Assembled CA tubes were diluted to 10uM in 50mM Tris pH 8 and negative-stained with uranyl acetate onto holey carbon grids after glow-discharge. Grids were examined on an FEI Talos L120C microscope and images at 57,000X magnification were collected using TEM Imaging and Analysis software (Gatan).
Talos l120c microscope
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Talos L120C is a high-performance transmission electron microscope (TEM) designed for advanced materials characterization. It features a 120 kV electron beam and a range of advanced imaging and analytical capabilities for detailed structural and compositional analysis of samples.
Automatically generated - may contain errors
Lab products found in correlation
Vitrobot mark 4, by Thermo Fisher Scientific (7 mentions)
Gatan 950 solarus plasma cleaning system, by Thermo Fisher Scientific (3 mentions)
Gatan 950 solarus plasma cleaning system, by Ted Pella (2 mentions)
55 20 mm blotting paper, by Ted Pella (2 mentions)
Tecnai spirit, by Thermo Fisher Scientific (1 mentions)
Talos f200x g2, by Thermo Fisher Scientific (1 mentions)
Quanta 250 feg microscope, by Thermo Fisher Scientific (1 mentions)
Oca20, by Dataphysics (1 mentions)
Ultrospec 2100 spectrophotometer, by Cytiva (1 mentions)
R1.2 1 3 holey carbon grid, by Quantifoil (1 mentions)
Gatan 950 solarus plasma cleaning system, by Ametek (1 mentions)
Leica em hpm100, by Leica camera (1 mentions)
Leica em afs2, by Leica camera (1 mentions)
Baltec med 020 device, by Leica Microsystems (1 mentions)
Ultrospec 2100, by Cytiva (1 mentions)
Linear polystyrene standards, by Waters Corporation (1 mentions)
Zetasizer pro, by Malvern Panalytical (1 mentions)
Ceta cmos camera, by Thermo Fisher Scientific (1 mentions)
26 protocols using talos l120c microscope
1
Negative Staining of CA Tubes
2
Cryo-EM Imaging of Macromolecular Complexes
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400-mesh Quantifoil R2/1 holey grids with additional ~4–5 nm continuous thin carbon layer were incubated with 5 μl of sample and vitrified with FEI Vitrobot Mark IV. Different glow-discharging, incubation and blotting times were tested and the grids were inspected either at the electron microscope FEI Tecnai Spirit at the Max Planck Institute for Molecular Genetics (Berlin, Germany) or FEI Tecnai G2 F20 in CEITEC (Brno, Czech Republic). 5,238 micrographs were collected with a FEI Titan Krios microscope in CEITEC (Brno, Czech Republic) at 300 kV equipped with a FEI Falcon II detector from a grid vitrified after 15 sec glow-discharging, 5 min sample incubation on ice and 2 sec blotting time. Each micrograph is composed of 7 frames, with a total exposure time of 1.5 sec per micrograph and total dose of 25 e-/Å2. Pixel size was 1.38 Å, corresponding to a magnification of about 100,000 fold. Nominal underfocus value was set randomly in the interval 1.5–4 μm.
Negative-stained micrographs of needle complex from Shigella mutants have been collected with a FEI Talos L120C microscope at the cryo-EM facility of the Centre for Structural System Biology (Hamburg, Germany). Samples were stained with 1% uranyl acetate.
Negative-stained micrographs of needle complex from Shigella mutants have been collected with a FEI Talos L120C microscope at the cryo-EM facility of the Centre for Structural System Biology (Hamburg, Germany). Samples were stained with 1% uranyl acetate.
3
Structural and Compositional Analysis of Sample
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XRD (Rigaku Corporation, Japan) was used to study the crystal structure of the sample. The voltage was 40 kV, and the current was 30 mA (Cu radiation) with a scanning rate of 10° min−1. SEM was equipped with a FEI Quanta FEG 250 microscope, and TEM was equipped with a FEI Talos L120C microscope. An EDS analyzer (EMAX Energy EX‐200) was utilized to investigate the elemental distribution. FEI Talos F200X G2 was used to collect HRTEM images. The XPS results were obtained from an AXIS ULTRA DLD. The N2 adsorption isotherm was conducted with a Quantachrome Autosorg‐iQ3 analyzer. TGA was executed using the Mettler‐Toledo instrument (Model TG50). The contact angle of the sample was analyzed by Dataphysics OCA20.
