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Glow discharged continuous carbon grid

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The Glow-discharged continuous carbon grid is a specialized lab equipment used in electron microscopy. It features a continuous carbon film surface that has been subjected to glow discharge treatment. This treatment modifies the surface properties of the carbon film, enhancing its ability to interact with and support samples for electron microscopy analysis.

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Lab products found in correlation

Tecnai t12 electron microscope, by Thermo Fisher Scientific (6 mentions) Cm10 electron microscope, by Philips (2 mentions) Tecnai f20 tem, by Thermo Fisher Scientific (1 mentions)

7 protocols using glow discharged continuous carbon grid

1

Ligase Complex Nanodisc Imaging

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The ligase complex reconstituted in nanodiscs or in digitonin at a concentration of 0.02 mg ml–1 was applied to glow-discharged continuous carbon grids (Electron Microscopy Sciences, Inc.). After 1 min of adsorption, the grids were blotted with filter paper to remove excess sample, immediately washed twice with 4 μl of 1.5% freshly made uranyl formate solution and incubated with 4 μl of 1.5% uranyl formate solution for an additional 30 s. The grids were then further blotted with filter paper to remove the uranyl formate solution, air dried at room temperature and examined with a Tecnai T12 electron microscope (Thermo Fisher Scientific) equipped with an LaB6 filament and operated at 120 kV acceleration voltage, using a nominal magnification of ×52,000 at a pixel size of 2.13 Å.
Feng P., Wu X., Erramilli S.K., Paulo J.A., Knejski P., Gygi S.P., Kossiakoff A.A, & Rapoport T.A. (2022). A peroxisomal ubiquitin ligase complex forms a retrotranslocation channel. Nature, 607(7918), 374-380.
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2

Structural Characterization of neddylated CRL2Lrr1

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rCRL2Lrr1, rCRL2Lrr1ΔPH or neddylated CRL2Lrr1 at concentrations of 0.01 mg/ml were applied onto glow-discharged continuous carbon grids (Electron Microscopy Sciences, Inc.). After 1 min of adsorption, the grids were blotted with filter paper to remove excess sample, immediately washed twice with 4 μl of 1.5% uranyl formate solution and incubated with 4 μl of 1.5% uranyl formate solution for an additional 90 s. The grids were then further blotted with filter paper to remove the uranyl formate solution, air dried at room temperature and examined with a Tecnai T12 electron microscope (Thermo Fisher Scientific) equipped with an LaB6 filament and operated at 120 kV acceleration voltage, using a nominal magnification of 69 000× at a pixel size of 1.68 Å.
Zhou H., Zaher M.S., Walter J.C, & Brown A. (2021). Structure of CRL2Lrr1, the E3 ubiquitin ligase that promotes DNA replication termination in vertebrates. Nucleic Acids Research, 49(22), 13194-13206.
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3

Cryo-EM Analysis of NTS-NTSR1-Gαi1β1γ1-cND Complex

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Three microliters of NTS-NTSR1-Gαi1β1γ1-cND complex at a concentration of 0.02 mg/mL was applied onto a glow-discharged continuous carbon grid (Electron Microscopy Sciences, Inc.). After two minutes of adsorption, the grid was blotted with filter paper to remove the excess sample, immediately washed twice with 50 μL of MiliQ water, once with 50 μL 0.75% uranyl formate solution and incubated with 50 μL of 0.75% uranyl formate solution for an additional one minute. The grid was then further blotted with filter paper followed by vacuum aspiration to remove excess stain, and finally examined with a Tecnai T12 electron microscope (Thermo Fisher Scientific) equipped with an LaB6 filament and operated at 120-kV acceleration voltage, using a nominal magnification of 52,000× at a pixel size of 2.13 Å.
Zhang M., Gui M., Wang Z.F., Gorgulla C., Yu J.J., Wu H., Sun Z.Y., Klenk C., Merklinger L., Morstein L., Hagn F., Plückthun A., Brown A., Nasr M.L, & Wagner G. (2021). Cryo-EM structure of an activated GPCR-G protein complex in lipid nanodiscs. Nature structural & molecular biology, 28(3), 258-267.
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4

