1:2500; monoclonal mouse anti-β-actin antibody (A1978, Sigma) at 1:10000; polyclonal rabbit anti-Glutathione-S-Transferase (GST) antibody (G7781, Sigma) at 1:2500; monoclonal mouse anti-ubiquitin (Ub) Antibody (P4D1) (sc-8017, Santa Cruz Biotech) at 1:500; and polyclonal rabbit anti-MARK4 antibody (bs-4659R, Bioss) at 1:3000. All primary antibodies were diluted in 5% BSA in 1 × TBST and incubated with the membrane at 4 °C overnight. Secondary antibodies goat anti-mouse IgG HRP conjugate (G21040, Life Technologies) and goat anti-rabbit IgG HRP conjugate (G21234, Life Technologies) were diluted in 1 × TBST and incubated with the membrane at room temperature for 1 h.

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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for Phospho-Tau (Ser396) (PHF13) Mouse mAb #9632 below.

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