Nativepage 5 g 250 sample additive

Purified thylakoid membranes were used for two-dimensional Phos-tag SDS-PAGE. Purified thylakoid membranes were resuspended in buffer [25 mM Bis–Tris, 20% (w/v) glycerol, pH 7.5] to a concentration of 2 mg/ml chlorophyll. To solubilize thylakoid membranes, an equal volume of n-dodecyl ß-D-maltoside was added to a final concentration of 1% (w/v). After centrifugation at 14,000 × g for 5 min, NativePAGE™ 5% G-250 Sample Additive (Thermo Fisher Scientific Inc.) was added to the supernatant according to the manufacturer’s instructions, and samples were loaded onto a NativePAGE™ 4–16% Bis–Tris Protein gel (Thermo Fisher Scientific Inc.) Electrophoresis was performed at 4°C overnight at 50 V. The gel lane was then excised from the gel and incubated in equilibration buffer [50 mM Tris–HCl, 6 M urea, 2% (w/v) SDS, 0.05% (w/v) BPB, 10 mM dithiothreitol] for 30 min at 37°C. Then proteins were separated using Phos-tag SDS-PAGE or conventional SDS-PAGE.

Recombinant proteins (5 μg) were diluted in 1× NativePAGE sample buffer (Thermo Fisher) with 0.25 μL of NativePAGE 5% G250 sample additive (Thermo Fisher) and fractionated in precast NativePAGE 4-to-16% Bis-Tris 1.0-mm mini-protein gels according to the manufacturer’s protocol (150 V for 110 min; Thermo Fisher). NativeMark unstained protein standards (ThermoFisher) were used to determine the molecular weights of the Ftl protein complexes. After electrophoresis, the gels were stained with CBB and then destained (40% methanol, vol/vol, and 10% glacial acetic acid, vol/vol) for 2 to 3 h with gentle rocking. The buffer was changed every 10 min, and the gels were imaged using a ChemiDoc imaging system (Bio-Rad).

Samples (5 μL of crude paired helical filaments (PHF) fraction or 20 μL of sucrose gradient fractions) were diluted in NativePAGE™ Sample Buffer and NativePAGE™ 5% G-250 Sample Additive loaded on 3–12% native PAGE gel (Thermo Scientific) according to the manufacturer’s instructions. After the electrophoresis, the gels were incubated 20 min in Tris/Glycine/SDS transfer buffer and blotted on a polyvinylidene difluoride (PVDF) membrane. After destaining in 100% methanol to remove excess of Coomassie dye, that was present during the run, membranes were rinsed in Tris-buffered saline-Tween (TBS-T) and blocked in TBS-T containing 5% non-fat dried milk. Blots were incubated with HRPO-labelled Tau antibody (hTau10 or AT120) for 2 h at RT or with primary antibodies followed by a secondary HRPO-labelled anti-mouse antibody. Detection was done using enhanced chemiluminescence (West Dura; Thermo Scientific) using a Amersham Imager 600 RGB (GE Healthcare). Images were captured in automated exposure modus.