Novex wedgewell 4 20 tris glycine gel

Western blot analysis was performed as previously described (38 (link)). Equal amounts of cells were diluted in 100 μl of Laemmli sample buffer and boiled for 10 min at 95°C in a heating-block. Thereafter, samples were separated by SDS-PAGE (Novex WedgeWell 4-20% Tris-Glycine Gels, Thermo Fisher Scientific). Gels were transferred onto Immobilon-FL PVDF membranes (EMD-Millipore), followed by blocking in Odyssey Blocking buffer (LI-COR, Lincoln, NE) mixed with an equal volume of TBS or PBS, prior to overnight-incubation at 4°C with one of the following primary antibodies (1:1000; in blocking buffer): anti-histone 2A (H2A), H2B, H4, H3K4me, H3K9me3, H3K27me3, H2B-S14p or H3-S10p antibody. Thereafter, membranes were washed (3 × 10 min) with PBS or TBS/0.1% Tween-20, followed by a final wash (10 min) with PBS or TBS and then by incubation with secondary fluorescent antibodies (1:15,000; in TBS or PBS). The secondary antibodies were from LI-COR (IRDye 800 CW Donkey Anti-Rabbit IgG, IRDye 680 CW Donkey Anti-Mouse IgG, and IRDye 800 Donkey; LI-COR). After one hour of incubation at room temperature, the membranes were washed and scanned by using the Odyssey CLx Imaging System (LI-COR). Western blots were quantified and analyzed using Image Studio Lite Software (LI-COR). The targeted proteins were normalized to the loading control (β-actin).

Total, soluble, and Sarkosyl-insoluble protein samples of mouse brains and HEK293 P301S tau-venus-seeded cell homogenates were run on a Novex WedgeWell 4–20% Tris-Glycine gels (catalog #XP04205BOX, Thermo Fisher Scientific) and transferred onto PVDF membranes (catalog #1704156, BIO-RAD). The blots were then blocked in 5% bovine serum albumin in PBS plus 0.2% Tween and incubated overnight at 4°C with primary antibodies diluted in blocking buffer, followed by either goat anti-mouse (1:5000; catalog #1706516, BIO-RAD) or goat anti-rabbit (1:4000; catalog #1706515, BIO-RAD) HRP-conjugated secondary antibodies, Dylight 680 goat-conjugated anti-mouse (1:10,000; catalog #5470, Cell Signaling Technologies), or Dylight 800 4× polyethylene glycol-conjugated goat anti-rabbit (1:10,000; catalog #5151, Cell Signaling Technologies) secondary antibodies. Signal was visualized using enhanced chemiluminescence (GE Healthcare) with x-ray film or using near-infrared fluorescence detection (ChemiDoc MP, BIO-RAD), and band intensities were quantitated using ImageJ.
For immunoblot analysis, the n values were as follows at: 3 and 12 months, n = 3 (2 M, 1 F); and 6 and 18 months, n = 3 (1 M, 2 F).