Liposome and Protein Visualization

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A 3 µL drop of solutions containing liposomes and proteins was placed on a glow-discharged continuous carbon grid (Electron Microscopy Science) and stained with 2% uranyl acetate. Grids of the samples from the lipid extraction experiments (Figure 2—figure supplement 2) were imaged with a Morgagni transmission electron microscope (TEM) operating at 80 keV at a magnification of 57,000 ×. Grids of the uranyl acetate-stained samples containing WIPI1 and PI3P-containing liposomes were imaged with an FEI Tecnai F20 TEM operating at 200 keV at a magnification of 11,500 ×.
Maeda S., Otomo C, & Otomo T. (2019). The autophagic membrane tether ATG2A transfers lipids between membranes. eLife, 8, e45777.
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5

Negative Staining Electron Microscopy of Bovine DMT

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A 4 μL aliquot of bovine DMT sample at an A280 reading of ~2 was applied onto a glow discharged continuous carbon grid (Electron Microscopy Sciences). After one minute of adsorption, the grid was blotted with filter paper to remove the excess sample, immediately washed twice with 4 μL of 1.5% uranyl formate solution and incubated with 4 μL of 1.5% uranyl formate solution for an additional one minute. The grid was then further blotted with filter paper to remove the uranyl formate solution, air-dried at room temperature, and examined with CM10 electron microscope (Phillips) or Tecnai T12 electron microscope (Thermo Fisher Scientific). The CM10 is operated at 100 kV acceleration voltage with a tungsten filament and is equipped with a Gatan UltraScan 894 (2k × 2k) CCD camera. The T12 is operated at 120 kV acceleration voltage with an LaB6 filament and is equipped with a Gatan UltraScan 895 (4k × 4k) CCD.
Gui M., Farley H., Anujan P., Anderson J.R., Maxwell D.W., Whitchurch J.B., Botsch J.J., Qiu T., Meleppattu S., Singh S.K., Zhang Q., Thompson J., Lucas J.S., Bingle C.D., Norris D.P., Roy S, & Brown A. (2021). De novo identification of mammalian ciliary motility proteins using cryo-EM. Cell, 184(23), 5791-5806.e19.
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6

Negative Staining Electron Microscopy of Bovine DMT

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A 4 μL aliquot of bovine DMT sample at an A280 reading of ~2 was applied onto a glow discharged continuous carbon grid (Electron Microscopy Sciences). After one minute of adsorption, the grid was blotted with filter paper to remove the excess sample, immediately washed twice with 4 μL of 1.5% uranyl formate solution and incubated with 4 μL of 1.5% uranyl formate solution for an additional one minute. The grid was then further blotted with filter paper to remove the uranyl formate solution, air-dried at room temperature, and examined with CM10 electron microscope (Phillips) or Tecnai T12 electron microscope (Thermo Fisher Scientific). The CM10 is operated at 100 kV acceleration voltage with a tungsten filament and is equipped with a Gatan UltraScan 894 (2k × 2k) CCD camera. The T12 is operated at 120 kV acceleration voltage with an LaB6 filament and is equipped with a Gatan UltraScan 895 (4k × 4k) CCD.
Gui M., Farley H., Anujan P., Anderson J.R., Maxwell D.W., Whitchurch J.B., Botsch J.J., Qiu T., Meleppattu S., Singh S.K., Zhang Q., Thompson J., Lucas J.S., Bingle C.D., Norris D.P., Roy S, & Brown A. (2021). De novo identification of mammalian ciliary motility proteins using cryo-EM. Cell, 184(23), 5791-5806.e19.
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7

Cryo-EM imaging of NT-NTR1-Ga i1 b 1 g 1 -cND complex

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3 μL of NT-NTR1-Ga i1 b 1 g 1 -cND complex at a concentration of 0.02 mg/mL was applied onto a glowdischarged continuous carbon grid (Electron Microscopy Sciences, Inc.). After two minutes of adsorption, the grid was blotted with filter paper to remove the excess sample, immediately washed twice with 50 μL of MiliQ water, once with 50 μL 0.75% uranyl formate solution and incubated with 50 μL of 0.75% uranyl formate solution for an additional one minute. The grid was then further blotted with filter paper to remove the uranyl formate solution, air-dried at room temperature, and examined with a Tecnai T12 electron microscope (Thermo Fisher Scientific) equipped with an LaB6 filament and operated at 120-kV acceleration voltage, using a nominal magnification of 52,000× at a pixel size of 2.13 Å.
Wagner G., Zhang M., Gui M., Wang Z., Gorgulla C., Yu J.B., Sun Z., Klenk C., Merklinger L., Morstein L., Hagn F., Plückthun A., Brown A., Nasr M.L., & Wu H. (2020). Cryo-EM structure of an activated GPCR-G protein complex in lipid nanodiscs.
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