Tecnai tf20 microscope

The morphology of the as-synthesized materials was examined via transmission electron microscopy (TEM) via Tecnai TF20 microscope (FEI Company, Eindhoven, Netherlands) at an operating voltage of 200 kV. The crystal structure was examined by X-ray diffraction (XRD) via an X’Pert Phillips diffractometer (Phillips-PANalytical, Almelo, Netherlands) equipped with Cu-kα radiation (λ = 1.54059 Å). The electronic structures and oxidation states were investigated by X-ray photoelectron spectroscopy (XPS) with Axis Ultra DLD XPS (Kratos, Manchester, UK) equipped with a monochromatic Al-Kα radiation source (1486.6 eV). All binding energies were corrected against standard C 1 s peak, i.e., 284.6 eV. The textural properties were examined via N₂ sorption experiments at liquid nitrogen temperature (77 K) using the Brunauer–Emmett–Teller (BET) method.
The zeta-potentials (ζ-potential) measurements were carried out using Zetasizer Nano ZSP instrument (Malvern Instruments Ltd., Worcestershire, UK) based on the electrophoretic mobility by applying Smoluchowski’s approximation. For each measurement, 5 milligrams of the sample were dispersed into 10 mL deionized water and sonicated for 10 min. After that, the pH value was adjusted by the addition of NaOH/HCl, and the steady state value was recorded.

Mouse ES cells were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4 for 1 h. Buffer rinsed cells were scraped in 1% gelatin and spun down in 2% agar. Chilled blocks were trimmed and postfixed in 1% osmium tetroxide for 1 h. The samples were rinsed three times in sodium cacodylate rinse buffer and postfixed in 1% osmium tetroxide for 1 h. Samples were then rinsed and stained in aqueous 2% uranyl acetate for 1 h followed by rinsing, dehydrating in an ethanol series, infiltrated with Embed 812 (Electron Microscopy Sciences) resin, and then baked overnight at 60 °C. Hardened blocks were cut using a Leica UltraCut UC7. Sections (60 nm) were collected in formvar/carbon-coated nickel grids and contrast stained with 2% uranyl acetate and lead citrate. They were viewed using a FEI Tencai Biotwin TEM at 80 Kv. Images were taken on a Morada CCD using iTEM (Olympus) software.
Purified human ATP synthase c-ring was stained with 2% uranyl acetate. Negative- stain electron microscopy images were taken by using FEI Tecnai TF20 microscope (FEG, 200 kV) (Yale Center for Cellular and Molecular Imaging).

TEM images were acquired on a FEI Tecnai TF20 microscope operating at 200 kV. TEM samples were prepared by drop casting 20 μl of the Pd nanocube solution onto a copper TEM grid (300 carbon mesh) and drying in ambient conditions.

Negative stain EM samples were prepared by applying polymerized CtpS to carbon-coated grids and staining with 0.75% uranyl formate (Ohi et al., 2004 (link)). 15 μM purified CTPs in 50 mM Tris HCl (pH 7.8) was incubated for 20 min at 37°C with 1 mM CTP and 5 mM MgCl₂, or without nucleotide as a control. Reactions were diluted 1/10 in the same buffer supplemented with 50% glycerol before being coated onto grids and stained with uranyl formate for analysis. Protein purifications for wild-type CTPs and mutants E155K and E277R were performed simultaneously. Negative stain EM was performed on a Tecnai TF20 microscope (FEI Co.) operating at 200 kV, and images were acquired on a 4 k × 4 k CCD camera (Gatan, Inc.). Micrographs all taken at 55,000X magnification.

Purified EmbB in nanodiscs was diluted to 0.005 mg/ml and applied onto copper grids (Ted Pella). These grids were overlaid by a thin (∼1.5 nm) layer of continuous carbon that had been plasma-cleaned (Gatan Solarus) for 30 s using a mixture of H₂ and O₂. Thereafter, filter paper (Whatman 4) was used to remove the protein solution. Three microliters of 2% uranyl formate was then added and immediately removed by absorbing with filter paper—this was repeated seven times. The grid was imaged on a Tecnai TF20 microscope (FEI) equipped with a Tietz F416 CCD camera (Tietz) at 1.10 Å per pixel, respectively, using the Leginon software package^{45 (link)}. Seventy seven images were collected and processed using the Appion software package^{46 (link)} to obtain 2D classes with Relion 2.1^{47 (link),48 (link)}. The micrographs showed good particle dispersion and homogeneity.