Nativemark protein standard

Agarose (BioGene Ltd) gels were prepared in 100 mM Tris (pH 8), 100 mM boric acid and 2 mM EDTA (TBE buffer) containing 100 ng ml⁻¹ of UltraPure ethidium bromide (ThermoFisher Scientific) and typically 1.0% (w/v) agarose. Samples were loaded in DNA gel loading dye (Invitrogen), alongside the 1 Kb plus DNA ladder (Invitrogen), run at 100 V for 1 h and visualized using ethidium bromide fluorescence on a ChemiDoc MP System (Bio-Rad).
SDS-PAGE analyses were performed using either Novex WedgeWell 10–20% tris–glycine gels or Novex 10–20% tris–glycine gels. Proteins were reduced with 100 µM DTT then approximately 10 µg of protein loaded per well, alongside the Precision Plus Protein Kaleidoscope prestained protein standards (Bio-Rad), and visualized using Coomassie R250. Blue native polyacrylamide gel electrophoresis (BN-PAGE) was performed using NativePAGE Novex 3–12% Bis-Tris gels (Invitrogen). Vesicles were solubilized using a 2 : 1 DDM : protein ratio, and approximately 8 µg samples loaded in each well alongside the NativeMark Protein Standard (Invitrogen). Gels were run as described previously [47 (link)] and visualized using Coomassie R250.

BN-PAGE analysis was performed as described (Schägger and von Jagow 1991 (link)). Gels were polymerized using ammonium persulfate (APS) and TEMED. After polymerization, the gels were stored overnight at 4 °C. Per gel lane, 100 μg of cytosol were applied after dilution with BNP-sample buffer (750 mM aminocaproic acid, 50 mM Bis–tris/HCl, 0.5 mM EDTA and 5% (v/v) glycerol, pH 7.0) in a ratio of 1:4. Cathode and anode buffers used for the separation consisted of 50 mM Tricine, 15 mM Bis–tris, 0.02% (w/v) Coomassie G250, pH 7.0, and 50 mM Bis–tris adjusted to pH 7.0 with HCl, respectively. The lanes were loaded horizontally onto 12.5% SDS-PA gels and electrophoresed. NativeMARK™ protein standard (Invitrogen) was used for the evaluation of protein size.

Blue Native-PAGE (BN-PAGE) was performed for the analysis of native protein complexes. Purified recombinant proteins in 1 × PBS, 1 mM DTT, 1 mM ATP were mixed with 4 × NativePAGE Sample Buffer (Thermo Fisher Scientific Inc.) followed by separation in Invitrogen Novex Native-PAGE 4–16% Bis-Tris Protein-Gels (Thermo Fisher Scientific Inc.). Electrophoresis was performed in the cold-room with pre-chilled buffers (cathode buffer: 50 mM Tricine, 15 mM Bis-Tris pH 7.0, 0.02% Coomassie G-250; anode buffer: 50 mM Bis-Tris pH 7.0) at 150 V for 4 h. Gels were stained with Coomassie Brilliant Blue and protein molecular masses were determined based on the NativeMark protein standard (Invitrogen). The ATPase activity assay for p97 either alone or in the presence of full-length or truncated UBXD9 proteins was carried out in phosphate-free sample buffer (10 mM Tris/HCl pH 7.4, 100 mM NaCl, 1 mM DTT, 1 mM MgCl₂) in the presence of 1 mM ATP according to the instructions of the manufacturer (Enzo Life Sciences, Farmington, NY). The ATPase activity of p97 was calculated based on the amount of the generated free phosphate. Sucrose density gradient sedimentation and pull-down assays were performed as described (Rijal et al., 2016 (link)).

MP experiments were performed using a OneMP mass photometer (Refeyn Ltd, Oxford, UK). Data acquisition was performed using AcquireMP (Refeyn Ltd. v2.3). MP movies were recorded at 1 kHz, with exposure times varying between 0.6 and 0.9 ms, adjusted to maximize camera counts while avoiding saturation. Microscope slides (70 × 26 mm²) were cleaned for 5 min in 50% (v/v) isopropanol (HPLC grade in Milli-Q H₂O) and pure Milli-Q H₂O, followed by drying with a pressurized air stream. Silicon gaskets to hold the sample drops were cleaned in the same manner and fixed to clean glass slides immediately prior to measurement. The instrument was calibrated using the NativeMark Protein Standard (Thermo Fisher Scientific) immediately prior to measurements. The concentration during measurement of Aq880, Aq880 mutants, or Hhal2243 during measurements was typically 100 nM. Each protein was measured in a new gasket well (i.e., each well was used once). To find focus, 18 µL of fresh buffer (10 mM Tris pH 8.0, 100 mM KCl, and 0.1 mM EDTA) adjusted to room temperature was pipetted into a well, the focal position was identified and locked using the autofocus function of the instrument. For each acquisition, 2 µL of diluted protein was added to the well and thoroughly mixed. For each sample, three individual measurements were performed. The data were analyzed using the DiscoverMP software.

Mass photometry experiments were performed using a OneMP mass photometer (Refeyn Ltd., Oxford, United Kingdom). Data acquisition was performed using AcquireMP (Refeyn Ltd. v2.3). Mass photometry movies were recorded at 1kHz, with exposure times varying between 0.6 and 0.9 ms, adjusted to maximize camera counts while avoiding saturation. Microscope slides (70 × 26 mm) were cleaned 5 min in 50% (v/v) isopropanol (HPLC grade in Milli-Q H₂O) and pure Milli-Q H₂O, followed by drying with a pressurized air stream. Silicon gaskets to hold the sample drops were cleaned in the same manner fixed to clean glass slides immediately prior to measurement. The instrument was calibrated using NativeMark Protein Standard (Thermo Fisher) immediately prior to measurements. Immediately prior to mass photometry measurements, protein stocks were diluted directly in HEPES buffer. Typical working concentrations of Vmp1 and Vmp2 were 25–50 nM for the actual measurement. Each protein was measured in a new gasket well (i.e., each well was used once). To find focus, 18 μl of fresh room temperature buffer was pipetted into a well, and the focal position was identified and locked using the autofocus function of the instrument. For each acquisition, 2 μl of diluted protein was added to the well and thoroughly mixed. The data were analyzed using the DiscoverMP software.