G-actin was polymerized for 1–2 hours in F-buffer (10mM HEPES, pH 7.4, 100mM KCl, 1mM MgCl2, 0.5mM ATP). For EM samples shown in Fig. 1c-g , 2 μM F-actin was incubated for 2 min in tube with 34 μM NTD, B-domain, A-domain, or G-domain. Samples were blotted and negatively stained with 2% (wt/vol) uranyl acetate. To obtain single actin filaments decorated with SEPT9 domains was 2 μM F-actin was incubated for 1 min on carbon coated glow discharged grids in a humidified chamber, gently blotted and incubated again with 34 μM NTD, B-domain, A-domain, or G-domain. Previous work has shown that decoration of actin filaments with cofilin/ADF results in the same binding modes independently of whether it is performed on carbon grid or in test tube43 (link),44 (link). Samples were negatively stained with 2% (wt/vol) uranyl acetate. A JEM-2100F (JEOL) Field Emission Transmission Electron Microscope, equipped with 11-megapixel Gatan SC1000 ORIUS CCD camera, was used at an accelerating voltage of 200 kV, and a nominal magnification of 60,000x to record micrographs at a raster of 1.58 Å/pixel.
Field emission transmission electron microscope
Manufactured by JEOL
Sourced in Japan
The Field Emission Transmission Electron Microscope (FE-TEM) is a high-resolution imaging and analytical tool. It uses a field emission electron source to generate a focused beam of electrons, which is then used to illuminate a thin specimen. The transmitted electrons are detected and analyzed, providing detailed information about the sample's structure and composition at the nanoscale level.
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2 protocols using field emission transmission electron microscope
1
Visualizing SEPT9 Binding to Actin Filaments
2
Photocatalyst Characterization and Photoelectrochemical Performance
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The phase purity of the photocatalyst was detected by X-ray diffractometer (XRD) (Bruker AXS, Karlsruhe, Germany). The morphology observation of the photocatalyst was performed by a field-emission scanning electron microscope (SEM) (JEOL Ltd., Tokyo, Japan) and field emission transmission electron microscope (TEM) (JEOL Ltd., Tokyo, Japan). The composition and surface chemical states of the photocatalyst were investigated though X-ray photoelectron spectroscopy (XPS) (Physical Electronics, Chanhassen, MN, USA). The UV-vis diffuse reflectance spectra of the photocatalyst were measured using a UV-vis spectrophotometer with BaSO4 as a reference (Beijing Purkinje General Instrument Co. Ltd., Beijing, China). The photoluminescence (PL) spectra of the photocatalyst were obtained on a fluorescence spectrophotometer with the excitation wavelength of ~265 nm (Shimadzu RF-6000, Kyoto, Japan). Photoelectrochemical measurements were performed on a CHI 660C electrochemical workstation (Shanghai Chenhua Instrument Co. Ltd, Shanghai, China) equipped with a three-electrode cell configuration. The working electrode preparation and measurement procedures were the same as those reported in the literature [52 (link)]. The used electrolyte was 1 mol L−1 Na2SO4 aqueous solution, and the light source was a 300 W xenon lamp with a 420 nm cut-off filter.
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