4
Characterization of Nanodrug Formulations
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Visible spectra of the nanodrugs were recorded using an UltroSpec 2100 spectrophotometer (Amersham Biosciences) and a 1-cm-path-length quartz cuvette. The concentration of Nps was calculated by interpolating the A530 nm values on a calibration curve obtained using uncoated nanogold in 5 mM sodium citrate buffer, pH 6.0 (stock solution: 3.3 × 1011 Nps/ml; A530 nm: 0.96 U/ml).
Dynamic light scattering (DLS) measurements were performed using a DLS DYNAPRO 99 instrument (Wyatt) operating with the laser intensity set to 20% power. The nanodrugs were diluted 1:10 in 5 mM sodium citrate buffer, pH 6.0 (1–3 × 1010 Nps/ml) and analyzed using 25–30 independent measurements of 10-s duration at 20 °C. The calculation of the hydrodynamic radius of the Nps was performed using DLS regularization analysis.
Transmission electron microscopy (TEM) analysis were performed using a TALOS L120C microscope (ThermoScientific) as described previously [23 (link)]. Morphometric analysis of nanoparticles shape and diameter was performed using the ImageJ software, essentially as previously reported [30 (link)].
Dynamic light scattering (DLS) measurements were performed using a DLS DYNAPRO 99 instrument (Wyatt) operating with the laser intensity set to 20% power. The nanodrugs were diluted 1:10 in 5 mM sodium citrate buffer, pH 6.0 (1–3 × 1010 Nps/ml) and analyzed using 25–30 independent measurements of 10-s duration at 20 °C. The calculation of the hydrodynamic radius of the Nps was performed using DLS regularization analysis.
Transmission electron microscopy (TEM) analysis were performed using a TALOS L120C microscope (ThermoScientific) as described previously [23 (link)]. Morphometric analysis of nanoparticles shape and diameter was performed using the ImageJ software, essentially as previously reported [30 (link)].
5
Negative-Stain TEM Analysis of Ferritin-Based Nanoparticles
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Transmission electron microscopy (TEM) Grids of SC-Ferritin, RBD-Ferritin and RBD-HR-chimeric-Ferritin nanoparticles were proceeded to negative-stain electron microscopy in Shuimu BioSciences Ltd. Briefly, 5 μL of each samples (0.5 μg/ul) were applied to glow-discharged electron microscopy grids covered by a thin layer of continuous film and stained with 2% uranyl acetate, followed by imaging on Talos L120C microscope (ThermoFisher) operating at an acceleration voltage of 120kV. Images were recorded at a magnification of 73,000 × and a defocus of 1.5 μm. The particles were manually picked and two-dimensional classification was performed with EMAN2.
6
Preparation of Negative Staining and Cryo-EM Grids
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For negative staining EM grid preparation, samples (5 µL at a concentration of ~0.06 mg/mL) were applied onto glow-discharged copper grids supported by a continuous thin layer of carbon film for 60 s before negative staining by 2% (w/v) Uranyl Acetate solution at room temperature. The grids were prepared in the Ar/O2 mixture for 15 s using a Gatan 950 Solarus plasma cleaning system with a power of 35 W. The negatively stained grids were loaded onto a Thermo Fisher Scientific Talos L120C microscope equipped with a Ceta CCD camera and operated at 120 kV at a nominal magnification of ×92,000, corresponding to a pixel size of 1.58 Å on the specimen.
For cryo-EM grid preparation, samples (4 μL at a concentration of ~0.6 mg/mL) were applied to freshly glow-discharged Quantifoil R1.2/1.3 holey carbon grids. After incubation for 5 s at 6 °C and 100% humidity, the grids were blotted for 1 s with blot force 2 in a Thermo Fisher Scientific Vitrobot Mark IV and plunge-frozen in liquid ethane at liquid nitrogen temperature. The grids were prepared in the H2/O2 mixture for 60 s using a Gatan 950 Solarus plasma cleaning system with a power of 5 W. The ø 55/20 mm blotting paper is made by TED PELLA used for plunge freezing.
For cryo-EM grid preparation, samples (4 μL at a concentration of ~0.6 mg/mL) were applied to freshly glow-discharged Quantifoil R1.2/1.3 holey carbon grids. After incubation for 5 s at 6 °C and 100% humidity, the grids were blotted for 1 s with blot force 2 in a Thermo Fisher Scientific Vitrobot Mark IV and plunge-frozen in liquid ethane at liquid nitrogen temperature. The grids were prepared in the H2/O2 mixture for 60 s using a Gatan 950 Solarus plasma cleaning system with a power of 5 W. The ø 55/20 mm blotting paper is made by TED PELLA used for plunge freezing.
