Gloqube glow discharge system

Manufactured by Quorum Technologies

The GloQube is a glow discharge system designed for surface treatment and cleaning applications. It generates a plasma discharge to modify the surface properties of materials. The core function of the GloQube is to provide a controlled environment for plasma-based surface treatments.

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Lab products found in correlation

Lacey formvar carbon 200 mesh copper grids, by Ted Pella (1 mentions) K2 summit direct electron detector, by Ametek (1 mentions) Titan krios electron microscope, by Ametek (1 mentions) Talos f200c, by Thermo Fisher Scientific (1 mentions) Em grid, by Electron Microscopy Sciences (1 mentions) Tecnai 12 biotwin transmission electron microscope, by Thermo Fisher Scientific (1 mentions) Velita 2kx2k ccd camera, by Olympus (1 mentions) Falcon 4 direct electron detector, by Thermo Fisher Scientific (1 mentions) Titan krios g4 cryo tem, by Thermo Fisher Scientific (1 mentions) Vitrobot mark 4 plunger, by Thermo Fisher Scientific (1 mentions) Epu 2, by Thermo Fisher Scientific (1 mentions)

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GloQube (12 citations) GloQube system (9 citations) GloQube® Plus Glow Discharge System (2 citations)

5 protocols using gloqube glow discharge system

Cryo-TEM Imaging of ssDNA-Amphiphiles

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Lacey formvar/carbon 200 mesh copper grids were purchased from Ted Pella Inc. (Redding, CA) and glow discharged for 1 min to make the grids more hydrophilic. ssDNA-amphiphiles (500 μM; 4.5 μl) in Milli-Q water were deposited onto the grid and vitrified in liquid ethane using a Vitrobot (Vitrobot parameters: blot time, 5 s; wait time, 3 s; relax time, 3 s; offset, 0; humidity, 95%; 25°C). The grids were transferred to and kept under liquid nitrogen until imaged on a Tecnai G2 Spirit TWIN 20-120 kV/LaB₆ TEM operated at an accelerating voltage of 120 kV using an Eagle 2k charge-coupled device (CCD) camera at the University of Minnesota Characterization Facility.
For the room temperature TEM experiments, 5 μl of ssDNA-amphiphiles was used at a concentration of 50 μM for the DOTA-labeled amphiphiles, 47 μM for the nuclease stability, and 27 μM for the serum stability experiment. Pure carbon 300 mesh copper grids (Ted Pella Inc.) were glow discharged in a GloQube glow discharge system (Quorum). Samples were then deposited on the grid. After 5 min, the excess sample was wicked off with filter paper. The grids were then imaged in a FEI Talos 200SC FEG TEM with a CETA scintillated 4k CMOS camera at the Integrated Imaging Center at the Johns Hopkins University.

Harris M.A., Kuang H., Schneiderman Z., Shiao M.L., Crane A.T., Chrostek M.R., Tăbăran A.F., Pengo T., Liaw K., Xu B., Lin L., Chen C.C., O’Sullivan M.G., Kannan R.M., Low W.C, & Kokkoli E. (2021). ssDNA nanotubes for selective targeting of glioblastoma and delivery of doxorubicin for enhanced survival. Science Advances, 7(49), eabl5872.

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Cryo-EM structure determination protocol

Cited 2 times

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UltrAuFoil R1.2/1.3 300-mesh grids were glow discharged at 40 mA for 5 min using a GloQube glow discharge system (Quorum) and coated with graphene oxide flakes (Sigma). Next, 4 μl of 40 ng μl^–1 DDK was applied to each grid for 30 s before double-sided blotting for 4 s and plunge-freezing in liquid ethane using a Vitrobot Mark IV (FEI) operated at room temperature with 90% humidity. A total of 9,469 videos were collected at ×165,000 magnification (yielding a pixel size of 0.84 Å at specimen level) on a Titan Krios electron microscope equipped with a K2 Summit direct electron detector (Gatan Inc.).
A total of 2,967,226 particles were picked from motion-corrected video sums using crYOLO^{65 (link)}. CTF was estimated using Gctf^{64 (link)}, and 2D classification was performed after extraction of particles in a 240-pixel box in RELION-3.0.7 (ref. ³⁹).

Greiwe J.F., Miller T.C., Locke J., Martino F., Howell S., Schreiber A., Nans A., Diffley J.F, & Costa A. (2021). Structural mechanism for the selective phosphorylation of DNA-loaded MCM double hexamers by the Dbf4-dependent kinase. Nature Structural & Molecular Biology, 29(1), 10-20.

