For cryo-EM, the inactive CASP complex was diluted to 1 μM in a final buffer containing 20 mM Tris pH 7.5, 100 mM NaCl, 0.5% glycerol, and the active CASP complex was used at 1.6 μM in its final storage buffer. Quantifoil R1.2/1.3 300 mesh Cu holey carbon grids (Quantifoil, Germany) were glow-discharged (EMS 100, ElectronMicroscopy Sciences) at 25 mA for 1 min. 3 μl of each sample was applied to glow-discharged grids, blotted for 5 s using Standard Vitrobot Filter Paper (Ted Pella), and plunge-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific) at 4°C and 100% humidity.
Ems 100
Manufactured by Electron Microscopy Sciences
Sourced in Germany
The EMS 100 is a high-performance electron microscope designed for scientific and industrial applications. It is capable of producing high-resolution images of small-scale structures and features.
Automatically generated - may contain errors
Lab products found in correlation
Vitrobot mark 4, by Thermo Fisher Scientific (4 mentions)
R1.2 1 3 300 mesh cu holey carbon grids, by Quantifoil (2 mentions)
Standard vitrobot filter paper, by Ted Pella (2 mentions)
Cf200 cu, by Electron Microscopy Sciences (2 mentions)
Tecnai spirit biotwin microscope, by Thermo Fisher Scientific (2 mentions)
Em grade paraformaldehyde, by Ted Pella (1 mentions)
Whatman filter paper, by Cytiva (1 mentions)
5 protocols using ems 100
1
Cryo-EM Specimen Preparation for CASP
2
Cryo-EM Specimen Preparation for CASP
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For cryo-EM, the inactive CASP complex was diluted to 1 μM in a final buffer containing 20 mM Tris pH 7.5, 100 mM NaCl, 0.5% glycerol, and the active CASP complex was used at 1.6 μM in its final storage buffer. Quantifoil R1.2/1.3 300 mesh Cu holey carbon grids (Quantifoil, Germany) were glow-discharged (EMS 100, ElectronMicroscopy Sciences) at 25 mA for 1 min. 3 μl of each sample was applied to glow-discharged grids, blotted for 5 s using Standard Vitrobot Filter Paper (Ted Pella), and plunge-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific) at 4°C and 100% humidity.
3
SARS-CoV-2 Neutralization by Fu2 Compound
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Concentrated SARS-CoV-2 was incubated in the absence or presence of 20 µg/ml of Fu2 for 1 h under agitation. Samples were fixed in EM-grade paraformaldehyde (Ted Pella) and used to prepare negative staining grids. Four microliters of sample was loaded on the grids (EM Resolutions (EMR), 200 squares, carbon support film on copper) that had been glow-discharged with 25 mA for 1 min using an EMS ×100 (Electron Microscopy Sciences) glow-discharge unit. The samples were incubated for 4 min on the grids and subsequently stained with 1% uranyl acetate solution (Ted Pella). The grids were imaged in Talos 120 C G2 (Thermo Scientific) equipped with a CETA-D detector in the Karolinska Institutet’s 3D-EM facility (https://ki.se/cmb/3d-em ). Micrographs were collected using Serial-EM46 (link) at a magnification of ×45,000, corresponding to a pixel size of 3.14 Å/pixel.
4
Nup133-NTD Liposome Adsorption and Staining
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Nup133NTD-liposome mixtures at 1 mg/ml were loaded on glow-discharged (EMS 100, Electron Microscopy Sciences) continuous carbon film grids (CF200-Cu, Electron Microscopy Sciences). After 45 s of adsorption on grids, the samples were blotted with Whatman filter paper and the specimen on the grid was immediately stained with 2% w/v uranyl acetate for 30 s. The specimen was blotted, stained once more, reblotted, and air dried. Electron micrographs were recorded on a FEI Tecnai Spirit BioTwin microscope (FEI) operated at 80 keV and equipped with a tungsten filament and an AMT XR16 CCD detector.
5
Negative Staining of TorsinA Filaments
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Overall, 5 µl of TorsinA filaments or TorsinA–liposome mixtures were loaded on glow-discharged (EMS 100, Electron Microscopy Sciences) continuous carbon film grids (CF200-Cu, Electron Microscopy Sciences) immediately after diluting them. TorsinA filaments were diluted in the same buffer which they were dialyzed against (typically 20 mM HEPES/NaOH pH 8.0, X mM NaCl, 10 mM MgCl2, 0.5 mM ATP). TorsinA–liposome mixtures were also diluted in dialysis buffer (20 mM HEPES/NaOH pH 8.0, 100 mM NaCl, 10 mM MgCl2, 0.5 mM ATP), except lacking sucrose to prevent interference with negative staining. Samples containing only liposomes were prepared identically to TorsinA–liposome mixtures, and the final sucrose concentration after dilution was about 1% w/v. After 45 s of adsorption on grids, the samples were blotted and the specimen on the grid was immediately stained with 2% w/v uranyl acetate for 30 s. The specimen was blotted, stained once more, re-blotted, and air dried. Electron micrographs were recorded on an FEI Tecnai Spirit BioTwin microscope (FEI) operated at 80 keV and equipped with a tungsten filament and an AMT XR16 CCD detector.
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