Nativepage novex 3 12 bistris gels

Soluble fractions were thawed on ice and mixed with 4× NativePAGE sample buffer comprising 50 mM Bis-Tris, 50 mM NaCl, 10% (w/v) glycerol, and 0.001% Ponceau S, pH 7.2. Ten-microgram samples per well were loaded onto NativePAGE Novex 3–12% BisTris gels (ThermoFisher). The proteins were fractionated for 90–115 min at 150 V using an XCell SureLock Mini-Cell system (ThermoFisher, Carlsbad, CA). The fractionated proteins then were transferred to PVDF membranes for 1 h at 25 V using the XCell II™ Blot Module (ThermoFisher). After the transfer, the membranes were incubated in 20 mL of 8% acetic acid for 15 min to fix the proteins. The membranes were probed with anti-human tau antibody HT7 (ThermoFisher) or anti-phospho-tau antibody AT8 (ThermoFisher). The intensity of the protein bands was quantified densitometrically using Image J [31 ].

For BN PAGE (Schägger and von Jagow, 1991 (link)), mitochondrial membranes were solubilized using 1% dodecyl-β-D-maltoside (DDM) (Glycon, Germany) for 30 min, centrifuged to remove insoluble materials (32,000 × g, 30 min), and run on Native PAGE™ Novex 3–12% Bis-Tris gels (ThermoFisher Scientific) according to the manufacturer’s instructions, except that the gel was run for 30 min. at 100 V, then the inner buffer was exchanged for one containing 1/10th of the standard cathode buffer and run for a further 1.5 h at 180 V. Proteins were visualized using colloidal Coomassie R250, or the gels de-stained with MilliQ water for in-gel complex I activity assays using 100 μM NADH and 1 mg mL⁻¹ nitroblue tetrazolium (NBT) (Wittig et al, 2007 (link)).

NativePAGE™ Novex® 3–12% Bis-Tris Gels were purchased from Thermo-Fisher Scientific. We first prepared 10 tubes with different concentrations of AMP-PNP ranging from 0 to 0.5 mM in buffer containing 20 mM Tris, pH 8.0, 200 mM NaCl, 10 mM MgCl₂, 10% glycerol, 0.05% DDM. Then, 6 µg of Bcs1 was added to each tube followed by 30 min incubation at 4 °C. All samples were loaded into native page gel wells and run at 150 V for 60 minutes (at 4 °C), and then run at 250 V for another 60 minutes (at 4 °C). The gel was then fixed using the following steps: (i) Place the gel in fixing solution (40% methanol, 10% acetic acid) and microwave (950–1100 watts) for 45 seconds, (ii) decant the fix solution after shaking for 15 minutes at room temperature, (iii) add 100 mL 8% acetic acid solution and microwave (950–1100 watts) for 45 seconds, (iv) shake the gel at room temperature until the desired background is obtained. The gel was imaged using a gel imaging systems (Bio-Rad). The images were analyzed in ImageJ. The data was fitted with the following equation^{36 (link),57 (link)}, $y=\frac{y_{\max }{C}^{n_H}}{{{EC}_{50}}^{n_H}+C^{n_H}}$ where y_max is the maximum value of AMP-PNP bound conformational state at saturating ligand concentration, C is the concentration of AMP-PNP, EC₅₀ is the concentration at half activation, and n_H is the Hill coefficient.