7
Cryo-EM imaging of SARS-CoV-2 spike protein
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Spike protein was diluted to 0.05 mg/ml in PBS before grid preparation. A 3-μl drop of diluted protein was applied to previously glow-discharged, carbon-coated grids for ~60 s, blotted and washed twice with water, stained with 0.75% uranyl formate, blotted, and air-dried. Between 30 and 50 images were collected on a Talos L120C microscope (Thermo Fisher Scientific) at ×73,000 magnification and a pixel size of 1.97 Å. RELION 3.1 (37 (link)) or cryoSPARC v3.3.2 (38 (link)) was used for particle picking, two-dimensional (2D) classification.
8
Cryo-EM and Negative Staining Grid Preparation
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For negative staining EM grid preparation, samples (5 µL at a concentration of ~0.035 mg/mL) were applied onto glow-discharged copper grids supported by a continuous thin layer of carbon film for 60 s before being negatively stained by 2% (w/v) uranyl formate solution at room temperature. The grids were prepared in the Ar/O2 mixture for 15 s using a Gatan 950 Solarus plasma cleaning system with a power of 35 W. The negatively stained grids were loaded onto a Thermo Fisher Scientific Talos L120C microscope equipped with a Ceta CCD camera and operated at 120 kV at a nominal magnification of 92,000×, corresponding to a pixel size of 1.58 Å on the specimen.
For cryo-EM grid preparation, samples (4 μL at a concentration of ~1.5 mg/mL) were applied to freshly glow-discharged Quantifoil R1.2/1.3 holey gold grids. After incubation for 5 s at 4 °C and 100% humidity, the grids were blotted for 8.5 s with force 13 in a Thermo Fisher Scientific Vitrobot Mark IV and plunge-frozen in liquid ethane at liquid nitrogen temperature. The grids were prepared in the H2/O2 mixture for 20 s using a Gatan 950 Solarus plasma cleaning system with a power of 5 W. The ø 55/20 mm blotting paper (TED PELLA) was used for plunge freezing.
For cryo-EM grid preparation, samples (4 μL at a concentration of ~1.5 mg/mL) were applied to freshly glow-discharged Quantifoil R1.2/1.3 holey gold grids. After incubation for 5 s at 4 °C and 100% humidity, the grids were blotted for 8.5 s with force 13 in a Thermo Fisher Scientific Vitrobot Mark IV and plunge-frozen in liquid ethane at liquid nitrogen temperature. The grids were prepared in the H2/O2 mixture for 20 s using a Gatan 950 Solarus plasma cleaning system with a power of 5 W. The ø 55/20 mm blotting paper (TED PELLA) was used for plunge freezing.
9
High-Pressure Freezing for Ultrastructural Analysis
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C6 and C14 culture (100 ml) were collected at 1,000 × g using a Stat Spin Microprep 2 table top centrifuge. Cells were transferred to aluminium platelets (150 µm depth) containing 1-hexadecene94 . Platelets were frozen using a Leica EM HPM100 high-pressure freezer (Leica). Frozen samples were transferred to a Leica EM AFS2 automatic freeze substitution unit and substituted at −90 °C in a solution containing anhydrous acetone and 0.1% tannic acid for 24 h, and in anhydrous acetone, 2% OsO4 and 0.5% anhydrous glutaraldehyde (Electron Microscopical Science) for a further 8 h. After further incubation over 20 h at −20 °C, samples were warmed to +4 °C and subsequently washed with anhydrous acetone. Samples were embedded at room temperature in Agar 100 (Epon 812 equivalent) at 60 °C for 24 h. Thin sections (80 nm) were counterstained using Reynolds lead citrate solution for 7 s and examined using a Talos L120C microscope (Thermo Fisher).
10
Structure of Dimerization Domain-Antibody Complex
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Dimerization domain protein in complex with antibodies 3C3 and 2F4 was diluted to 0.001 mg/ml in PBS prior to grid preparation. A 3μL drop of diluted protein was applied to previously glow-discharged, carbon coated grids for ∼60 sec, blotted, and washed twice with water, stained with 0.75% uranyl formate, blotted, and air dried. 50 images were collected on a Talos L120C microscope (Thermo Fisher) at 73,000 magnification and 1.97 Å pixel size. Cryosparc v3.3.2 (Punjani et al., 2017 (link)) was used for particle picking, 2D classification and 3D reconstruction. Models corresponding to Nucleocapsid dimerization domain (PDB: 6WZO) and Fab region of an IgG were docked into NS-EM density map using UCSF ChimeraX (Pettersen et al., 2021 (link)).
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