C-Flat 2/2-3C grids (Protochips) were glow discharged for 30 seconds at 25 mA, and a 5 ul sample of the tube assembly reaction was applied 3 times to the grid with 30 s incubation and blotting between each sample application. Grids were washed with 5 µl H2O before blotting and plunge-freezing in liquid ethane using an FEI Vitrobot. Grids were stored in liquid nitrogen until imaging. Imaging was performed on an FEI Titan Krios operated at 300kV, equipped with a Gatan K2-summit direct detector using a 20 eV slit width operated in zero-loss mode. 2347 2D images were acquired using SerialEM-3.7.0 35 (link) with a nominal magnification of 105000 X, giving a pixel size of 1.128 Å at the specimen level.
C flat 2 2 3c grids
Manufactured by Protochips
Sourced in United States
The C-Flat 2/2 3C grids are a type of lab equipment produced by Protochips. The grids are designed for use in electron microscopy applications. They provide a stable, consistent support structure for samples during imaging and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Vitrobot, by Thermo Fisher Scientific (4 mentions)
Titan krios, by Ametek (2 mentions)
Titan krios electron microscope, by Thermo Fisher Scientific (2 mentions)
K2 summit, by Ametek (1 mentions)
Vitrobot mark 4, by Thermo Fisher Scientific (1 mentions)
Talos arctica microscope, by Thermo Fisher Scientific (1 mentions)
K3 bioquantum, by Thermo Fisher Scientific (1 mentions)
5 protocols using c flat 2 2 3c grids
1
Cryo-EM Grid Preparation and Imaging
2
Cryo-ET Imaging of Virus Particles
3
Cryo-EM Analysis of AnaS Samples
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The geometry of AnaS samples subjected to precollapse pressures was characterized using cryo-EM as described before (12 (link)). A 3-μL volume of a sample with OD500 = ∼5 was applied to C-Flat 2/2-3C grids (Protochips, Cary, NC, USA) that were freshly glow discharged (Pelco EasiGlow, 10 mA, 1 min, Pelco, Fresno, CA, USA). GV samples were frozen using a Mark IV Vitrobot (FEI, now Thermo Fisher Scientific) (4°C, 100% humidity, blot force 3, blot time 4 s). Micrographs were collected on a 200 kV Talos Arctica microscope (FEI, now Thermo Fisher Scientific) equipped with a K3 6k × 4k direct electron detector (Gatan, Pleasanton, CA, USA). Multiframe images were collected using SerialEM 3.39 software (25 (link)) with a pixel size of 1.17 Å (36,000× magnification) and a defocus of −2.5 μm. Super-resolution movies were corrected for gain reference, binned by a factor of 2, and motion corrected using MotionCor2 (26 (link)). GV dimensions were measured using IMOD 4.12 (27 (link)). Statistical analysis was performed in GraphPad PRISM.
4
Cryo-EM Imaging of SARS-CoV-2 Spike Proteins
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Grid preparation and image collection were performed for S-R/x3 and S-R/x3/D614G spike proteins essentially as described in Xiong et al. 2020 [4 (link)]. C-Flat 2/2 3C grids (Protochips) were glow-discharged for 45 seconds at 25 mA. 3 μl of freshly purified protein at 0.6 mg/ml supplemented with 0.01% octyl-glucoside (OG) was applied to the grids, which were plunge-frozen in liquid ethane using a Vitrobot (Thermo Fisher Scientific).
An aliquot of S-R/x3 freshly purified spike at 1.0 mg/ml was stored at 4°C for 40 days. 10 μl of the stored protein was subjected to plunge-freezing at 1.0 mg/ml following the same procedure as for the freshly purified S trimers. Another 10 μl of the stored protein was incubated with 1 μl of citrate acid (pH 4.8) overnight at 4°C and then plunge-frozen.
Grids were stored in liquid nitrogen and loaded into a Titan Krios electron microscope (Thermo Fisher Scientific) operated at 300 kV. Movies with 48 frames were collected with a Gatan K3 BioQuantum direct electron detector with the slit retracted. Three shots per hole were achieved with beam-image shift controlled in SerialEM-3.7.0 [34 (link)]. An accumulated dose of 50 electrons/Å2 were acquired in counting mode at the magnification of 81,000 X, corresponding to a calibrated pixel size of 1.061 Å/pixel. Detailed data acquisition parameters are summarized inS1 Table .
An aliquot of S-R/x3 freshly purified spike at 1.0 mg/ml was stored at 4°C for 40 days. 10 μl of the stored protein was subjected to plunge-freezing at 1.0 mg/ml following the same procedure as for the freshly purified S trimers. Another 10 μl of the stored protein was incubated with 1 μl of citrate acid (pH 4.8) overnight at 4°C and then plunge-frozen.
Grids were stored in liquid nitrogen and loaded into a Titan Krios electron microscope (Thermo Fisher Scientific) operated at 300 kV. Movies with 48 frames were collected with a Gatan K3 BioQuantum direct electron detector with the slit retracted. Three shots per hole were achieved with beam-image shift controlled in SerialEM-3.7.0 [34 (link)]. An accumulated dose of 50 electrons/Å2 were acquired in counting mode at the magnification of 81,000 X, corresponding to a calibrated pixel size of 1.061 Å/pixel. Detailed data acquisition parameters are summarized in
5
SARS-CoV-2 Spike Protein Cryo-EM Sample Preparation
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