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Cryo-EM Imaging of Macromolecular Complexes

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EM grids (400 mesh copper with continuous carbon, Electron Microscopy Sciences) were glow discharged for 15 s at 10 mA using a GloQube glow discharge system (Quorum) before applying 4 μl of sample diluted to approximately 0.1 mg ml⁻¹ in SEC buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.005% (w/v) LMNG, 0.0005% (w/v) CHS). After 1 min of incubation, the grid was washed with distilled, deionized water, and stained with 1% uranyl acetate. After drying completely, the grids were imaged in a Talos F200C equipped with a 4k × 4k Ceta 16M Camera (Thermo Fisher Scientific) using SerialEM Version 3.9.0. Images were recorded at a nominal magnification of 45,000× (3.2 Å per pixel).

Kayagaki N., Stowe I.B., Alegre K., Deshpande I., Wu S., Lin Z., Kornfeld O.S., Lee B.L., Zhang J., Liu J., Suto E., Lee W.P., Schneider K., Lin W., Seshasayee D., Bhangale T., Chalouni C., Johnson M.C., Joshi P., Mossemann J., Zhao S., Ali D., Goldenberg N.M., Sayed B.A., Steinberg B.E., Newton K., Webster J.D., Kelly R.L, & Dixit V.M. (2023). Inhibiting membrane rupture with NINJ1 antibodies limits tissue injury. Nature, 618(7967), 1072-1077.

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Negative Staining Nanoparticle Imaging

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All the negative stain EM samples were prepared using staining methods described in previous studies (36 (link), 37 (link)). Initially, glow discharge of the copper grids was carried out for 40-60 seconds at 20 mA current using GloQube glow discharge system (Quorum technologies) for uniform settling of the particles on the grid. Following this, 3.5 µl of nanoparticle (0.4 mg/mL) in 20 mM Tris was adsorbed on the grids for 30-60 secs. Excess buffer was removed by blotting with a filter paper and negative stain was performed using 1% uranyl acetate. Negative stain imaging experiment was carried out at room temperature using a FEI Tecnai 12 BioTwin transmission electron microscope equipped with a LaB6 (lanthanum hexaboride crystal) filament at a voltage of 120 Kv. Datasets were collected using a side-mounted Olympus VELITA (2Kx2K) CCD camera at a calibrated magnification of 75000 x and a defocus value of −1.3 µm. The final images were recorded at a pixel size of 2.54 Å on the specimen level. Following this, properly sized particles were manually chosen and extracted using e2boxer.py and e2projectmanager.py EMAN 2.1 software (38 (link)). Finally, a 2-D reference free classification of particles was carried out using e2refine2d.py.

Kar U., Khaleeq S., Garg P., Bhat M., Reddy P., Vignesh V.S., Upadhyaya A., Das M., Chakshusmathi G., Pandey S., Dutta S, & Varadarajan R. (2022). Comparative Immunogenicity of Bacterially Expressed Soluble Trimers and Nanoparticle Displayed Influenza Hemagglutinin Stem Immunogens. Frontiers in Immunology, 13, 890622.

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Cryo-EM of SARS-CoV-2 HKU1 Hemagglutinin-Esterase

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Three μl of purified HKU1 HE (4 mg/ml) was dispensed on Quantifoil R1.2/1.3 200-mesh grids (Quantifoil Micro Tools GmbH) that had been freshly glow discharged for 30 seconds at 20 mA using GloQube Glow Discharge system (Quorum Technologies). Grids were blotted for five seconds using Whatman No. 1 filter paper and immediately plunge-frozen into liquid ethane cooled by liquid nitrogen using a Vitrobot Mark IV plunger (Thermo Fisher Scientific) equilibrated to ~95% relative humidity, 4 °C. Movies of frozen-hydrated HKU1 HE were collected using Titan Krios G4 Cryo-TEM (Thermo Fisher Scientific) operating at 300 keV and equipped with a Falcon 4 Direct Electron Detector (Thermo Fisher Scientific). All cryo-EM data were acquired using the EPU 2 software (Thermo Fisher Scientific). Microscope was aligned to produce fringe-free imaging (FFI) allowing five acquisition areas within a hole and aberration-free image shift (AFIS) was used to acquire images from up to 21 holes per single stage move. Movies were collected in electron counting mode at 96,000× corresponding to a calibrated pixel size of 0.805 Å/pix over a defocus range of −1.0 to −2.5 μm. 6,029 movies were collected using a dose rate of 5 e⁻/pix/s for a total of 5.6 s (207 ms per fraction, 27 fractions), resulting in a total exposure of ~40 e⁻/Å (1.5 e⁻/Å²/fraction).

Hurdiss D.L., Drulyte I., Lang Y., Shamorkina T.M., Pronker M.F., van Kuppeveld F.J., Snijder J, & de Groot R.J. (2020). Cryo-EM structure of coronavirus-HKU1 haemagglutinin esterase reveals architectural changes arising from prolonged circulation in humans. Nature Communications, 11, 4646.

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